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University of North Florida eBook Collection 

Structure-based Drug Design 

by Veerapandian, Pandi. 

New York Marcel Dekker, Inc., 1997. 

ISBN: 0824798694 

eBook ISBN: 0585157448 

Subject: Drugs--Design. Drugs--Structure-activity 

relationships. Drugs--Conformation. Drug Design. 

Structure-Activity Relationship. 

Language: English 

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Table of Contents 

Structure-Based Drug Design 

Preface 

Contents 

Contributors 

1 Inhibitors of HIV-1 Protease 

Help 

2 Structural Studies of HIV-1 Reverse 

Transcriptase and Implications for 

Drug Design 

3 Retroviral Integrase: Structure as a 

Foundation for Drug Design 

4 Bradykinin Receptor Antagonists 

5 Design of Purine Nucleoside 

Phosphorylase Inhibitors 

6 Structural Implications in the Design 

of Matrix-Metalloproteinase Inhibitors 

7 Structure—Function Relationships in 

Hydroxysteroid Dehydrogenases 

8 Design of ATP Competitive Specific 

Inhibitors of Protein Kinases Using 

Template Modeling 

9 Structural Studies of Aldose 

Reductase Inhibition 

10 Structure-Based Design of 

Thrombin Inhibitors 

11 Design of Antithrombotic Agents 

Directed at Factor Xa 

12 Polypeptide Modulators of Sodium 

Channel Function as a Basis for the 

Development of Novel Cardiac... 

13 Rational Design of Renin Inhibitors 

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14 Structural Aspects in the Inhibitor 

Design of Catechol O- 

Methyltransferase 

15 Antitrypanosomiasis Drug 

Development Based on Structures of 

Glycolytic Enzymes 

16 Progress in the Design of 

Immunomodulators Based on the 

Structure of Interleukin-1 

17 Structure and Functional Studies of 

Interferon: A Solid Foundation for 

Rational Drug Design 

18 The Design of Anti-Influenza Virus 

Drugs from the X-ray Molecular 

Structure of Influenza Virus Ne... 

19 Rhinoviral Capsid-Binding 

Inhibitors: Structural Basis for 

Understanding Rhinoviral Biology and 

f... 

20 The Integration of Structure-Based 

Design and Directed Combinatorial 

Chemistry for New Pharmaceut... 

21 Structure-Based Combinatorial 

Ligand Design 

22 Peptidomimetic and Nonpeptide 

Drug Discovery: Impact of Structure- 

Based Drug Design 

Index 

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1 

Inhibitors of HIV-1 Protease 

Krzysztof Appelt 

Agouron Pharmaceuticals, Inc., San Diego, California 

I. Introduction 

Page 1 

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired 

immunodeficiency syndrome (AIDS), perhaps the largest and most powerful consortium of scientists 

ever assembled to tackle a single disease has been brought to bear on the problem of AIDS and its 

treatment. From an unprecedented wealth of information regarding the molecular biology and virology 

of HIV collected in recent years, it became possible to identify numerous intervention points in the viral 

life cycle that could be exploited in the development of drugs for AIDS therapy (for reviews see 

Reference 1, 2, and 3). Among these, the virally-encoded enzymes, in particular reverse transcriptase 

and protease, have emerged as the most popular targets. A separate chapter of this book is dedicated to 

the description of reverse transcriptase and its inhibitors [4]. For the purpose of introduction only, it 

should be noted that nucleoside inhibitors of reverse transcriptase (AZT, ddI, ddC, d4T, and 3TC) have 

been widely used in clinical practice since 1987. Since then it has become apparent that this class of 

agents, while slowing progression of disease in HIV-infected patients, is limited in both activity and the 

duration of the clinical responses produced. Therefore in the search for better anti-HIV agents, the focus 

of effort was expanded to include the search for clinically useful inhibitors of a second viral enzyme, 

namely the protease. In contrast to reverse transcriptase, for which activity is required prior to the 

integration of viral genetic information into the host cell chromosomes, the viral protease plays a key 

role late in the virus life cycle and inhibitors of this enzyme display equal anti-viral activity in chronic 

and acute infection models in vitro [5]. 

The HIV protease (HIV PR) is encoded by the 5' portion of the retroviral pol gene, which encodes all 

replicative enzymes. Viral structural proteins (p24, 

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p17, p9, and p7) and replicative enzymes (protease, reverse transcriptase/ RnaseH, and integrase) are 

translated as either polyprotein P55-GAG, or a larger frameshift product P160-GAG-POL. In the 

process of virus assembly these polyproteins are proteolytically cleaved by the protease and this 

processing step, both in its timing and accuracy, is essential for the formation of infectious particles of 

HIV [6]. It was also shown early on that the inactivation of HIV PR, either by chemical inhibition or 

certain mutations, leads to the production of immature, noninfectious viral particles [7,8]. 

Page 2 

Structurally HIV PR is a 99-amino-acid protein translated initially as a central part of the P160-GAG- 

POL polyprotein precursor. The autocatalytic processing from the 160 kDa precursor is poorly 

understood, but most likely occurs during the process of budding of pre-formed viral particles from the 

host cell [9]. After release from the precursor polyprotein, HIV PR forms a homodimer and acts in trans 

to correctly process GAG and GAG-POL polyproteins—a process required for formation of the viral 

capsid and nucleoprotein core. 

Retroviral proteases such as HIV PR are the latest additions to the wellstudied family of aspartic 

proteases. This family of enzymes, which includes, among others, proteases such as pepsin, renin, and 

cathepsins D and E, has been intensely studied in the past, and the knowledge gained from studies of 

these enzymes allowed early inferences as to the structure and function of the dimeric HIV PR. 

Moreover, the intensive effort over the past two decades to make inhibitors of human renin provided 

impetus for the early design of inhibitors of HIV PR. In fact, some of the renin inhibitors have turned 

out to be effective inhibitors of retroviral aspartic proteases as well and have served as the starting point 

for drug design. As a result of this many early inhibitors of HIV PR were peptidyl in nature and the best 

known example of such compounds is Ro31-8959, better known as saquinavir, a hydroxyethylaminecontaining 

mimetic of a hexapeptide substrate [10]. This potent inhibitor of HIV PR was discovered 

using a substrate-based rational approach to drug design and displays extremely high in vitro activity 

against clinical isolates and laboratory strains of HIV. Saquinavir has been recently approved by the 

FDA for the treatment of AIDS in combination with nucleoside inhibitors of reverse transcriptase, and 

the discovery of this compound was the first breakthrough and the starting point for many other 

innovative designs. 

Determination of the crystal structures of HIV PR gave new impetus to the design of novel inhibitors. 

One measure of the intensity with which new inhibitors were designed or discovered is the total number 

of crystal structures of inhibitory complexes, currently exceeding 250, that have been determined over 

the past 5 years. Very detailed crystallographic analysis combined with extensive biochemical 

characterization and site-specific mutagenesis studies made HIV PR perhaps the best characterized 

enzyme to date. 


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Page 3 

Based on the avalanche of papers describing the structure-based design of various HIV PR inhibitors, it 

would be reasonable to assume that, with the exception of saquinavir, all other HIV PR inhibitors that 

entered the stage of preclinical or clinical development were discovered using the elements of a structurebased 

approach. From the long list of more than 30 inhibitors considered as clinical candidates [11], 

currently there are three compounds (saquinavir, ritonavir, and indinavir) already approved by the FDA 

as anti-HIV drugs. Many factors that are requisite for in vivo activity in AIDS patients can only be 

predicted a priori in a very general sense. For instance, erratic oral bioavailability in humans, first-pass 

metabolism, binding to plasma proteins or tissue distribution may disqualify a perfect in vitro inhibitor 

of HIV replication and such properties can be very poorly predicted by any process of drug design. A 

potential answer to these problems is the parallel design of several chemically distinct compounds that 

may have similar in vitro activity but significantly different in vivo properties. The application of protein 

structure-based design offers such possibilities and in this text the discovery and optimization of 

different series of potent inhibitors of HIV PR will be discussed. In order to familiarize the reader with 

the architecture of HIV PR and the properties of its active site, the first paragraphs are devoted to the 

detailed description of the x-ray structures of the enzyme followed by several examples of inhibitors in a 

bound conformation. 

A. Three-Dimensional Structure of HIV PR 

Retroviral proteases such as HIV PR were tentatively assigned to the aspartic protease family on the 

basis of putative active-site sequence homology [12]. Mammalian aspartic proteases are bilobal, singlechain 

enzymes in which each lobe (or domain) contributes an aspartic acid residue to the active site [13]. 

The active site itself is formed at the interface on the N- and C-terminal domains and exhibits 

approximate two-fold symmetry. Since the retroviral proteases are only about one-third the size of the 

two-domain eukaryotic enzymes, they were hypothesized to function as dimers in which each monomer 

contributes a single aspartic acid to the active site [14]. Obligate homodimeric proteases, in addition to 

providing a regulatory mechanism to control activation of the enzyme, represent the most efficient use 

of genetic information which, in retroviruses, is naturally parsimonious. 

The crystal structures of HIV PR confirmed the predicted dimeric character of the enzyme [15,16] 

(Figure 1). In all published crystallographic investigations of the unliganded form of the enzyme, the 

monomers are related to each other by crystallographic two-fold symmetry and are necessarily identical. 

The general topology of the HIV PR monomer is similar to that of a single-domain 


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Figure 1 

Stereo view of the α-carbon backbone of HIV PR dimer. (a) The apoenzyme 

with flaps in the “open” conformation. (b) Inhibited form of HIV PR with flaps in a 

“closed” conformation. For clarity, the inhibitor is removed from the active site. 

pepsin-like aspartic protease and consists of antiparallel β-strands and a short, two-turn α-helix 

connected by loops of varying length. The dimer interface is formed by an antiparallel β-sheet 

comprising two strands from each monomer. The hydrophobic residues from those β-strands and two 

symmetry-related α-helices form the core of the dimer. The dimer is further stabilized by a net of 

hydrogen bonds involving the residues around the catalytic aspartic acids. The active site is formed by 

the dimer interface and is composed of equivalent contributions of residues from each monomer. The 

substrate-binding cleft is bound on one side by the active site aspartic acid (Asp25/25') and on the other 

side by a pair of two-fold related, antiparallel β-hairpin structures, commonly referred to as “flaps.” 

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The conserved active-site residues (Asp25, Thr26, and Gly27 from both monomers) form a symmetrical 

and highly hydrogen-bonded arrangement virtually identical to that described for pepsin [17]. The two 

aspartates are nearly coplanar with the “inner” carboxylate oxygens hydrogen bonded to the amide 

hydrogens of Gly27/27'. This designation (e.g. Gly 27/27') will be used throughout this text to indicate 

equivalent residues of the dimer. The two threonines are inaccessible to solvent and are hydrogenbonded 

to the main-chain amide groups of the other monomer, forming a rigid network called a 

“fireman's grip” [17]. As in the case of the structures of eukaryotic pepsins, there is electron density for 

a water molecule bound between the two carboxylates of the active-site aspartates. 

Page 5 

In the structure of the apo-form of HIV PR, the flaps from both monomers are related by 

crystallographic two-fold symmetry and can be considered as being in an open conformation. In the 

structures of related proteases from Rous Sarcoma Virus and HIV-2, the flaps are either 

crystallographically disordered or in a partly closed conformation [18]. This suggests that, in solution, in 

the absence of ligands, the flaps are rather flexible and that the stable conformation of the flaps observed 

in the crystal structure of the apo-enzyme of HIV PR could be considered to result from kinetic trapping 

during the crystallization process. 

In the apo-form of HIV PR, the active site residues are located at the bottom of a rather shallow groove. 

Upon binding an inhibitor, the protease undergoes significant structural changes, particularly apparent in 

the flap region. As a result, a tunnel-like site is formed, which runs diagonally across the dimer 

interface. The tunnel has a volume of approximately 1140 Å 3 and is 23 Å long. Because of the dimeric 

nature of HIV PR, the active site has approximate two-fold symmetry with the dyad axis intersecting the 

plane of the catalytic aspartates. Along the active site tunnel, starting from the central aspartates, there 

are distinct subsites S1, S2, S3, and S4, and corresponding symmetry related subsites S1', S2' S3', and 

S4' (Figure 2). It should be noted that in this chapter, the convention of Schechter and Burger [19] will 

be used to describe enzyme specificity subsites (S1, S1', etc.) and the corresponding side chains of 

inhibitors (P1, P1', etc.). The boundaries of the subsites are formed by residues from both monomers of 

HIV PR. All subsites, with the exception of S4/S4', which are exposed to solvent, are bounded by mostly 

aliphatic side chains and have hydrophobic character. The borders of the S1/S1' subsites are formed by 

the side chains of Ile23/23', Ile50/50', Ile84/84', Pro81/81', the γ carbon of Thr80/80', carboxylates of the 

active site Asp25/25', and the carbonyl oxygens of Gly27/27'. The S2/S2' subsites are bounded by 

Val32/32', Ile50/50', Ile47/47', Leu76/76', Ala28/28', and the carboxylates of Asp30/30'. The S3/S3', 

subsites are partly exposed to solvent and are bordered by the side chains of Leu23/23', Val82/82', 

Pro81/81', and the guanidinium groups of Arg8/8', which form a salt bridge with the carboxylates of 

Asp29/29'. Most of 


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Figure 2 

Schematic representation of the specificity subsites of the HIV PR active site with bound 

peptidic inhibitor JG-365. Amino acids forming the boundaries of the particular 

subsites are shown. 

the hydrogen bond donor and acceptor functional groups of the active site are located in an approximate 

plane that lies along the long axis of the tunnel and is somewhat perpendicular to the plane of the 

subsites. The hydrogen-bonding functionalities include the carboxylates of the catalytic aspartates, the 

carbonyl oxygens of Gly27 and Gly48, the amide nitrogens of Asp29' and Gly48, the carboxylate of 

Asp29', and the dimer symmetry-related groups on the other side of the active site. Additional groups 

capable of forming hydrogen bonds with ligands are located in the outer part of the S2/S2' subsite and 

include the amide nitrogens and the carboxylates of Asp30/30'. There are five conserved water 

molecules in the active site of HIV PR. Four of the waters are symmetrically distributed in the S3/S3' 

subsites and one, hereafter called Wat301, is located near the two-fold axis of the dimer and, in the 

presence of most inhibitors, is approximately tetrahedrally coordinated by the hydrogen bonds formed 

between carbonyl oxygens of the ligand(s) and the amide nitrogens of Ile50/50' of the flaps. In the 

ligand-bound form of HIV PR, Wat301 is completely inaccessible to solvent, and it has been speculated 

that its functional substitution could be energetically favorable [18] or at least may lead to discovery of 

novel nonpeptidic inhibitors [20]. Thus, there are 18 hydrogen bond donors or acceptors in 


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Page 7 

the active site of HIV PR-16 that could form hydrogen bonds directly and two in which the interaction is 

mediated by the conserved Wat301. The total solvent accessible surface area of the eight subsites of the 

HIV PR active site is approximately 1150 Å 2. Because of the large number of groups with hydrogen-bondforming 

potential, 450 Å 2 of the surface has a polar character, and the nonpolar area of the subsites is 

slightly larger, approximately 700 Å 2. 

B. Structural Flexibility of HIV PR 

In the process of viral assembly, HIV PR specifically cleaves nine cleavage sites on GAG and GAG-POL 

polypeptides [21]. Examination of the amino acid composition of the recognized substrate sites (Table 1) 

indicates their hydrophobic character and significant sequence variability. The loose specificity of HIV PR 

most likely reflects its functions in a world of reduced complexity within the confines of the budding 

virion. The length of the viral protein precursors (approximately 1500 amino acids) reduces the number of 

potential sequences the protease must discriminate from in selecting its nine cleavage sites. Therefore, 

HIV PR and other retroviral proteases are not enzymes that have evolved to carry out a single reaction at a 

rapid rate, but rather enzymes with minimum specificity required to cleave the viral precursors in a 

specific and orderly manner. 

The loose specificity requirements demonstrated by effective binding and catalytic processing of all nine 

sequences, albeit at different rates [22], was the 

Table 1 The Sequences of the Proteolytic Processing Sites of HIV-1 

HIV-1 PR 

Cleavage sites 

Scissile bond 

P17/P24 V S Q N Y P I V Q N 

P24/P2 K A R V L A E A M S 

P2/P7 S A T I M M Q R G N 

P7/P1 E R Q A N F L G K I 

P1/P6 R P G N F L Q S R P 

TF/PR V S F S F P Q I T L 

PR/RT C T L N F P I S P I 

RT/RN G A E T F Y V D G A 

RN/IN I R K V L F L D G I 

Schechter-Berger 

notation 

P5 P4 P3 P2 P1 P1' P2' P3' P4' P5' 

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TF—transframe, PR—protease, RT—reverse transcriptase, RH—RNAse H, IN—integrase. The location of the 

processing sites in HIV-1 were determined by protein sequencing of HIV-1 virion proteins. 


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first indication that the recognition subsites of the HIV PR can display flexibility upon binding of 

substrates or inhibitors. Early crystal structures of the HIV PR apo-enzyme and complexes with peptidic 

inhibitors showed several conformations of the active site forming flaps, which include the residues 

Met46/46' to Ile54/54' [15,16]. Increased availability of coordinates of HIV PR complexed with various 

inhibitors and crystallized in different crystallographic space groups allowed for more rigorous 

examination of domain movements and structural changes in the active site. 

The alignment of several crystal structures of HIV PR in a common frame of reference, which most 

commonly includes the region around the symmetryrelated active site triad Asp25/25'-Thr26/26'- 

Gly27/27', will highlight those regions of the backbone where significant displacement occurs upon 

accommodating the individual inhibitors. Examination of the aligned structures, which included 

examples of all classes of inhibitors, indicated only small variation of the backbone and limited 

movements in the two binding loops, comprising residues Leu76-Ile84 from both monomers. The loops 

form the outer walls of subsites S1/S1' and S3/S3' with inward-facing hydrophobic side chains of 

isoleucines and valines. The flexibility of these loops, which in some cases can move outward by as 

much as 2.5 Å, has a significant impact on the volume of the specificity subsites, which in turn can 

accommodate corresponding P1/P1' and P3/P3' moieties of various sizes. Interestingly, the predominant 

resistance-causing mutations are located on the same loops and involve changes in residues Val82/82' 

and Ile84/84' (see below). It should be noted, that while the alignment of several crystal structures 

provides information about the flexible regions, the extent of flexibility of the residues around the HIV 

PR active site can be limited by crystal packing forces and may represent a crystallographic artifact. In 

all characterized crystal forms of HIV PR [23] the loops 76–84 and 46–56 participate in crystal lattice 

formation and the particular conformation of these loops can be driven by crystallization conditions or 

interactions with other molecules related by the crystallographic symmetry. 

C. Inhibitors of HIV PR 

In general, inhibitors of HIV PR can be divided into three distinct groups. The first group includes 

peptidic inhibitors that utilize various transition-state dipeptide analogs such as statine, hydroxyethylene, 

and hydroxyethylamine incorporated into peptidic frameworks of differing lengths. Several crystal 

structures of this type of inhibitor complexed with HIV PR were solved and the structural information 

provided a wealth of information as to the minimum size of inhibitors, geometry of hydrogen bonds 

formed within the active site, and the structural flexibility of the subsites (for reviews see Reference 18 

and 23). The second and perhaps largest group of HIV PR inhibitors includes peptidomimetic 


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compounds that utilize similar transition-state analogs and retain at least one peptide bond with a side 

chain corresponding to a naturally occurring amino acid. Several compounds from this group have 

excellent pharmacokinetic and antiviral properties and, in fact, all three HIV PR inhibitors approved for 

clinical use (saquinavir, ritonavir, and indinavir) belong to this class of compounds. The last and the 

smallest group of HIV PR inhibitors has a distinct nonpeptidic character. Compounds from this class 

were discovered either by screening libraries of existing compounds or by structure-based de novo 

design. Illustrative examples of inhibitors belonging to all three classes and a brief description of the 

discovery of selected compounds are presented below. 

D. Peptidic Inhibitors of HIV PR 

Page 9 

The concept of peptidic inhibitors of HIV PR can be exemplified by the crystal structure of the statinecontaining 

peptidic compound AG1002 (Figure 3) [23]. In AG1002, the statine moiety replaces the 

scissile dipeptide while the flanking amino acids were derived from the natural substrate cleaved by HIV 

PR. The inhibitor binds to the active site in an extended conformation with the central hydroxyl group of 

the statine moiety forming hydrogen bonds with the active-site aspartic acids 25/25'. The peptidic 

backbone and the side chains of the 

Figure 3 

Stereo view of the peptidic inhibitor AG1002 bound to the active site of HIV PR. 

The distribution of the specificity subsites S and S' is similar to that shown in 

Figure 2. The boundaries of the HIV PR active site are indicated by the dotted 

surface. 


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inhibitor form 16 hydrogen bonds and occupy subsites from S4 to S1, S2', and S3'. The carbonyl 

oxygens of P2 and P1' accept two hydrogen bonds from the flap water Wat301, which in effect is nearly 

tetrahedrally coordinated. Due to the structural nature of statine, which lacks the P1' side chain, the S1' 

pocket remains unoccupied. The S1 subsite is only partially filled by the P1 side chain of leucine. The 

P2 and P2' side chains of asparagine and glutamine form hydrogen bonds with Asp30' and 30, while the 

aliphatic carbons of both side chains make several hydrophobic contacts in the S2 and S2' pockets 

respectively. Despite the large number of hydrogen bonds formed within the HIV PR active site, 

AG1002 has rather low inhibitory potency with a binding constant of 0.55 μM The low binding constant 

most likely reflects the absence of the P1' group, the free energy required for desolvation of the 

hydrophilic side chains, and the charged N- and C-termini as well as entropic effects caused by the 

flexible nature of the heptapeptide. 

Other interesting examples of peptidic inhibitors are compounds utilizing other transition-state analogs, 

e.g. reduced amide-containing hexapeptide MVT-101 [24], hydroxyethylene-containing octapeptide U- 

85548e [25], and hydroxyethylamine-containing heptapeptide JG-365 [26]. All these compounds bind to 

the active site of HIV PR in a similar extended conformation and the small differences in the geometry 

of hydrogen bonds formed with HIV PR can be attributed to the different character and length of the 

transition-state analogs. The chemical structures and inhibition constants of these inhibitors are 

summarized in Table 2. Note that the inhibition constants cited throughout this chapter and in Tables 2, 

3, and 6 were determined in different laboratories—often using significantly different assay 

conditions—and therefore might not be meaningfully comparable. 

Due to their substantial size and peptidic nature, inhibitors from this class were not suitable for clinical 

application. Nevertheless, the structural information derived from many crystal structures of peptidic 

inhibitors bound to the HIV PR active site was critical for subsequent modeling and design of the next 

generation of peptidomimetic and nonpeptidic inhibitors of HIV PR. 

E. Peptidomimetic Inhibitors of HIV PR 

Design and Structure of Ro-31-8959 (Saquinavir) 

The strategy of designing saquinavir was based on the transition-state mimetic concept, an approach that 

has been used successfully in the design of potent inhibitors of renin and other aspartic proteases [10]. 

From the variety of nonscissile transition-state analogs of a dipeptide, the hydroxyethylamine mimetic 

was selected because it most readily accommodates the amino acid moiety characteristic of the Phe-Pro 

and Tyr-Pro cleavage sequence of the 


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retroviral substrates. In the first step of design, the dipeptide analog consisting of 

Phe[CH(OH)CH 2N]Pro was used to determine the minimum sequence required for potent inhibition. 

From this study a compound was selected that included benzyloxycarbonyl at the N-terminal side of the 

inhibitor followed by the P2 asparagine, the hydroxyethylamine isostere with side chains of 

phenylalanine and proline in the P1 and P1' positions respectively and the NH-t-butyl group at the Cterminal 

part. In the following design, the side chain of proline was consequently modified to a 

piperidine and finally to a decahydroisoquino- 


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line moiety, and the N-terminal benzyloxycarbonyl group was replaced by the quinoline-2-carbonyl. The 

resulting compound, Ro-31-8959, was one of the first peptidomimetic inhibitors with very high antiviral 

potency and became a benchmark for further design of HIV PR inhibitors [10]. 

The high-resolution crystal structure of saquinavir bound to the active site of HIV PR was solved in 

many laboratories [23,27]. The incorporation of decahydroisoquinoline moiety, which can be considered 

as a conformationally restrained mimic of cyclohexylalanine, has some important consequences. First, 

the length of the C-terminal part of the inhibitor has been restricted to the P2' residue which, in 

saquinavir, consists of a NH-t-butyl group. Second, it restrained the conformational freedom of the 

otherwise peptidic backbone, minimizing the entropic penalty to the free energy of binding. In the 

crystal structure of saquinavir with HIV PR (Figure 4), the decahydroisoquinoline in the preferred chairchair 

conformation, makes extended hydrophobic contacts in the S1' subsite. The bond between the 

methylene carbon and the nitrogen of decahydroisoquinoline is in the low-energy equatorial 

conformation and the nitrogen, even if protonated, is not in a position to form a hydrogen bond with the 

active-site residues. The central hydroxyl group is in the R(syn) conformation and is within the 

hydrogen-bond-forming distance with both carboxylates 

12640-0012a.gif 

Figure 4 

Stereo view of the peptidomimetic inhibitor Ro 31-8959 (saquinavir) bound 

to the active site of HIV PR. The distribution of the specificity subsites S and 

S' is identical to that shown in Figure 2. Note the stacking interaction 

between the quinoline moiety and the P1 side chain of phenylalanine. 


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of the active-site aspartates. Similar to Ag1002 and other peptidic inhibitors, the carbonyl oxygens of the 

P2 and P1' amides are within hydrogen-bonding distance of the flap water molecule; however, the 

geometry of the second hydrogen bond is distorted due to the additional spacing between both carbonyl 

groups. The nitrogen of the t-butylamide is displaced from the normal P2' position by approximately 1.8 

Å and, as a result, cannot form a direct hydrogen bond with the carbonyl oxygen of Gly27. Instead the tbutylamide 

nitrogen interacts via highly ordered water molecules with the amide nitrogen of Asp29 and 

the carbonyl oxygen of Gly27. The aliphatic t-butyl moiety occupies the S2' subsite and the position of 

the backbone in this region prohibits any further extension into the S3' pocket. The P1 and P2 side 

chains of phenylalanine and asparagine, respectively, occupy the corresponding subsites and have a 

similar conformation to the equivalent groups observed in peptidic inhibitors. In the crystal structure, the 

N-terminal quinoline-2-carboxylate is moved to the side and, as a result, the carbonyl oxygen forms 

hydrogen bonds with the ordered water molecule and with the amide nitrogen of Asp29'. The quinoline 

ring is in a low-energy conformation with respect to the preceding carbonyl oxygen, which places the 

aromatic nitrogen in unfavorable close contact (3.3 Å) to the carbonyl oxygen of the flap Gly48. 

Because of the absence of any further contacts with the HIV PR active site residues, the contribution of 

the quinoline moiety to the free energy of binding remains unclear. Perhaps in solution, a stacking 

interaction of the P1 phenyl ring and the aromatic quinoline restricts the conformational freedom of Ro- 

31-8959, in effect diminishing the free-energy loss due to the entropic and desolvation effects. 

Saquinavir, despite its distinct peptidomimetic character is a very potent inhibitor of HIV PR with an 

inhibition constant of 0.9 nM and an antiviral IC50 in vitro of 0.020 μM [10]. Although it suffers from a 

low oral bioavailability (5–10% in humans), it became an important starting point for the design of 

second generation, less-or nonpeptidic inhibitors. Saquinavir became the first HIV PR inhibitor 

approved by the FDA for treatment of AIDS. 

Design and Structure of ABT-538 (Ritonavir) 

An interesting concept for designing specific HIV PR peptidomimetic inhibitors with internal two-fold 

symmetry was first formulated by John Erickson and his colleagues from Abbott Laboratories [28]. 

They reasoned that if HIV PR incorporates symmetry into its active site structure, compounds that 

mimic this symmetry might be novel, more specific, and potent inhibitors and, furthermore, due to the 

bidirectionality of peptide bonds, might be sufficiently less peptidic in character and pharmacologically 

superior to the classical peptide-based compounds. The crystal structure of one of the first compounds 

from this series (A74704) verified the assumption of symmetrical binding conformation in the 

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active site of HIV PR. The inhibitor consists of the central diamino alcohol moiety with symmetrically 

distributed phenylalanine side chains and two flanking, Cbz-blocked, valine residues. Except for the 

asymmetric hydroxyl group, A74704 binds to the active site in a symmetric mode and the inconsistent 

distribution of the terminal Cbz groups is most likely caused by crystal lattice contacts and may not 

reflect the binding mode in solution [28]. 

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The design of symmetrical inhibitors was further extended to include a series of diamino, diol core units, 

in which the C2 axis bisects the bond connecting the two hydroxy-bearing carbon atoms [29]. Such 

inhibitors consistently showed greater potency than A74704, but the relative potencies of the diols 

differed for different diastereomers, and they did not exhibit a uniform dependence on the 

stereochemistry at the hydroxymethyl position. Surprisingly, high-resolution crystal structures of HIV 

PR with all possible diol diastereomers, (S,S, R,R and R,S) revealed that most of the inhibitors bind in a 

clearly asymmetric fashion placing only one of the diol hydroxyl groups on the C2 axis dissecting the 

active site of HIV PR and the catalytic carboxylates of Asp25/25'. The asymmetric placement of diols 

causes translation of inhibitors along the long axis of the active site and, as a result, the midpoint of the 

compounds is displaced by up to 0.9 Å from the two-fold axis of the HIV PR. Nevertheless, the dihedral 

angles of the symmetry-related bonds are in most cases within 10°, and the inhibitors maintain overall 

symmetry in the bound conformation [23,29]. 

The ABT-538 design was a direct consequence of the pioneering work with peptidomimetic compounds 

with the internal C2 symmetry [30]. Since the high-resolution crystal structures of a family of diolcontaining 

compounds indicated that in most cases only one of the diol hydroxyls interacts with the 

catalytic aspartic acids 25/25', in subsequent designs the noninteracting hydroxyl group was replaced by 

a hydrogen. This substitution reduced the free energy penalty required for desolvation of the hydroxyl 

group and increased the inhibitory potency without perturbing the binding mode of the compounds [30]. 

In the further search for related inhibitors with improved oral bioavailability, the focus of effort 

concentrated on the effect that molecular size, aqueous solubility, and hydrogen-bonding capability has 

on pharmacokinetic behavior. This study resulted in a smaller compound, A-80987, in which the P2' 

side chain of valine was eliminated and the terminal 2-pyridinyl group was replaced by 3-pyridinyl 

moiety that makes van der Waals contacts in the S2' subsite and forms a hydrogen bond with the amide 

nitrogen of Asp30 [31]. The pharmacokinetic properties of A-80987 were significantly improved over 

larger, symmetrical compounds from this series and, at the same time, the high antiviral activity typical 

for these inhibitors was largely unaffected. In subsequent optimization, which focused on the metabolic 

stability of these inhibitors in vivo, the electron-rich and oxidation-prone pyridinyl groups were replaced 

by thiazoles. 


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Thiazoles are less electron-rich isosteres of pyridines and therefore it was speculated that compounds 

with such substitution may have improved metabolic stability [30]. The modeling of A-82200 in which 

the N-terminal pyridinyl group was substituted by a 4-thiazolyl moiety indicated that the 5-membered 

ring binds in the S3 subsite and can be further derivatized at the 2 position by an isopropyl group. The 

isopropyl functionality makes van der Waals contacts with Val82 and fills the hydrophobic part of the 

S3 subsite in nearly optimal fashion. 

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The resulting compound, ABT-538 (Table 3), binds to the active site of HIV PR in an extended 

conformation. The central, asymmetric hydroxyl group is within hydrogen-bonding distance of the 

catalytic aspartates 25/25', and the P1/P1' phenylalanine side chains are symmetrically distributed in the 

corresponding subsites. The nitrogens of the symmetric amide bonds on both sides of the central 

aminoalcohol are barely within the hydrogen-bonding distance of the carbonyl oxygens of Gly27/27' 

(3.4 Å) and the carbonyl oxygens of these amide bonds participate in the tetrahydral coordination of the 

flap water molecule Wat301. On the N-terminal side of the compound, the P2 side chain of valine fills 

the S2 subsite and the terminal 2-isopropyl-4-thiazolyl makes hydrohobic contacts with the residues in 

the S3 pocket and has a stacking interaction with the P1 phenylalanine. On the C-terminal side, the 5thiazole 

is positioned to interact within the S2' subsite, and the nitrogen on the 5-membered ring is 

within hydrogen-bonding distance of the amide of Asp30'. 

Despite two peptide bonds present in ABT-538, this compound has substantial oral availability in 

humans and a very high antiviral activity in vivo [30]. Recently, ABT-538, better known as ritonavir, 

has been approved by the FDA for treatment of AIDS in combination with inhibitors of the reverse 

transcriptase. 

Design and Structure of L-735,524 (Indinavir) 

Indinavir is another example of very potent peptidomimetic compound discovered using the elements 

the crystal structure-based design [32] and SAR (structure activity relationship). The starting point for 

the design was a series of compounds containing the hydroxyethylene isostere of a scissile dipeptide 

[33]. An example of compounds from these series is L-685,434, which consists of a tert-butylcarbamate 

group forming the P2 moiety, symmetrically distributed phenylalanine side chains in the P1/P1', and the 

indanol group in the P2' portion of the inhibitor. Although very potent, the optimized molecules from 

this series lacked aqueous solubility and an acceptable pharmacokinetic profile [32]. The Merck group 

hypothesized that incorporation of a basic amine-containing functionality, such as the 

decahydroisoquinoline group of saquinavir, into the backbone of L-685,434 series might improve the 

solubility and bioavailability of this type of compound. Also the replacement of the P2/P1 

functionalities, the tert-butylamide and phenylalanine side chain by the decahydroisoquinoline tertbutylamide, 

would generate a novel class of hydroxylaminepentanamide isostere with potentially 

improved metabolic stability in vivo. An additional strong argument for using decahydroisoquinoline as 

an isostere of P1/P2 moieties was the restricted conformational freedom of the enclosed-into-a-ring 

basic amine, which should decrease the entropy change upon binding to HIV PR in a similar fashion to 

that observed in saquinavir. In 


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the resulting chimeric inhibitor the central hydroxyl group forms hydrogen bonds with the catalytic 

aspartic acids 25/25' and the hydrophobic side chains of the P1/P1' decahydroisoquinoline and 

phenylalanine respectively are separated from the central hydroxyl-bearing carbon by the methylene 

linkers forming a pseudosymmetrical arrangement. In the subsequent optimization of inhibitors from 

this novel series, a smaller piperazine group was substituted for the decahydroisoquinoline group, which 

offered a possibility to expand from the N4 position to the partially lipophilic S3 subsite. One of the first 

compounds from the piperazine series possessed a benzyloxycarbonyl moiety attached to the piperazine 

ring and the additional hydrophobic interaction in the S3 subsite was reflected by substantial increase in 

both intrinsic potency and in the ability to inhibit viral spread in infected cells in vitro. Finally, the 

replacement of the benzyloxycarbonyl group by the 3-pyridylmethyl moiety (Table 3) provided both 

lipohilicity for binding to the HIV PR active site and a weakly basic nitrogen that increased aqueous 

solubility and oral bioavailability. The crystal structure of L-735,524 (indinavir) bound to the active site 

of HIV PR [34] indicates that the 3-pyridylmethyl group attached to the N4 position of the piperazine 

ring makes hydrophobic contacts with the residues in the S3 and S1 pockets and the tert-butyl moiety 

fills the S2 subsite in the fashion previously observed in the structure of saquinavir. The positions of the 

P2 and P1' carbonyls maintain the proper alignment to form hydrogen bonds with the flap water 

Wat301. The terminal indanol group of indinavir occupies the S2' subsite with the hydroxyl group 

within hydrogen-bonding distance of the amide nitrogen of Asp29. 

The high aqueous solubility and largely nonpeptidic character of indinavir may be responsible for the 

good oral bioavailability, respectable pharmacokinetic profile, and high antiviral activity observed with 

this compound. Similar to saquinavir and ritonavir, indinavir has been recently approved by the FDA for 

treatment of AIDS. 

F. Nonpeptidic Inhibitors of HIV PR 

The nonpeptidic inhibitors of HIV PR can be divided into two subclasses. Compounds that belong to the 

first group maintain the general binding mode of the peptidomimetic inhibitors including formation of 

the key hydrogen bonds with the active site residues. An example of such nonpeptidic inhibitors of HIV 

PR is AG1343 (nelfinavir). The second group of nonpeptidic HIV PR inhibitors includes compounds 

with a binding mode significantly different from that described for the peptidomimetic compounds. 

Most inhibitors in this latter class were initially discovered by screening natural-products libraries or by 

structurebased de novo design. The most interesting examples of the nonpeptidic inhibitors from this 

group are the independently discovered but structurally 


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related 4-hydroxypyrans and 4-hydroxycoumarins, the cyclic urea-based DMP323 series, and AG1284. 

Design and Structure of AG1343 (Nelfinavir) 

Analysis of the crystal structure of saquinavir with HIV PR indicated that while the nonpeptidic 

components of the ligand, namely the decahydroisoquinoline and the t-butylamide moieties fill the S1' 

and S2' subsites nearly optimally, the N-terminal portion offered the possibility for remodeling, aimed at 

the elimination of the peptidic character. Also, the contribution of the quinoline to the binding affinity to 

HIV PR was difficult to rationalize. Since the removal of quinoline resulted in a nearly 1000-fold loss in 

binding constant, it was concluded that the stacking interactions of the P1 phenyl ring and the P3 

aromatic moiety of quinoline are necessary for the conformational stability of Ro 31-8959. In an attempt 

to redesign the N-terminal part of the ligand, the nonpeptidic portions of the P1' and P2' were maintained 

but for reasons of synthetic accessibility, the decahydroisoquinoline moiety was replaced by an orthosubstituted 

benzylamide [35]. Crystallographic analysis of both compounds showed that saquinovir and 

the modified LY289612 bind essentially identically to the active site of HIV PR and their inhibition 

constants and antiviral activity were very similar (Table 4 and Table 3). 

In the first attempt to functionally substitute the P2 side chain of asparagine, the isophthalic-acidcontaining 

compound was modeled and the low-energy conformation of the aromatic ring, required for 

binding in the S2 subsite, was stabilized by a tertiary carboxamide in the P3 region of the inhibitor [36]. 

The analysis of the binding mode and interactions of the isophthalic ring in the S2 subsite indicated a 

lipophilic pocket deep on the border between the S2 and S1' subsites, which could be conveniently filled 

with a methyl group extending from the 2 position of the ring. The resulting compound II in Table 4 lost 

most of the peptidomimetic character of LY289612 but retained its inhibitory potency. 

In an independent line of design, the relationship between the P1 phenylalanine side chain and the P3 

quinoline was investigated. In the crystal structure of saquinavir bound to the active site of HIV PR 

(Figure 4), the aromatic ring of the P1 phenylalanine makes several van der Waals contacts with 

residues forming the S1 subsite. Computer modeling indicated that an extension of the phenylalanine 

side chain to phenethyl (homophenylalanine) would lead to prohibitive close contacts of the phenyl ring 

with the aliphatic side chains of HIV PR. On the other hand, replacement of the γ-carbon of the 

homophenylalanine by sulphur, which has a more acute C-S-C bond angle, would direct the aromatic 

ring into the neighbouring S3 subsite without changing the desired lipophilic nature of the P1 side chain. 

The increased area of hydrophobic interactions in the S1 and S3 subsites by compounds with the Sphenylcysteine 

and 


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S-naphthylcysteine derived side chains in P1 resulted in a substantial increase in the inhibition constants 

[37]. The increase in the binding affinity to the low picomolar range in enzyme inhibition assay, allowed 

for subsequent truncation of the P3 quinoline moiety. The final compound from this miniseries 

(compound III in Table 4) consisted of the ortho-substituted benzamide in the P1' and P2', Snaphthylcysteine 

in P1 and asparagine in P2. Despite reduced molecular weight, the inhibition constant 

of this compound for HIV PR was comparable to LY289612. 

The observation that a larger, nonpeptidic moiety in the P1 could eliminate the need for the P3 side 

chain led to hybrid molecules that incorporated ring structures as the P2 component and maintained the 

P1 S-naphthylcysteine side chain of compound III. In this miniseries several bicyclic functionalities 

were modeled as the P2 substituents and one example, compound IV utilizing a tetrahydroquinoline 

group, is shown in Table 4 [38]. In subsequent modeling, it was noticed that the P2 bicyclic functionality 

might be replaced by 2,3-disubstituted phenyl rings. In particular, a methyl substitution in position 2 

would increase the area of hydrophobic interaction in a manner previously observed in the isophthal 

series. Addition of a hydrophilic functionality attached at position 3 could increase the solubility of the 

compound and contribute to the binding constant by forming a hydrogen bond with the carboxylate 

oxygen of Asp30. A compound with a 2-methyl-3-hydroxy substitution pattern was synthesized and 

showed an improved inhibition constant of 3 nM in the HIV PR enzyme assay (Table 4). The crystal 

structure of compound V with HIV PR was solved and indicated the predicted binding mode with the 

possibility of a stacking interaction between the P2 phenyl and the P1 thio-naphthyl groups and the 

expected hydrophobic and hydrogen-bonding interactions of the P2 moiety with the protein side chains 

in the corresponding specificity pocket [38]. 

As with the optimized compounds from other series, compound V suffered from low aqueous solubility. 

The replacement of the P1' aryl group by the basic amine-containing decahydroisoquinoline 

dramatically increased the solubility and allowed for truncation of the P1 S-naphthylcysteine side chain 

to S-phenylcysteine without any loss of inhibitory activity. The resulting compound VI, AG1343 or 

nelfinavir, has an inhibition constant of 1.9 nM in the HIV PR enzyme assay and respectable antiviral 

activity with an IC 90 of 60 nM [39]. The nonpeptidic character, pH-dependent solubility profile, and the 

small molecular weight of nelfinavir may contribute to its good pharmacokinetic profile in humans 

[40,41]. Currently, this compound is being tested and is in the advanced phase of clinical trials. 

The crystal structure of nelfinavir bound to the active site of HIV PR is shown in Figure 5. The general 

binding mode of this compound, in particular the path of the backbone, is similar to the binding mode of 

peptidyl inhibitors. Nevertheless, the lack of any peptide bonds utilizing naturally occurring amino 


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Figure 5 

Stereo view AG1343 (nelfinavir) bound to the active site of HIV PR. 

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acids qualifies nelfinavir to be a member of the group of nonpeptidic inhibitors of HIV PR. The unique, 

and perhaps crucial hydrogen-bonding interaction of the P2 hydroxyl group with the carboxylate oxygen 

of Asp30, combined with the smaller area of hydrophobic contacts in the S1 and S3 specificity subsites 

are the principal differences from other clinically active HIV PR inhibitors and may contribute to a 

distinct resistance pattern and point to additional utility of nelfinavir in the treatment of AIDS. 

Design and Structure of DMP323 

A cyclic urea-containing HIV PR inhibitor, DMP323, was discovered using de novo structure-based 

design principles. Similar to the concept of Erickson and his co-workers from Abbott Laboratories, the 

group from DuPont-Merck attempted to take advantage of the two-fold symmetry of HIV PR in 

designing compounds that maintained the interaction of the diol with the catalytic aspartic acids 25/25' 

and at the same time were able to functionally displace the ubiquitous flap water molecule Wat301. 

They hypothesized that incorporation of the binding features of this structural water molecule into an 

inhibitor would be beneficial because of the entropic gain due to its displacement and because the 

conversion of a flexible linear inhibitor into a rigid, cyclic structure with restricted conformation should 

provide an additional, positive entropic effect. In the initial design, a cyclohexanone with the ketone 

oxygen as the structural 


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water mimic was used and in subsequent synthetic targets the cyclohexanone ring was enlarged to a 7membered 

ring to incorporate a diol functionality. This target was further modified to a cyclic urea, 

which can be symmetrically substituted from both nitrogens without creating unnecessary stereocenters. 

The crystal structures of about 10 cyclic-urea-based inhibitors with HIV PR were solved [42]. In all 

cases, the C2 symmetric inhibitors bind to the HIV PR active site with the diad symmetry axes of the 

protease and the compounds being nearly coincident. The 7-membered ring of the inhibitors is roughly 

perpendicular to the plane of the catalytic aspartates 25/25' and both hydroxyl groups of the diol are 

positioned to interact with their carboxylates. The carbonyl oxygen of the inhibitors accepts hydrogen 

bonds from backbone amides of symmetrically distributed residues Ile50/50' of the flap. In the structure 

of DMP323, symmetrically substituted moieties of hydroxymethylbenzyls and phenylalanines extend 

towards the S2/S2' and S1/S1' subsites respectively and are involved in van der Waals interactions with 

the hydrophobic residues of these pockets [42]. 

The interaction of DMP323 with the residues of HIV PR are restricted to the central four specificity 

subsites of the active site. Despite this limited area of hydrophobic interaction and hydrogen bonding 

restricted to the central cyclic urea functionality, DMP323 is a very potent inhibitor of HIV PR with 

good antiviral activity in vitro (Table 5). The limited solubility of this compound was perhaps 

responsible for erratic oral availability in humans, and after short trials, DMP323 was withdrawn from 

the clinical investigation. Nevertheless, the discovery of this class of compounds represents a very 

interesting and, by now, classical example of de novo structure-based drug design. 

Design and Structure of AG1284 

Another compound discovered by the application of de novo structure-based design is AG1284 [43]. In 

the initial design of a lead compound, the nonpeptidic hydroxyethyl-t-butylbenzylamide portion of 

LY289612 occupying the S1' and S2' subsites was retained as a “starting module.” In attempting to fill 

the pockets related by the dimer two-fold symmetry it was discovered that, by extending a two-carbon 

fragment from the central hydroxyl carbon, the S1 subsite could be accessed by an aromatic ring. The 

ring was oriented orthogonal to the observed P1 phenyl group of the classical inhibitors and this allowed 

further extension off the ortho position towards the S2 subsite. In order to maintain the critical hydrogen 

bond to the flap water Wat301, in the initial compounds an acylated amino group was used, replaced in 

subsequent designs by a benzamide functionality. In this model, the geometry of the hydrogen bonds to 

the flap water was somewhat perturbed, and the nitrogens of the t-butyl amides on both sides of the 

compound were in a position to interact favorably with solvent, 


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potentially lowering the desolvation penalty. The absence of hydrogen-bonding interactions with the 

carbonyl oxygens of Gly27/27' was viewed as a positive factor, since the accumulated structural and 

mechanistic information suggested that formation of these hydrogen bonds may not be energetically 

favorable [23]. The compound was synthesized as a racemic mixture of two enantiomers of the central 

hydroxymethyl group and had the inhibition constant of 24 μM. Despite the modest binding constant of 

compound II (Table 6) and very low water solubility, the co-crystal structure with HIV PR was solved at 

2.3 Å resolution, providing a critical starting point for further design. The inhibitor was found to bind 

largely as anticipated with the two aromatic rings occupying the S1 and S1' subsites and the two 

benzamide carbonyls forming hydrogen bonds to the flap water Wat301. Both benzamide nitrogens 

interact via a string of highly ordered water molecules with the amide nitrogens of Asp29/29'. The 

crystal structure of the complex indicated that the S enantiomer was the more active component of the 

racemic mixture and this was confirmed by stereoselective synthesis of subsequent compounds [44]. 

In the subsequent designs, the ortho-substituted benzyl rings were consecutively replaced by larger 

naphthyl groups that occupied more of the S1–S3 and S1'–S3' subsites. The increased area of 

hydrophobic interactions with the residues in these subsites was reflected in a substantial improvement 

in the 


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binding constant and also in reduced aqueous solubility. Also, due to the very tight fit of both naphthyl 

moieties in the S1 and S1' subsites, subsequent design targeting the S3/S3' subsites proved to be difficult 

and synthetically challenging [44]. In the search for a simpler solution, the di-tertiary amides were 

designed using the crystal structure of compound II (Table 6) as a starting model. Branching from the 

amide nitrogens provided an interesting possibility to access S2–S3/S2'–S3' subsites while 

simultaneously increasing the solubility and stability of the compounds. In the first design, the 

hydroxyethyl moieties were fused to the amide nitrogens and the hydroxyl groups were intended to form 

hydrogen bonds with the amide nitrogens of Asp29/29' (compound III in Table 6). The addition of both 

hydroxyethyl groups resulted in a rather significant increase in the binding constant, and the racemic 

mixture had the K i of 1.1μM. When the crystal structure of compound III complexed with HIV PR was 

solved at 2.2 Å resolution, it was observed that the inhibitor had undergone an inversion in binding 

mode relative to the secondary amide series. The phenyl groups of compound III occupied the S2/S2' 

subsites, switching positions with the t-butyl groups, which were in turn occupying the S1/S1' pockets 

(Figure 6). Due to this change in binding mode, the R enantiomer would be expected to be preferred 

relative to S. The final position of the hydroxyethyl moieties was less effected by the change, and both 

hydroxyls were within hydrogen-bonding distance from the amide nitrogens of Asp 29/29'. In the S2/S2' 

pockets, the phenyl groups occupied only a fraction of subsites, but the interaction was strengthened by 

highly ordered water molecules involved in electrostatic interaction with the aromatic rings and by 

forming hydrogen bonds to Asp30/30'. Interestingly the position of the hydrogen bonds with respect to 

the flap water was significantly disturbed in the new binding mode, and the conserved Wat301 was no 

longer tetrahydrally coordinated [43,45]. 

This unanticipated change in binding mode presented a potential for new avenues of design different 

from those of the secondary amides. The ability to design into neighboring subsites depends to a large 

extent on the positions of bond vectors suitable for substitution in the bound conformation of a given 

inhibitor. These vectors in the crystallographically discovered new binding mode of compound III were 

positioned ideally to access unfilled space in the S3/S3' pockets. The discovery of this new conformation 

of compound III highlighted the power of crystallographic feedback in the process of inhibitor design 

and, without this structural information, further design in this series would have been severely impeded. 

Inspection of the crystal structure of compound III bound to the active site of HIV PR revealed 

lipophilic cavities extending off the S1/S1' subsites adjacent to the t-butyl groups of the benzamidine 

moiety. The cavities are bordered by flexible loops around Pro81/81' and previous crystallographic 

studies indicated that both loops can move back by up to 2.5 Å, extending the size and 


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Figure 6 

Change of the binding mode of compound III observed during 

iterative design of AG1284. (a) Crystallographically determined 

binding mode of compound II. Pseudosymmetrically distributed aryl groups 

are bound in the S1 and S1' specificity subsites. (b) Crystallographically 

determined binding mode of compound III. Note the inversion of the binding 

mode. The ortho-substituted benzyl groups bind in a pseudosymmetric 

fashion in the S2 and S2' subsites. 


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volume of the active site. With this in mind, the dimethylbenzyl group was attached to compound III and 

the additional phenyl ring was accommodated well in the lipophilic pocket of the S1'/S3' sides. As the 

S1 pocket was not fully occupied, a Monte Carlo-based De Novo Ligand-Generating program 

(MCDNLG) [46] was used to identify other amide substituents that might fill this subsite more 

effectively. From several moieties identified by the MCDNLG program, a larger cyclopentylethyl group 

showing very good shape complementarity to the S1/S3 subsite was selected for synthesis. In addition, 

due to the asymmetrical nature of this compound, additional space was identified at the bottom of the 

S2' pocket that was conveniently filled with either a methyl or a chlorine group on the 5 position of the 

benzamidine ring. The inhibition constant of the resulting compound (compound IV in the Table 6) was 

0.008 μM, which represents approximately a 2500-fold improvement over the first compound from this 

series. 

The crystal structure of compound IV or AG1284 complexed with HIV-1 PR was solved, revealing 

excellent complementarity between the ligand and protein. The ligand forms only 4 hydrogen bonds 

with either protein functional groups or ordered water molecules, in contrast to the nine hydrogen bonds 

formed by peptidomimetic LY289612, despite their similar binding affinities. The nonpeptidic character 

of AG1284 may have contributed to good oral bioavailability and pharmacokinetics in three animal 

species [43]. 

Despite very good inhibitory potency on the enzyme level, AG1284 has rather modest antiviral activity 

in vitro (Table 6). The reason for this discrepancy is unclear but could be related to the low water 

solubility and higher affinity for membranes, which may effect cell partitioning. A similar lack of 

correlation between the potency of enzyme inhibition and antiviral activity has been previously observed 

with other HIV PR inhibitors [11]. 

Hydroxypyrans and Hydroxycoumarins 

The lead compounds for the 4-hydroxypyran and 4-hydroxycoumarin series were discovered in 

biological screens as low potency inhibitors of HIV PR [47–49]. Successful structure solution of both 

lead compounds with HIV PR enabled rapid optimization of their enzyme inhibitory potencies and anti- 

HIV activities, and one of these compounds, U96988, has already entered Phase I clinical testing 

[49,50]. The binding mode of this type of inhibitor differs substantially from the classical 

peptidomimetic compounds and is somewhat similar to de novo-designed compounds from the cyclic 

urea series. In the case of 4-hydroxycoumarin, the two oxygen atoms of the lactone functionality are 

positioned within hydrogen-bonding distance of the two NH amides of Ile50/50' on the flap, replacing 

the ubiquitous water molecule Wat301. The 4-hydroxyl group (Table 5) is located within hydrogenbonding 

distance of the two catalytic 


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aspartic acid residues Asp25/25' and this hydrogen-bonding network of the 4-hydroxycoumarin defines 

the essential pharmacophore of this new class of inhibitors. In the structure of U96988, this 

pharmacophore is pseudosymmetrically subsituted by an ethyl and a phenyl group at the C-3a and an 

ethyl and a benzyl group at the C-6a positions. These four substituents extend into the central core of 

S2/S2' subsites, where they make van der Waals contacts with the hydrophobic residues of the active site 

[49]. With a molecular weight of 362 U96988 is the smallest inhibitor of HIV PR in clinical testing. It 

suffers from rather low antiviral activity (ED90 of ~ 10 μM)but can be considered as the first in a series 

of this promising class of nonpeptidic HIV PR inhibitors. 

II. Structural Basis of Resistance of HIV PR Inhibitors 

The dimeric character and the two-fold symmetry of the active site, in which the monomers contribute 

equivalent residues to symmetrically distributed specificity subsites, led to early speculations that HIV 

PR may be less susceptible to resistance than, for example, reverse transcriptase. In the case of retroviral 

proteases, a single base mutation in the viral genome corresponds to two changes in the threedimensional 

structure and two structurally identical changes in the active site could result in an enzyme 

with a drastically modified specificity profile and impaired catalytic activity. Identification of HIV PR 

variants in cell-culture experiments clearly indicated, however, that this class of drugs is not immune to 

the challenge of viral resistance. It should be stressed that HIV, unlike other human viruses, is 

characterized by a dynamic viral turnover in the steady state [51,52]. The rapid replication rate coupled 

with the lengthy duration of infection will favor the emergence of resistant mutants to targeted antiviral 

agents [53]. 

The accumulated data from cell-culture sequential-passage experiments with several structurally 

different inhibitors and from the resistant variants identified during clinical exposure to four HIV PRtargeting 

drugs indicate a very complex pattern of mutations in the structure of HIV PR. In contrast to 

mutations in the reverse transcriptase, which frequently cause multihundredfold resistance [54], single 

base changes in the HIV PR gene (i.e., two identical substitutions per protease dimer) lead in most cases 

to 5–10-fold decrease in the antiviral potency of a given drug [11]. It has been shown for the most 

clinically studied HIV PR inhibitors, such as indinavir and ritonavir, that the clinical manifestation of 

resistance (increase in the viral load and decrease in the CD4 count) requires the simultaneous 

appearance of several mutations [55,56]. For example the resistant HIV strain isolated from patients 

exposed for 40 weeks to indinavir carried mutations at residues 10/10'L > R, 46/46'M > I, 63/63'L > P, 

82/82'V > T, and 84/84'I > V [59,60]. However, the combination of these five 


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Figure 7 

Cartoon representation of the HIV PR dimer. The sites of primary 

resistance-causing mutations in the active site are indicated. For clarity, the names of the 

residues are shown for one monomer only. 

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mutations (ten assuming the dimeric nature of HIV PR) changed the susceptibility of the resistant strain 

to indinavir by only eight-fold if compared to the wild type HIV. 

The resistance-causing mutations are localized in a few “hot spots” in the structure of HIV PR and can 

be divided into two groups. The first group consists of the primary mutations located directly in the 

active site and includes changes at residues Val82/82', Ile84/84', somewhat less frequently at Gly48/48' 

and, in the case of nelfinavir, Asp30/30' (Figure 7). Residues 82/82' and 84/84' are located on the 

flexible loops that form the outer walls of the S3/S3' and S1/S1' subsites, respectively. In the resistant 

variants, valine 82/82' is most frequently substituted by the smaller side chain of alanine or the larger 

side chains of phenylalanine or isoleucine [57,58]. The change in position 82/82' is usually accompanied 

by a substitution of Ile84/84', most commonly to the smaller amino acids alanine or valine [57]. From 

the clinically tested compounds, ritonavir and indinavir, which were optimized to form strong 

hydrophobic interactions with the side chain of Val82/82' in the S3 subsite, suffer most significantly 

from any change at this position. On the other hand, the antiviral activities of saquinavir, and nelfinavir, 

which do not form any interaction in the S3/S3' subsite are not affected by mutations at Val82/82' and 

are only marginally cross resistant to changes involving Ile84/84' [57,58,62]. The resistance-causing 


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mutation of Asp30/30' to asparagine seems to be specific for nelfinavir and was initially observed in cellculture 

sequential passage experiments [62]. Recently, the same phenotypic change was confirmed as 

the predominant mutation in the resistant variants appearing in AIDS patients exposed to low doses of 

this HIV PR inhibitor [64]. The molecular basis of resistance involving this mutation can be rationalized 

as follows: in the crystal structure of nelfinavir with the wild type HIV PR, the 3-hydroxyl group of the 

2,3-substituted phenyl group is within hydrogen-bonding distance of the carboxylate oxygen of Asp30 in 

the S2 subsite. Due to the expected coulombic character of this interaction, the hydrogen bond formed 

with the negatively charged carboxylate of Asp30 would be expected to be a relatively strong one. The 

change of the negatively charged carboxylate of Asp30/30' to the amide oxygen of the asparagine side 

chain should reduced the strength of this interaction. Apparently the loss in the enthalpic contribution to 

the free energy of binding is only partially balanced by the entropic gain caused by the difference in 

desolvation of a charged vs. neutral side chain of the receptor, leading to decreased binding affinity of 

nelfinavir and eventually to viral resistance. 

An additional resistance-causing mutation that qualifies as a primary mutation involves the change of 

Gly48/48' to valine. This particular mutation seems to be specific for saquinavir and was observed both 

in cell-culture sequential passage experiments and in AIDS patients exposed to this inhibitor [61,65]. 

Located on the lower strands of the active-site forming flaps, Gly48/48' can be considered a part of the 

S4/S4' subsites. The replacement of the glycine hydrogen by the rigid side chain of valine has most 

likely a dual effect: first it has a direct impact on the interaction of the quinoline moiety of saquinavir 

with the active site of HIV PR, and second it may change the mobility of the flaps, which in turn will 

effect the binding kinetics of the natural substrates or inhibitors. Although none of the other clinically 

tested inhibitors form any interaction with this part of the flap, the HIV variants with mutation of 

Gly48/48' seem to be cross-resistant to all compounds, which is reflected by a 3–5-fold reduction of 

their antiviral activity [55,62]. 

While the effect of primary mutations on reduced binding affinities of inhibitors can be at least partially 

explained in view of the accumulated structural data, the function of secondary, or compensatory 

mutations in the resistant HIV PR is difficult to rationalize as yet. The predominant compensatory 

mutations observed in the resistant variants involve residues Leu63/63', Ala71/71', Met46/46', 

Asn88/88', Leu10/10', and Leu90/90' (Figure 8) [60,63]. Changes of these residues alone do not confer 

viral resistance, but their appearance increases the viability of the virus carrying the primary mutations 

in the active site of protease. All these residues are located far away from the active site of HIV PR do 

not participate in any apparent way in the inhibitor binding and it seems unlikely that they form a longrange 

interaction with the natural 


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Figure 8 

Cartoon representation of the HIV PR dimer. The sites of compensatory 

mutations are indicated. 

substrate. Also, the reported compensatory mutations are conservative in nature and have no effect on 

the overall distribution of atomic charges on the surface of HIV PR. 

Page 31 

Sequence polymorphism at the Leu63/63' position, located on the surface at the base of HIV PR, has 

been observed in clinical isolates of the virus not exposed to any HIV PR inhibitors. Variations of 

Ala71/71', where the side chains are buried very close to Leu63/63', are less commonly found in clinical 

isolates. After a prolonged challenge by HIV PR inhibitors, Leu63/63' changes to proline and Ala71/71' 

to valine. 

The side chains of Met46/46' are fully exposed to solvent and these residues are located on the βhairpins 

that form the active side flaps. It has been speculated that the compensatory change of 

Met46/46' to isoleucine or phenylalanine may affect the dynamics of the flap movement, which in turn 

could influence the rates of catalytic activity of HIV PR impeded by the primary mutations in the active 

site [58]. 

Any changes to Asn88/88' and Leu90/90', buried in the body of HIV PR, most likely affect the structural 

stability of the enzyme. The side chains of Asn88/88' form buried hydrogen bonds and replacement of 

this residue by aspartic acid or serine not only eliminates some of these bonds but also introduces 

unfavorable interactions in the core of HIV PR. Similarly, Leu90/90' is buried in a tight hydrophobic 

space close to the “fireman's grip” motif, which 


Document 

involves the catalytic asparates 25/25'. The structural effect of a mutation of Leu90/90' to the larger 

methionine is rather difficult to predict since it can either rigidify or destabilize the HIV PR dimer or it 

may have an effect on the catalytic efficiency of the “resistant” enzyme. 

Page 32 

The complicated pattern of HIV resistance to protease inhibitors, in particular the appearance of 

compensatory mutations that alone do not confer any resistance, suggests that the key to understanding 

the basis of decreased susceptibility of the virus to a given drug is the kinetics of specific processing of 

the GAG and GAG-POL polyproteins. The reduction in sensitivity of a mutant HIV PR towards any 

inhibitor can be conveniently reflected by the ratio of K i mutant/K i wild type. However, this reduced 

inhibitor sensitivity is only one component that distinguishes mutant-form from wild-type proteases. For 

virus encoding of a mutant HIV PR to be viable, the mutant protease must be capable of a minimal 

(although not yet quantified) level of enzymatic activity towards all substrates required for maturation of 

the virions. This proteolytic efficiency is reflected in the specificity constants (K cat/K m) as determined for 

mutant and wild type HIV PRs. In order to rationalise these potentially conflicting relationships between 

enzymes, substrates, and inhibitors, Gulnik and his colleagues [66] introduced the term “Vitality 

Factor,” in which 

In order for the “Vitality Factor” to be predictive for the level of resistance expected for a particular drug 

or combination of drugs for a given resistant strain of HIV, the determination of the specificity constants 

(K cat/K m for mutants) must be repeated for all nine known substrates processed by HIV PR. The 

inhibition constants of a given compound should not depend on the substrate, but the K cat/K m ratios do 

and therefore vitality values will differ for different substrates. It will be expected that the mean for all 

nine “vitality” values will be predictive for the change in antiviral activity for a particular compound. 

Although those data will be derived from in vitro experiments and are clearly not without some 

limitations, they may help in understanding the molecular basis of resistance and may contribute some 

value to possible multidrug strategies for the clinical management of AIDS. 

III. Perspective 

HIV PR inhibitors with acceptable oral availability and pharmacokinetic properties offer great promise 

for the treatment of HIV infection and AIDS. Efficacy studies of indinavir, ritonavir, or nelfinavir using 

plasma viral RNA as a marker have demonstrated up to three log reductions in RNA copy numbers that 

are 


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Page 33 

sustained in many patients [67–69]. In contrast, nucleoside antiretroviral therapy that targets reverse 

transcriptase rarely results in more than one log reduction of viral RNA, indicating fairly poor inhibition 

of viral replication by this class of compounds. One of the reasons for the apparent greater in vivo 

antiviral activity of HIV PR inhibitors could be the mechanistic difference of the two enzymes and their 

respective activities in the viral life cycle. However, growing evidence of retroviral resistance to 

protease inhibitors remains a concern. The availability of several chemically distinct HIV PR inhibitors, 

including the second generation of compounds currently under preclinical development, offers a 

possibility of combining two or more drugs that share little cross-resistance. Also, it seems reasonable to 

evaluate these compounds in combination with various nucleoside and nonnucleoside reverse 

transcriptase inhibitors. Early clinical data from such combination therapy indicates reduction of 

retroviral RNA in plasma to levels lower than the currently available limit of detection [70]. This is the 

first indication that the application of well-chosen combination therapy can place AIDS patients in 

prolonged virologic and clinical remission. 

Undoubtedly, protein crystallography and other elements of structure-based drug design were widely 

applied in the discovery of HIV PR inhibitors. It will be prudent to assume that, in the absence of 

structural feedback, rapid discovery of several chemically different and potent inhibitors of HIV PR 

would have been severely impeded if not even impossible. However, structure-based drug design still 

remains a new and developing technology. Further success of this drug discovery technique largely 

depends on the development of methods of computational chemistry. Several computational approaches 

such as ALADDIN [71], DOCK [72], and MCDNLG [46] have been applied with a limited degree of 

success in a search for novel inhibitors of HIV PR and these methods will be developed further. The 

most difficult and challenging computational task required for full implementation of structure-based 

drug design involves assigning a priority to designed compounds before their synthesis, by computation 

of the absolute free energy of binding or by prediction of the relative difference in the binding constants 

of chemically related compounds. While the former approach is technically very difficult, due to the size 

of configurational space that must be sampled and the limited accuracy of the force field that describes 

atomic interactions in the molecular system [73], the latter approach has had some successes [74,75]. 

Nevertheless, owing to the various assumptions and approximations that underlie these techniques, such 

methods are useful only as order-of-magnitude estimates [74]. Further improvements of these methods 

heavily depend on the availability of structural and thermodynamic data for several closely related 

compounds that could be used to calculate parameters required for the implementation of such 

thermodynamic-integration cycles. The large number of high-resolution crystal structures of HIV PR 


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Page 34 

complexed with various inhibitors offers a unique opportunity for the development of such 

computational methods if the structural data can be coupled with thermodynamic measurements of 

inhibitor-protein binding. These include direct measurements by microcalorimetry of the association 

constant, K, and in addition the enthalpy, entropy, heat capacity, and stoichiometry of binding. The 

combination of such thermodynamic and structural data will lead to a more precise understanding of the 

factors that influence binding and, ultimately, will lead to new general design principles that can be 

applied to drug discovery in the area of AIDS as well as other challenging diseases. 

Acknowledgments 

I wish to thank all my co-workers from Agouron who contributed to these studies, in particular J. 

Davies, S. Reich, M. Melnick, V. Kalish, A. Patick, L. Musick, and B-W. Wu. Steven Kaldor from Ely 

Lilly is acknowledged for his contribution in designing AG1343 (nelfinavir). I would like to thank 

Richard Ogden for critical reading of the manuscript and D. Olson for expert assistance in preparing the 

manuscript. 

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8:677–691. 


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2 

Structural Studies of HIV-1 Reverse Transcriptase and Implications for 

Drug Design 

Jianping Ding, Kalyan Das, Yu Hsiou, 

Wanyi Zhang, and Edward Arnold 

Center for Advanced Biotechnology and Medicine, and Rutgers University, 

Piscataway, New Jersey 

Prem N. S. Yadav 

University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 

Stephen H. Hughes 

ABL-Basic Research Program, NCI-Frederick Cancer Research and 

Development Center, Frederick, Maryland 


Page 41 

Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional 

enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either 

an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of singlestranded 

viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell 

chromosomes [1–4]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately 

20,000 nucleotides. Chemotherapeutic agents have been identified that target virtually all stages of the 

HIV-1 replication cycle (see review [5]). Since both the polymerase and RNase H activities of HIV-1 

RT are essential, inhibiting either step blocks viral replication. Therefore, HIV-1 RT is an important 

target for the treatment of AIDS. Two major classes of antiviral agents that inhibit HIV-1 RT 

polymerization have been identified; these are nucleoside RT inhibitors (NRTIs) (Figure la) and 

nonnucleoside RT inhibitors (NNRTIs) (Figure 1b). Nucleoside analogs, such as 3'-azido-2',3'dideoxythymidine 

(AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-3'thiacyti- 


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Page 42

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Figure 1 

(a) Chemical structures of representative nucleoside analog 

inhibitors of HIV-1 RT. AZT: 3'-azido-2',3'-dideoxythymidine; d4T: 

2',3'-didehydro-2',3'-dideoxythymidine; ddI: 2',3'-dideoxyinosine; 

ddC: 2',3'-dideoxycytidine; 3TC: 2',3'-dideoxy-3'-thiacytidine; 

PMEA: 9-(2-phosphonylmethoxylethyl)adenine. 


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(b) Chemical structures of representative nonnucleoside inhibitors of HIV-1 RT. 

Nevirapine: 11 -cyclopropyl-5,11 -dihydro-4-methyl-6H-dipyrido (3,2-b:2'3'-e)(1,4) 

diazepin-6-one; α-APA: αanilinophenylacetamine; TIBO: 

tetrahydroimidazo-(4,5,1-jk) 

(1,4)-benzo-diazepin-2(1H)-one and thione; 

pyridinones; HEPT: 1-{(2-hydroxyethoxy) 

methyl}-6-(phenylthio)thymine; 

BHAP: bis(heteroaryl)piperazine; TSAO: 

{2',5'-bis-O-(tert-butyldimethylsilyl)}-3' 

-spiro-5''-(4''-amino-1",2"-oxathiole)-2", 

2"-dioxide; L-737,126: 5-chloro- 

3-(phenylsulfonyl)indole-2-carboxamide; TBA: 

1-(2',6'-difluoro-phenyl)-1H,3H-thiozolo -(3,4-a) -benzimidazole; 

quinoxaline S-2720: 6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl) 

-3-4-dihydroquinoxalin-2(1H)-thione. 


Page 43

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dine (3TC), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), have been widely used in the treatment of 

HIV-1 infections [5–7]. However, the effectiveness of these drugs is limited by their cytotoxicity and the 

rapid emergence of drug-resistant viral strains [5,8–12]. Nonnucleoside inhibitors, e.g., the HEPT 

derivatives [13], TIBO derivatives [14], nevirapine [15], pyridinones [16], BHAP derivatives [17], TBA 

derivatives [18,19], TSAO derivatives [20], α-APA [21], and quinoxalines (HBY) [22,23], are potent 

inhibitors of HIV-1 RT (see reviews [5,11,12]). While these inhibitors differ considerably in chemical 

structure, all of them are quite specific for HIV-1 RT and inhibit neither HIV-2 RT nor variety of 

cellular polymerases. Challenging a virus with these drugs 


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Figure 

Continued 

Page 45 

rapidly selects viral strains containing drug-resistance mutations [9,24,25]. HIV-1 viral variants are 

known whose RT is resistant to all of the currently available drugs/inhibitors (see reviews 

[5,9,11,12,26,27]). In some cases, drug-resistant variants can be selected in very short periods of time 

[9], a consequence of the high viral load and rapid turnover of viral populations in infected individuals 

[28–30]. A better understanding of how these viral variants confer resistance should provide insight into 

the limitations of their genetic flexibility. In the past few years, substantial progress has been made in 

understanding the three-dimensional structure of HIV-1 RT. This paper will discuss the recent 

biochemical, genetic, and clinical data of HIV-1 RT in the context of the crystal structure of HIV-1 RT 

and prospects for development of more effective inhibitors of HIV-1 replication. 

II. Three-Dimensional Structures of HIV-1 RT 

Three-dimensional crystal structures of HIV-1 RT have been determined both for the unliganded form of 

the protein and for complexes with either template-primer substrate or nonnucleoside inhibitors (Figure 

2 and Table 1). Structures of HIV-1 RT have been determined in complexes with a series of NNRTIs, 

including nevirapine [31–33], 1051U91 (a nevirapine analog) [33], α-APA R95845 [34], α-APA 

R90385 [33], HEPT [33], 8-Cl TIBO (R86183) [35], and 9-Cl TIBO (R82913) [36,37]. The structure of 

HIV-1 RT in a ternary complex with a 19-mer/18-mer double-stranded DNA (dsDNA) template-primer 

and an antibody Fab fragment has been described [38]. In addition, structures of unliganded HIV-1 RT 

have also been solved in multiple crystal forms [39–43]. The structure of a polypeptide corresponding to 

the fingers and palm subdomains of the HIV-1 RT polymerase domain has also been determined [44]. 

HIV-1 RT is an asymmetric heterodimer consisting of the p66 (66 kDa) and p51 (51 kDa) subunits. The 

N-terminal 440 residues of the p66 subunit constitute the polymerase domain and the C-terminal 120 

residues of p66 form the RNase H domain; the p51 subunit has the same amino acid sequence as the 

polymerase domain of the p66 subunit [1,2]. The polymerase domain of the p66 subunit has been 

likened to a human right hand. On this basis the subdomains of both p66 and p51 have been designated 

as fingers, palm, thumb, and connection (Figure 2) [31,38]. In the p66 subunit, the fingers, palm, and 

thumb subdomains form a large cleft that can accommodate the DNA substrate. The polymerase active 

site, which contains three strictly conserved aspartic acid residues (Asp110, Asp185, and Asp186), is 

located in the DNA-binding cleft and is part of the p66 palm subdomain (Figure 3) [31,38]. In the p51 

subunit, however, the thumb is rotated away from the fingers and the connection subdomain is folded 

over onto the palm subdomain between the fingers and thumb subdomains. As a 


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Table 1 Crystal Structures of HIV-1 Reverse Transcriptase 

PDB entry HIV-1 RT Structure 

Resolution 

Range (Å) 

R-Factor/Free R-Factor 

(%) 

Data Completeness 

(%) 

Page 46 

Temperature 

(°C) References 

1hmv unliganded RT 6.0-3.0 25.4/29.7 85 -165 41 

1rtj unliganded RT 25.0-2.35 21.9 89.5 -173 42 

1dlo unliganded RT 8.0-2.7 23.0/33.6 99.5 -165 43 

1hmi RT/DNA/Fab 15.0-3.0 26.0 88.1 -10 38 

3hvt RT/nevirapine 8.0-2.9 26.6 95.6 -165 31,32 

1vrt RT/nevirapine 25.0-2.2 18.6 87.1 16 33 

1rth RT/1051U91 25.0-2.2 21.4 81.4 16 33 

1hni RT/α-APA (R95845) 10.0-2.8 25.5/36 78.5 -15 34 

1vru RT/α-APA (R90385) 25.0-2.4 18.7 86.5 16 33 

1hnv RT/8-Cl TIBO (R86183) 10.0-3.0 24.9/35.6 81 -10 35 

1rev RT/9-Cl TIBO (R82913) 25.0-2.6 22.4 80.7 -173 36 

1tvr RT/9-Cl TIBO (R82913) 10.0-3.0 25.9 72 -165 37 

1rti RT/HEPT 25.0-3.0 23.6 86.3 14 33 

1hrh RT (RNase H domain) 20.0-2.4 20.0 93.1 4 94 

1rdh RT (RNase H domain) 20.0-2.8 21.5 95.6 4 95 

1har RT (fingers and palm subdomains) 7.0-2.2 20.8/27.0 96 4 44 


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Figure 2 

Ribbon diagrams of the three types of HIV-1 RT crystal structures. (a) Structure of the HIV-1 RT/DNA/Fab ternary complex 

[38]. The bound nucleic acid is shown as double-stranded helices with template strand in black and primer strand in gray. The Fab 

fragment is not shown. (b) Structure of the HIV-1 RT complexed with the NNRTI TIBO R86183 [34]. For the sake of clarity, the bound 

TIBO inhibitor is shown as an atomic model. (c) Structure of unliganded HIV-1 RT [41,43]. (d) A schematic diagram showing the 

resemblance of the HIV-1 RT p66 subunit to a human right hand. The RNase H domain, which has no counterpart for a human hand, is 

shown as an oval below the thumb. When a template-primer binds to HIV-1 RT, the fingers, palm, and thumb subdomains of p66 form a 

large cleft to bind the DNA. The polymerase active site (shown as a small circle) lies at the bottom of the DNA-binding cleft. The 

NNRTI binds in the highly hydrophobic NNIBP (shown as a large circle), which is located in the vicinity of the polymerase active site. 

The p66 thumb subdomain in the NNRTI-bound HIV-1 RT structures is in an upright position extended beyond that observed in the 

structure of RT with bound DNA. In the absence of any bound nucleic acid or NNRTI, however, the p66 thumb folds down into the 

DNA-binding cleft (shown as dashed drawing). 


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Figure 

Continued 

consequence, the p51 submit has no cleft for binding nucleic acid substrates and hence no polymerase 

activity. 

Page 48 

There is considerable evidence showing that HIV-1 RT is quite flexible and that this flexibility is 

essential for DNA polymerization. Comparisons among DNA-bound, inhibitor-bound, and unliganded 

HIV-1 RT structures provide a demonstration of the enzyme's flexibility. When a DNA template-primer 

binds to HIV-1 RT, structural elements of the fingers, palm, and thumb subdomains of the p66 subunit 

form a clamp-like structure that holds the nucleic acid (Figure 2) [38]. The template-primer substrate 

interacts with amino acid residues of the fingers, palm, and thumb subdomains, especially in the regions 

denoted as “primer grip” and “template grip,” believed to position the template-primer precisely relative 

to the polymerase active site [38]. The primary contacts between the template-primer and the protein are 

along the sugar-phosphate backbone of the DNA and thus are not sequence-specific [38]. In the absence 

of nucleic acid template-primer or NNRTI, the thumb subdomain of p66 is folded down into the DNAbinding 

cleft and lies near the fingers subdomain (Figure 2) [40,41,43]. As a consequence, the DNAbinding 

cleft is closed. However, even in the absence of a bound nucleic acid, binding of an NNRTI 

induces both short-range and long-range structure distortions, including a hinge-like movement near the 

base of the p66 thumb that constrains the p66 thumb in a conformation that is extended beyond the 

upright 


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Figure 3 

Overall structure of HIV-1 RT p66/p51 heterodimer [38] showing the locations 

of the major target sites for anti-HIV-1 RT inhibitors. The NRTIs target the dNTP-binding 

site/the polymerase active site (shown as a small striped circle) that lies at the 

floor of the DNA-binding cleft. The NNRTIs bind to the NNIBP (shown as a large 

dotted circle), which is near, but distinct from, the polymerase active site. The RNase H 

domain is located at the C-terminal of the p66 subunit. The RNase H catalytic site 

(shown as a medium circle) is an attractive target site for anti-HIV-1 drugs. The HIV-1 

RT p66/p51 heterodimer interface is shown as a dashed line. Since HIV-1 RT functions 

as a heterodimer, any inhibitors that could interfere with the dimerization process might 

also be potential drugs for treating HIV-1 infection. 

position of the thumb observed in the HIV-1 RT/DNA/Fab structure [31,33–37,43]. 

In the unliganded structure of HIV-1 RT reported by Esnouf et al. [42], the p66 thumb subdomain is in 

an upright conformation different from that observed in the other unliganded HIV-1 RT structures but 

similar to that found in the DNA-bound HIV-1 RT and NNRTI-bound HIV-1 RT structures. Esnouf et 

al. [42] contend that the upright conformation of the p66 thumb subdomain in their unliganded RT 

structure is appropriate for unliganded HIV-1 RT and that the binding of an NNRTI does not affect the 

conformation of the p66 thumb. However, we believe that the conformation of the p66 thumb in the 

Esnouf et al. structure may well be a result of the method used to prepare the 


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Page 50 

crystals, which were produced by soaking out a weakly bound NNRTI (HEPT) from pregrown crystals. 

When the crystals were grown in the presence of HEPT, the p66 thumb was presumably in an upright 

position similar to that seen in all of the known NNRTI-bound HIV-1 RT structures. As the weakly 

bound HEPT diffused out, the molecular packing arrangement may have constrained the position of the 

p66 thumb subdomain. As a consequence, this unliganded HIV-1 RT structure may represent an 

intermediate between the other unliganded structures and the structure of HIV-1 RT with a bound 

NNRTI. It is evident that the p66 thumb subdomain has considerable flexibility and can adopt 

substantially different conformations during the binding of template-primer or inhibitors and, 

presumably during DNA polymerization as well. 

III. Polymerase Active Site of HIV-1 RT and the NRTIs 

Polymerization of DNA by HIV-1 RT involves a sequential stepwise binding of the template-primer and 

deoxynucleoside triphosphate (dNTP) substrates at the polymerase active site [45,46]. The incoming 

dNTP is covalently linked via the α-phosphorus to the 3'-oxygen of the primer strand, accompanied by 

the release of pyrophosphate. An essential requirement for the polymerization reaction is the presence of 

a 3'-OH group at the end of the primer strand. Nucleoside analogs contain a modified sugar moiety in 

which the 3'-OH group is replaced by another group (e.g., hydrogen, halogen, or azido) (Figure la). To 

exert their antiviral activity at the level of RT, the NRTIs must be phosphorylated successively to the 5'monophosphate, 

5'-diphosphate, and 5'-triphosphate forms by a series of kinases. Once the NRTI is 

converted to the triphosphate form and interacts with RT, it can inhibit polymerization in two possible 

ways. One possibility is that the NRTI binds preferentially to the dNTP-binding site and competitively 

inhibits the binding of natural dNTPs. Another possibility, which seems to be the predominant mode of 

inhibition, is that the NRTI is incorporated into the growing chain and acts as a terminator of chain 

elongation. Once an NRTI is incorporated, no additional nucleotides can be added to the DNA chain 

since the primer terminal 3'-OH group (the site of phosphodiester bond formation) is absent. 

Phosphorylation is a crucial step in the intracellular metabolism of the NRTI and often is a limiting 

factor for the antiviral activity of the NRTI [47]. In an attempt to bypass the first phosphorylation step, 

several acyclic nucleoside phosphonates have been developed in which the sugar moiety of normal 

NRTIs is replaced with an acyclic phosphonate group, such as 9-(2-phosphonylmethoxyethyl)adenine 

(PMEA), (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), and (S)-9-(3-fluoro-2phosphonylmethoxyethyl) 

adenine (FPMPA) (Figure la) (see reviews [5,11]). These acyclic nucleoside 

phosphonates are dideoxynucleoside 


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monophosphate analogs which can easily be converted to the active triphosphate form by adding two 

additional phosphates [48,49], and can inhibit HIV replication [50,52]. 

Page 51 

Analysis of the structure of the HIV-1 RT/DNA/Fab complex showed that the dNTP-binding site is 

composed of both protein and nucleic acid. In addition to the 5'-terminus of the template nucleotide and 

three carboxylate residues (Asp110, Asp185, and Asp186), the amino acid residues Asp113, Tyr115, 

Phe116, Gln151, Phe160, and possibly Met184 and Lys219 form part of the putative dNTP-binding site 

[12,53] (Figure 4 and Table 2). It is important to realize that the precise composition, position, and 

conformation of the template-primer can influence the recognition and incorporation of incoming 

nucleotides at the polymerase catalytic site. In the wild-type HIV-1 RT, the dNTP-binding 

Figure 4 

Stereoview of the polymerase active site of HIV-1 RT [38]. The amino acid 

residues that compose the putative dNTP-binding site, including the three catalytically 

essential aspartic acids, are shown with side chains. The double-stranded nucleic acid is 

shown with the atomic model in the HIV-1 RT/DNA/Fab complex. The dNTP-binding 

site consists of structural elements from both protein and nucleic acid. The precise 

composition, position, and conformation of the template-primer can affect the recognition of 

incoming dNTPs at the polymerase active site. 


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Table 2 HIV-1 RT Amino Acid Residues Composing the Putative dNTP-Binding Sites and the Locations of NRTI- 

Resistance Mutations (see also [12]) 

dNTP-Binding Site Nucleoside Drug-Resistance Mutation Site 

Residue Location Mutation Location Possible Effects References 

Asp110 β6 Met41Leu αA template binding 55 

Asp113 β6-αC loop Ile50Thr β2 unclear 154 

Try115 αC Lys65Arg β3–β4 template binding 154 

Phe116 αC Asp67Asn β3–β4 template binding 155 

Gln151 β8-αE Thr69Asp β3–β4 template binding 156 

Phe160 αE Lys70Arg β3–β4 template binding 155 

Asp185 β9–β10 Leu74Val β4 template binding 8 

Asp186 β9–β10 Val75Thr β4 template binding 163 

Lys219 β11 Glu89Gly β5 dsDNA binding 157 

Tyr115Phe αC dNTP binding 170 

Ile135Thr β7–β8 unclear 158,159 

Gln151Met β8-αE dNTP binding 

Met184Val, Ile β9–β10 dNTP-binding/fidelity 154,160,161 

Thr215Tyr, Phe, 

Cys 

β11 indirect effect/dNTP 

binding 

155,162 

Lys219Gln β11 dNTP binding 155 

Page 52 

site can accommodate both the normal dNTP substrates and dideoxynucleoside analogs. The majority of 

mutations that confer resistance to NRTIs are not located at the dNTP-binding site; however, they 

appear to influence the geometry of the dNTP-binding site indirectly in a way that permits RT to 

discriminate between a normal dNTP and a modified nucleoside triphosphate (see discussion in the next 

section). 


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Analysis of various HIV-1 RT structures has revealed an unusual β-turn geometry for the YMDD motif 

at the polymerase catalytic site of p66 [33,34,41–43]. The energetically unfavorable main chain 

conformation of Met184 (torsion angles φ~60° and ϕ~-120°) is stabilized by a hydrogen bond of its 

carbonyl oxygen to the side chain of either Gln182 [33,41,43] or Gln161 [42]. It has been suggested that 

this novel β-turn geometry might be required to position the aspartic acids in precisely the correct way 

for catalysis [41]. 

IV. Mechanism of NRTI-Resistance Mutations 

Development of resistance to NRTIs has been a major problem with clinical use of these drugs. Careful 

analysis of mutations that confer resistance to different 


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Page 53 

NRTIs in light of available structural data might provide information that could be used in the 

development of improved NRTIs that are more effective against the commonly observed NRTI-resistant 

HIV-1 variants. A viable drug-resistant RT mutant should be able to recognize and incorporate normal 

nucleoside triphosphate, yet reject a nucleoside analog. The only difference between normal nucleotide 

substrates and the NRTIs is the modification of the sugar moiety. This alteration may affect sugar 

puckering and the conformation of the glycosyl bond. Recognition of these differences could render the 

triphosphate form of the nucleoside analog a good substrate for wild-type RT but a poor substrate for a 

drug-resistant variant of RT. 

Structural analysis of HIV-1 RT has shown that most of the NRTI-resistance mutations are not located 

close to the putative dNTP-binding site and are unlikely to have a direct impact on the binding of dNTP 

analogs (Figure 5 and 

Figure 5 

A close-up view showing the relative locations of the commonly identified 

drug-resistance mutations for NRTIs (in dark-gray) and for NNRTIs (in light-gray) with 

respect to the bound DNA. Most of the NRTI-resistance mutations are not located at the 

putative dNTP-binding site, but are at positions to have potential interactions with the 

nucleic acid template-primer. Conversely, all the NNRTI-resistance mutations are 

clustered around the NNIBP and have direct contacts with NNRTIs or have direct effect on 

inhibitor binding. 


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Page 54 

Table 2) [12,54]. For example, none of the five mutations, in HIV-1 RT Met41Leu, Asp67Asn, 

Lys70Arg, Thr215Tyr, and Lys219Gln [55] (Table 2) consistently associated with resistance to AZT, 

are at locations close to the dNTP-binding site. However, most (but not all, c.f. [56]) biochemical studies 

have failed to show that recombinant HIV-1 RT enzymes containing these mutations are more resistant 

to inhibition by AZT triphosphate than the wild-type HIV-1 RT [27,57,58]. Other mutations that confer 

resistance to NRTIs have been identified at positions 50, 65, 69, 74, 75, 89, 115, 135, 151, and 184 of 

HIV-1 RT (Figure 5 and Table 2). Most of these mutations do not lie close to the dNTP-binding site 

(Met184Val/Ile are the exception), but instead are located at positions where they could interact with the 

nucleic acid template-primer [12,59,60]. Biochemical data have shown that only when the 5'-template 

extension length is greater than three nucleotides does the wild-type RT begin to incorporate 

dideoxynucleotides effectively [54]. If the template extension is less than three nucleotides in length, 

wild-type HIV-1 RT is resistant to dideoxynucleotides. On the other hand, HIV-1 RT variants containing 

the mutations Leu74Val or Glu89Gly did not readily incorporate dideoxynucleotides either with short or 

long template extensions [54]. Based on both structural and biochemical data, it was proposed that 

mutations that cause HIV-1 RT to have reduced sensitivity to NRTIs exert their effects via interactions 

with the nucleic acid template-primer, which consequently alter the geometry of the polymerase active 

site [54]. It has been suggested that mutations that confer resistance to foscarnet might use a similar 

mechanism [61]. One possible exception to this mechanism might be the mutations of Met184Val and 

Met184Ile (see review [27]). Part of the highly conserved YMDD motif, Met184 is adjacent to residues 

Asp185 and Asp186, which are two of the three catalytically essential aspartic acid residues at the 

polymerase active site. In addition, Met184 appears to interact with the ribose moiety of the 3'-terminal 

nucleotide of the primer strand [12,38,53] (Figure 4). Therefore, mutations at this position could affect 

interactions with the incoming dNTP directly and/or alter the positioning of the nucleic acid. These 

mechanisms are not mutually exclusive and which mechanism is responsible for resistance has not yet 

been resolved [62]. There are two recent reports suggesting that the Met184Val mutant HIV-1 RT has 

approximately three-fold higher fidelity than the wild-type enzyme [63,64]. Based on these data, it was 

suggested that this increase in fidelity might reduce the overall rate of generation of viral variants in 

patients treated with 3TC or other dideoxynucleosides [64]. However, owing to both theoretical and 

technical problems with these analyses, these conclusions are controversial. Determination of crystal 

structures of both wild-type and mutant HIV-1 RT complexed with individual NRTIs in the presence of 

a variety of template-primers and/or dNTP substrates should provide a better understanding of the 

mechanisms of dNTP selection and drug resistance.ed at Arial for catalysis [41]. 

IV. Mechanism of NRTI-Resistance Mutations 

Development of resistance to NRTIs has been 


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V. Drug Design Targeting at the Polymerase Active Site 

Page 55 

All of the existing NRTIs contain a modified sugar moiety that lacks the 3'-OH group that is essential 

for incorporation of the next nucleotide. Modification can also be made on other functional groups such 

as the base and the triphosphate moieties. It may be worthwhile to try to alter the base moiety of the 

NRTIs to produce compounds that will be more specific to HIV-1 RT (i.e., less cytotoxic to normal 

cellular polymerases) and more effective against both wild-type or drug-resistant viral variants. 

Structure-activity analysis indicates that the pyrimidine moiety of the NRTIs can be modified at the C5 

position. An AZT derivative that has a 3'-azido group on the sugar moiety and a methyl group at the C5 

position of the pyrimidine moiety showed potent antiviral activity [65]. Substitution of the methyl group 

with a chlorine atom at position C5 of AZT results in a compound that has strong anti-HIV-1 activity 

[66]. Other possible substitutions at the C5 position include other halogen atoms or an ethyl group. 

Another possible drug-design strategy would be to devise compounds that can interface with the binding 

of the metal ions (Mg 2+ or Mn 2+) at the polymerase active site. Metal ions appear to be important in 

DNA polymerase catalysis. Based on the structural and biochemical data, a two-metal dependent 

mechanism of polymerization has been postulated [53,67,68] that is similar to that proposed for other 

DNA polymerases [69–71]. In this model, the metal ions mediate interactions between the three 

catalytically essential aspartic acid residues (Asp100, Asp185, and Asp186) and the α-, β-, and γphosphates 

of the incoming dNTP and promote the nucleophilic attack on the α-phosphate by the 

oxygen atom of the 3'-OH group of the primer strand. In the structure of the fingers and palm 

subdomains of the RT of Moloney murine leukemia virus (MuLV), a single Mn 2+ ion was found bound 

to the two aspartic acid residues at the polymerase active site [72]. In the structure of the unliganded 

HIV-1 RT, an electron density peak was located at the polymerase active site with a good coordination 

geometry to the Oδ1 atoms of both Asp185 and Asp186 [43]. This electron-density peak is in a position 

similar to that of the Mn 2+ ion observed in the MuLV RT structure. It is possible that this position 

corresponds to a Mg 2+ ion-binding site [43]. It might be useful to design inhibitors that would influence 

the metal-ion coordination using either computer-based calculations (such as DOCK [73–75]) or based 

directly on an analysis of HIV-1 RT structure. Crystal structures of HIV-1 RT complexed with 

Mg 2+/Mn 2+ ion(s) at the polymerase catalytic site in the presence of template-primer and/or dNTP 

substrates would be helpful in defining the target sites of inhibitors of this type. Further studies on the 

structure activity relationship of HIV-1 RT complexes with these inhibitors, if active, might ultimately 

lead to a new type of HIV-1 RT drug that would not compete with the dNTP binding but would affect 

the DNA polymerization mechanism. 


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Page 56 

It is also attractive to consider developing agents that bind to the HIV-1 RT polymerase active site but 

are not nucleoside analogs. Since the amino acid residues at the dNTP-binding site are highly conserved, 

viral variants resistant to such inhibitors may be significantly impaired in their polymerase activity. 

However, there is a good chance that drug resistance could result from mutations in HIV-1 RT that 

influence the precise positioning of template-primer [54]. Initial attempts to use this approach starting 

from the crystal structure of the HIV-1 RT/DNA/Fab complex [38] (Ding, et al., in preparation) have 

uncovered some interesting lead compounds (Kuntz, Kenyon, Arnold, Hughes, et al., unpublished). 

VI. NNRTIs and the NNIBP 

Nonnucleoside RT inhibitors (NNRTIs) constitute the other major class of HIV-1 RT inhibitors. Many 

structurally distinct families of NNRTIs have been identified, including HEPT [13], TIBO [14], 

nevirapine [15], BHAP [17], TBA [18,19], TSAO [76], α-APA [21], pyridinones [16] and quinoxalines 

(HBY) [22,23] (Figure 1b). However, development of drug resistance is a major problem when NNRTIs 

are used to treat AIDS patients. An ideal drug should be able to block replication of all viable strains of 

HIV-1, but should not inhibit normal cellular enzymes. In this regard, the known NNRTIs may be too 

specific. While these inhibitors do not inhibit cellular polymerases, they are also inactive against HIV-2 

RT (which can be viewed as an extreme variant of HIV-1 RT). In addition, drug-resistant variants of 

HIV-1 RT emerge rapidly in the presence of most inhibitors. In contrast, the NRTIs inhibit a broad 

spectrum of polymerases including the host cellular polymerases. Though it appears to be more difficult 

for the virus to evade NRTIs than NNRTIs (in general, it takes longer for the virus to develop resistance 

to NRTIs than NNRTIs), NRTI toxicity is a serious problem. 

Structural and biochemical studies have shown that all NNRTIs bind in a highly hydrophobic pocket in 

the p66 subunit, located approximately 10 Å away from the polymerase active site (Figures 2 and 3) 

[31,33–37]. Nevertheless, in all known structures of HIV-1 RT/NNRTI complexes, the bound NNRTIs 

have not been found to have any direct interactions with residues that compose the putative dNTPbinding 

site. The nonnucleoside inhibitor binding pocket (NNIBP) contains primarily amino acid 

residues from the β5–β6 loop (Pro95, Leu100, Lys101, and Lys103), β6 (Val106 and Val108), the 

β9–β10 hairpin (Val179, Tyr181, Tyr188, and Gly190), and the β12–β13 hairpin (Phe227, Trp229, 

Leu234, His235, and Pro236) of the p66 palm subdomain, and β15 (Tyr318) of the p66 thumb 

subdomain, as well as the β7–β8 connecting loop (Glu138) of the p51 fingers subdomain (Figure 6 and 

Table 3). 


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Figure 6 

Superposition of the NNRTIs in the HIV-1 RT/TIBO complex [35], HIV-1 RT/α-APA complex [34], and HIV-1 

RT/nevirapine complex [32]. The side chains are shown for those amino acid residues that have close contacts with bound inhibitors 

and the three catalytically essential aspartic acids in the HIV-1 RT/TIBO complex. Most of the amino acid residues that form the 

NNIBP are hydrophobic. Though the NNRTIs are chemically and structurally diverse, the bound NNRTIs all assume a strikingly 

common butterfly-like shape. 


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Table 3 HIV-1 RT Amino Acid Residues Defining the Nonnucleoside Inhibitor-Binding Pocket (NNIBP) and the 

Locations of NNRTI-Resistance Mutations (see also [12]) 

NNIBP 

Residues 

Pro95 β5 

Location Mutation Possible Effects References 

Ala98 β5–β6 Gly less bulky 9,164 

Leu100 β5–β6 Ile β-branch 9,164-166 

Lys101 β5–β6 Glu charge change 164 

Lys103 β5–β6 Asn, Gln charge loss, less bulky 9,24,164 

Val106 β6 Ala less bulky 9,58,165 

Val108 β6 Ile bulkier 9,164 

Glu138 β7–β8(p51) Lys charge change 166 

Val179 β9 Glu, Asp charge gain, bulkier 164 

Tyr181 β9 Cys, Ile aromaticity loss, less bulky 24,58,164 

Tyr188 β10 His, Cys, Leu aromaticity loss, less bulky 9,167 

Gly190 β10 Glu charge gain, bulkier 9,22,168 

Phe227 β12 

Leu228 β12 Phe aromaticity gain, bulkier 133 

Trp229 β12 

Glu233 β13 Val charge loss, less bulky 169 

Leu234 β13 

Pro236 β13–β14 Leu increase flexibility, bulkier 133 

Lys238 β14 Thr charge loss, less bulky 133 

Tyr318 β15 


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Most of the amino acid residues that form the binding pocket are hydrophobic and five of them are 

aromatic residues. The hydrophobic interactions of the side chains of these residues, especially Tyr181, 

Tyr188, and Trp229, with the hydrophobic moieties of the NNRTIs appear to be important for inhibitor 

binding [32–34,59]. Since most of the NNRTIs also contain polar group(s), they have the potential to 

form hydrogen bonds with surrounding amino acid residues either directly or via water bridges [33–36]. 

In the structures of both liganded HIV-1 RT and the HIV-1 RT/DNA/Fab complex, there are two small 

surface depressions at the base of the NNIBP that are the putative entrances to the pocket [34,43]. One 

surface depression is located at the p66/p51 heterodimer interface and is composed of amino acid 

residues Leu100, Lys101, Lys103, Val179, Tyr181, and Tyr188 of p66, and Glu138 of p51 [34]. This 

putative entrance is narrow compared to the size of the NNRTIs. Another surface depression has been 

found at the location near the base of the p66 thumb subdomain between two adjacent structural 

elements: the β5–β6 connecting loop (Lys101 and Lys103) and the β13–β14 hairpin (Pro236 and 

Leu238) [43]. Since this site is also exposed to solvent, an NNRTI could approach the NNIBP from it. 

How- 


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ever, once an NNRTI is bound to RT, only the first putative entrance remains accessible; the second 

disappears due to the conformational change and repositioning of the β12-β13-β14 sheet [43]. 

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It is evident, from comparison of the various HIV-1 RT structures that the NNIBP has a highly flexible 

structure that apparently allows the enzyme to accommodate various types of NNRTIs with different 

shapes and sizes. Despite apparent differences in the structures of the bound inhibitors, comparison of 

structures of several HIV-1 RT/NNRTI complexes revealed remarkable similarity in the geometry of 

both the bound inhibitors and the NNIBP [33,35]. All these chemically diverse NNRTIs assume a 

strikingly similar butterfly-like shape (Figure 6). The binding of NNRTIs in the NNIBP can be likened 

to a butterfly sitting on the β6-β10-β9 sheet and facing toward the putative entrance to the pocket. The 

angle between the two wings of the “butterfly” is approximately 112–115° in the TIBO, α-APA, and 

nevirapine complexes [35]. This angle might be critical in inhibitor binding and could be a crucial 

parameter in the design of new NNRTIs. There are many other NNRTIs that are significantly larger or 

smaller in size than α-APA, TIBO, or nevirapine. It is very likely that the NNIBP can adopt other 

conformations. For example, BHAP appears to be too large to fit into the NNIBP in any of the reported 

HIV-1 RT/NNRTI complexes. The NNIBP in the HIV-1 RT/BHAP complex would need to be 

significantly larger than that observed in the structures of the known HIV-1 RT/NNRTI complexes. It is 

possible that the BHAP inhibitor may not conform to a butterfly-like shape. This underscores the 

importance of solving crystal structures for as many HIV-1 RT/NNRTI complexes as possible. 

Additional structural and biochemical data for other HIV-1 RT/NNRTI complexes should provide the 

insight needed to define the limits of the flexibility of HIV-1 RT in the NNIBP region. 

VII. Process of NNRTI Binding 

In crystal structures of unliganded HIV-1 RT [40,41,43] and of HIV-1 RT/DNA/Fab complex [38], the 

NNIBP does not exist (although a small cavity is found in the region of the NNIBP proximal to the 

polymerase active site in the unliganded HIV-1 RT structure described by Esnouf et al. [42]). In these 

structures, the side chains of Tyr181 and Tyr188 in p66 point away from the polymerase active site and 

toward the hydrophobic core. However, in the HIV-1 RT/NNRTI complex structures, the side chains of 

Tyr181 and Tyr188 point toward the polymerase active site, and the side chain of Tyr181 is in a position 

that prevents Trp229 from occupying the position it has in the unliganded or DNA bound HIV-1 RT 

structures. Binding an NNRTI also moves the β12-β13-β14 sheet away from the hydrophobic core 

[34,35,37]. These conformational 


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changes create the space in the pocket required to accommodate inhibitors. In other words, significant 

conformational changes occur during the process of inhibitor binding that lead to the formation of the 

NNIBP [33–35]. This observation also underscores the importance of determining structures of HIV-1 

RT with and without bound inhibitors. 

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An obvious question is, what forces initiate this series of conformational changes during NNRTI 

binding? One possibility is the contacts between the inhibitor and the protein. Though the NNIBP is 

hydrophobic, there are three hydrophilic amino acid residues (Lys101 and Lys103 of p66, and Glu138 of 

p51) at the rim of the putative entrance(s) to the pocket. The flexible and polar side chains of these 

residues could assist in steering an inhibitor into the pocket and/or could block the bound inhibitor from 

escaping out of the pocket. Mutagenesis studies have shown that these three residues are important in 

the binding of NNRTIs. Though the importance could be explained in terms of the interactions between 

these residues and the bound inhibitor in the final complexes, interactions at the initial stages of inhibitor 

binding might also be crucial. The flexible and polar side chains of these residues might help in directing 

the inhibitor toward the entrance to the pocket via electrostatic interactions, in part by replacing the 

original hydrogen bonds between the drug and the solvent molecules. Any initial energy gains from such 

polar interactions could potentially be replaced by hydrogen bonds or other types of interactions 

between the inhibitor and alternative residues as the inhibitor moves deeper into the binding pocket. In 

addition, significant portions of the aromatic rings of both Tyr181 and Tyr188 are exposed at the bottom 

of the surface depression and offer the potential for early π-π interactions with the inhibitor. This type of 

π-π interaction might also play an important role in the initial approach of inhibitors to the binding 

pocket. This hypothesis may provide a kinetic explanation for the ineffectiveness of NNRTIs against 

viral strains of HIV-1 that carry nonaromatic amino acids at positions 181 and 188. As the solvated 

inhibitor approaches the enzyme and proceeds to enter the binding pocket, most of the water molecules 

of solvation are lost. The few water molecules that remain in the NNRTI-bound complex are typically 

located at the entrance to the pocket, forming water bridges between the inhibitor and one or two polar 

residues around the entrance [33,35,36]. Once the inhibitor is in place, the surface residues close down 

around the drug preventing it from escaping by effectively sealing the entrance to the pocket. 

VIII. Mechanisms of Inhibition by NNRTIS 

Based on structural, biochemical, and genetic data several hypotheses have been postulated about the 

mechanism(s) of inhibition of HIV-1 RT by NNRTIs. It is 


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now clear that the binding of NNRTIs provokes substantial conformational changes in both secondary 

structural elements and in side chains of residues in the NNIBP. These conformational changes in the 

NNIBP could directly or indirectly affect the precise geometry and/or mobility of the nearby polymerase 

catalytic site, especially the highly conserved YMDD motif and/or the divalent metal ions 

[31,33,34,42,68]. The binding of NNRTIs appears to lock the flexible hinge-like structure between the 

palm and thumb subdomains and restrict mobility of the thumb subdomain, placing constraints on the 

geometry of the DNA-binding cleft [12,31,34,43]. The “primer grip” (i.e., β12-β13-β14 sheet), which 

has close interactions with the 3'-terminus of the primer strand [38], forms a part of the NNIBP and is 

involved in the binding of NNRTIs. It has become apparent that binding of NNRTIs can substantially 

alter the conformation of the primer grip; this could affect the precise positioning of the primer strand 

relative to the polymerase active site [34,37]. Displacement of the primer grip by NNRTI binding could 

lead to repositioning of the primer terminus. This could explain the observation that dNTP binding is 

largely unaffected by NNRTI binding while the rate of the chemical step of DNA polymerization is 

reduced [77]. Long-range distortions of the HIV-1 RT structure by NNRTI binding can potentially 

account for NNRTI inhibition of polymerization [39,41,43] and alteration of RNase H cleavage 

specificity [43,78]. These possible mechanisms are not mutually exclusive and the binding of inhibitors 

might have multiple influences on HIV-1 RT polymerization. The exact mechanism(s) of inhibition is 

still under investigation. 

IX. NNRTI-Resistance Mutations 

Analyses of the crystal structures of HIV-1 RT complexed with various NNRTIs have indicated that 

amino acid residues whose mutations confer high levels of resistance to NNRTIs [9,11,12,26,27] are 

located close to the bound inhibitors (Figure 5 and Table 2). Subunit-specific mutagenesis studies have 

confirmed that mutations that confer resistance to the NNRTIs act directly through the change in the 

NNIBP itself [60,79]. In these studies, recombinant HIV-1 RTs that contained amino acid substitutions 

only in the p66 subunit were resistant to NNRTIs, while those containing the same amino acid 

substitutions only in the p51 subunit remained susceptible to the drugs. There is one exception: the 

amino acid substitution of Glu138 to Lys, which confers resistance to inhibitors only when it is present 

in the p51 subunit. Amino acid residue 138 is located in the β7–β8 connecting loop of the fingers 

subdomain. In the p51 subunit this residue forms a part of the NNIBP, while its counterpart in the p66 

subunit is far away from the pocket [12,60]. 


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The mechanism(s) of resistance may depend on the specific amino acid change. It is likely that most 

NNRTI-resistance mutations exert their effects by altering interactions between protein side chains and 

the inhibitors [12,34,35]. Drug-resistance mutations that result in a decrease or increase in the size of 

side chains might lead to loss of favorable contacts or steric conflicts with bound inhibitors. Mutations 

that alter the local electrostatic potential, i.e., gain, loss, or inversion of charge, may change the affinity 

of the NNIBP for inhibitor binding. These altered interactions could interfere with the binding of 

NNRTIs to the hydrophobic pocket or conceivably could even relax the geometric distortion that the 

binding of an inhibitor causes in the vicinity of the polymerase active site. 

X. Design of Improved NNRTIs 

Different NNRTIs, even from the same class of compounds, show remarkable variations in their ability 

to inhibit HIV-1 replication and can give rise to different spectra of resistance mutations [9,11,26,27]. 

For example, biochemical studies showed that the 8-chloro TIBO derivative R86183 is quite potent in 

inhibiting an HIV-1 strain containing the Tyr181Cys mutation, which is one of the frequently occurring 

HIV-1 RT mutations that gives rise to a high level of resistance to almost all NNRTIs, including other 

TIBO derivatives [80]. There are several other reports of NNRTIs that are also relatively effective in 

inhibiting the HIV-1 RT Tyr181Cys variant [81–84]. These results suggest that although all the 

inhibitors appear to bind in the NNIBP, there are differences in their specific interactions with HIV-1 

RT. Structural analyses of HIV-1 RT/NNRTI complexes and computer modeling studies confirmed that 

the exact conformations of the amino acid residues forming the NNIBP appear to vary in different 

complexes and that there are specific interactions between individual inhibitor and surrounding residues 

[33,35,36,85]. However, these differences have not been sufficiently large to allow a successful 

combination therapy to be developed using two or more of the currently available NNRTIs (discussed in 

more detailed in a later section) [9,11,26,27,86]. Systematic analysis of wild-type and drug-resistant 

mutant HIV-1 RT structures in complexes with various NNRTIs should provide additional insights 

about constraints that could be used to optimize the design of NNRTIs. This knowledge could guide 

development of more effective inhibitors for AIDs treatment. 

As discussed earlier, the bound NNRTIs in HIV-1 RT complexes determined so far conform to a 

common butterfly-like shape (Figure 6). A close inspection of interactions between inhibitors and 

protein reveals that though 


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most of the amino acid residues forming the pocket adjust their side chains to make close contacts with 

the inhibitor, the inhibitor is not sufficient to fill all of the space in the pocket. There is space for 

additional nonpolar, polar, or charged groups. Modification of the inhibitor would result in adjustment 

of the orientation of the side chains and could improve interactions between the inhibitor and 

surrounding residues such as Leu100, Lys101, Lys103, Val106, or Leu234. Inhibitors designed to have 

more extensive interactions with essential elements in the pocket should minimize the chances of 

selecting resistant HIV-1 RT variants. From this point of view, NNRTIs that interact with the relatively 

conserved residues of the pocket, such as Trp229, Leu234, and Tyr318, may reduce the risk of 

encountering resistance mutations that do not have significant costs for the enzyme. In addition, 

compounds could be designed to contain functional groups (for example charged or polar groups) able 

to fill more of the available space of the NNIBP and also capable of specific hydrophilic interactions 

with the polar or charged side chains and/or with polypeptide backbone atoms of the NNIBP (for 

example the main chain amide nitrogens and carbonyl oxygens). The hydrophilic interactions between 

inhibitors and protein backbone atoms should be advantageous because mutations to any amino acid 

other than proline would not affect such contacts. In the structures of HIV-1 RT/NNRTI complexes, the 

bound inhibitors are located very close to the polymerase active site composed of the three catalytically 

essential aspartic acids Asp110, Asp185, and Asp186. It might be useful to design compounds that have 

a long and branched aliphatic group or a substituted aromatic group that could not only produce 

hydrophobic interactions with Tyr181, Tyr188, and Trp229, but could also be able to interact with the 

three aspartic residues or interfere with the metal ion(s) binding at the polymerase active site. 

XI. RNase/H-Active Site as a Potential Drug Target Site 

HIV-1 RT contains RNase H, which is responsible for degradation of viral RNA and removal of RNA 

primers for minus- and plus-strand DNA synthesis (see reviews [87–89]). The absolute requirement for 

virus-associated RNase H function [90–93] offers an additional target for antiretroviral drugs. The 

RNase H domain of HIV-1 RT is located at the C-terminus of the p66 subunit (Figures 2 and 3). In 

contrast to the polymerase domain of HIV-1 RT, the structure of the RNase H domain is quite similar in 

all known HIV-1 RT structures and conforms quite well with the structure of the isolated HIV-1 RNase 

H domain [94–95]. The relative stability of the structure of the RNase H domain suggests that the RNase 

H active site could be a relatively well-defined target for drug 


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design. Mutagenesis studies have demonstrated the interdependence of DNA polymerase and RNase H 

activities. Mutations that disrupt one of the two enzymatic activities of HIV-1 RT often also impair the 

second activity [96–99]. Indeed, according to the crystal structure of HIV-1 RT, the polymerase active 

site and the RNase H active site are separated by approximately 17–18 nucleotides [38] and the RNase 

H domain has many contacts with the polymerase domain, especially with the connection subdomain of 

p66 and the thumb and connection subdomains of p51 [31,38,100]. Interactions between the polymerase 

domain and nucleic acid can modulate RNase H activity. Because the predominant contacts of HIV-1 

RT with template-primer occur in the vicinity of the polymerase active site, precise placement of the 

template strand relative to the RNase H active site may be regulated by the sequence and composition of 

the template-primer. Mutagenesis experiments showed that mutations located at or near the “template 

grip” in the polymerase domain of HIV-1 RT can have a greater effect on RNase H than on polymerase 

activity [99,101,102]. It was also reported that binding of the NNRTI nevirapine alters the cleavage 

specificity of RNase H [78]. Structural distortions in the position and conformation of template-primer 

induced by NNRTI-binding may account for alteration of the cleavage specificity of RNase H [43]. 

Divalent metal ions such as Mg 2+ or Mn 2+ are essential for the RNase H activity [103–106]. The 

structure of the isolated RNase H domain crystallized in the presence of MnCl 2 revealed two tightly 

bound Mn 2+ ions in close proximity to four catalytically essential acidic residues, Asp443, Glu478, 

Asp498, and Asp549, that form the active site [94]. Biochemical data have shown that mutations of 

these conserved residues could either disrupt RNase H activity or lead to a highly unstable enzyme 

[107–109]. Based on the crystal structures, a two-metal ion-dependent catalytic mechanism for RNase H 

activity has been postulated [101], which is similar to that proposed for phosphoryl transfer reactions 

catalyzed by polymerases and their associated nucleases [67,69–71]. In contrast, in the structure E. coli 

RNase H reported by Katayanagi et al. [111] only one Mg 2+ ion was observed, and that led to the 

proposal of a single metal-ion catalyzed hydrolysis [112]. Interestingly, in the structure of unliganded 

HIV-1 RT reported by Rodgers et al. [41] and Hsiou et al. [43] only one Mg 2+ ion was found at the 

RNase H active site. The mechanism of RNase H cleavage and the exact role of metal ion(s) in the 

hydrolysis and formation of phosphodiester bonds are still under investigation (see review [89]). 

Very few inhibitors specifically target HIV-1 RNase H activity. Illimaquinone, a natural marine product, 

was shown to preferentially inhibit the HIV-1 RNase H activity [113,114]. However, this compound 

appears to react with a sulfhydryl group in the polymerase domain and not with RNase H itself. It may 

be possible to use the available information on structural and biochemi- 


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cal properties of the polymerase and RNase H of HIV-1 RT to design compounds that would bind at the 

RNase H active site or interfere with the metal ion(s) binding and inhibit the RNase H activity of HIV-1 

RT. However, for optimal utility, these compounds should selectively inhibit the RNase H activity of 

HIV-1 and not the RNase H activity of the host cells. 

XII. Other Possible Target Sites for HIV-1 RT Inhibitors 

Both polymerase and RNase H activities of HIV-1 RT require that the enzyme be in a dimeric form 

[115–118] (Figure 3). The exact role(s) of the p51 subunit in the enzymatic activities of HIV-1 RT are 

not yet known. The three-dimensional structure of HIV-1 RT shows that the interface between p66 and 

p51 primarily involves interactions between the p66 palm and the p51 fingers subdomains, between the 

p66 connection and the p51 connection and fingers subdomains, and between the RNase H and the p51 

thumb and connection subdomains [34,100,119] (Figure 3). A compound that would interfere with 

dimerization would be a potential candidate for an anti-AIDS drug. 

As discussed earlier, the flexibility of HIV-1 RT permits the enzyme to adopt different conformations. 

In the absence of bound DNA, the thumb and the fingers subdomains come together and close a major 

portion of the DNA-binding cleft [40,41,43]. Synthetic oligonucleotides that could interact with the 

specific or conserved regions of the DNA-binding cleft could potentially block binding of templateprimer 

substrates. An RNA pseudoknot has been reported to bind and specifically inhibit HIV-1 RT 

[120]. Chemical modification and substitution of specific groups in RNA ligands can change the 

structure of the pseudoknot, which could result in considerably more effective pseudoknot inhibitors 

with high binding specificity [121]. Studies employing the phosphorodithioate analogs of the primer 

sequence recognized by HIV-1 RT showed that these compounds can act as inhibitors and that inhibition 

is a function of both the sequence and length of these novel single-stranded nucleic acid oligomers 

[122,123]. 

A series of natural products, i.e., trihydroxyquinolone compounds isolated from Red Sea marine 

organisms, were reported to inhibit the DNA polymerase activity of HIV-1 RT [124,125]. This type of 

inhibitor appears to have a mechanism of inhibition that is different from either the NRTI inhibition 

mechanism or the NNRTI inhibition mechanism. The inhibition is reversible and noncompetitive with 

respect to both dNTP and template-primer [125]. This result indicates that there are other potential 

binding sites for inhibitors of HIV-1 RT. 


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XIII. Useful Tools in Structure-Based Drug Design 

Several computer modeling algorithms have been developed for structure-based drug design. Among 

them, DOCK [73–75] and 3D SEARCH [126] have been successfully applied in the design of HIV-1 

protease inhibitors. These programs search a target protein for invaginations, grooves, and recognition 

surfaces that could bind a potential receptor molecule. Compounds complementary to the putative 

receptor binding site in both shape and chemical properties can be identified through searching 

databases of small molecules, such as the Cambridge Crystallographic Database, the Fine Chemicals 

Directory, or other commercially available databases. 

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An important issue in analyzing HIV-1 RT is the flexibility of the enzyme. Comparison of structures of 

unliganded HIV-1 RT and NNRTI-bound HIV-1 RT complexes has shown that the NNIBP is not 

present in the unliganded form [34,41,43]. This underscores the importance of searching both the 

unliganded HIV-1 RT and the HIV-1 RT complexes with inhibitors and substrates in order to identify 

any potential inhibitor-binding sites. 

Many other approaches have been and are being developed for computeraided design of inhibitors. For 

example, pharmacophore analysis can identify the spatial arrangement of groups or atoms common to all 

active inhibitor molecules and then incorporate these elements into a single molecule [127,128]. 

Detailed analysis of the volumes occupied by different inhibitors bound to the same binding site could 

also provide new suggestions for inhibitor design. For example, the volume union of all known NNRTIs 

such as nevirapine, TIBO, α-APA, HEPT, and 1051U91 can be calculated. This type of analysis could 

be used to screen for new NNRTIs. Since the coordinates for a number of HIV-1 RT/NNRTI complex 

structures are now available in the Protein Data Bank, these approaches can be applied to the design of 

new or improved NNRTIs. Given the relatively high flexibility of the NNIBP region and the diversity of 

NNRTI structures, the NNIBP of HIV-1 RT could be a methodologically challenging yet extremely 

important target for structure-based drug design. 

XIV. Enzymatic Efficiency of Drug-Resistant HIV-1 RT Variants 

Analyses of viral population dynamics indicated that, although drug resistance cannot be seen as a 

positive outcome of chemotherapy, clinical progress can be made through the development of drugresistant 

viral variants (see review [30]).alyzed hy Arial H itself. It may be possible to use the available 

information on structural and biochemi- 


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Biochemical data show that HIV-1 replicates extremely rapidly in infected individuals and that the viral 

load is low in the early stages of the disease because the host immune system is initially successful in 

limiting viral replication [28,29]. When patients are treated with either RT and/or protease inhibitors, 

wild-type HIV-1 is rapidly replaced with drug-resistant variants. In fact, even in patients who have not 

received treatment with any anti-RT drugs, HIV-1 variants that contain residues corresponding to both 

NRTI- and NNRTI-resistance mutations in RT can be found as minor components of the viral 

population [129]. Similarly, viral variants that contain residues in protease sequence corresponding to 

protease inhibitor drug-resistance mutations have also been observed in patients prior to drug therapy 

(see review [130]). Enzymatic components found in a wild-type virus, such as RT or protease, are 

optimized for efficient viral replication [30]. In the absence of selective pressure (drug), the wild-type 

virus has a fitness advantage over drug-resistant viral variants. However, in the presence of drugs, drugresistant 

variants have a fitness advantage over the wild-type because the drug impairs efficiency of the 

target enzyme in the wild-type virus [30]. 

Binding of an NRTI or an NNRTI to wild-type HIV-1 RT interferes with the polymerization reaction. 

However, the presence of resistant variants in the population allows the virus to escape, and the variants 

to rapidly replace the wild-type virus. Nevertheless, this escape has a price. When the optimized wildtype 

virus is replaced by the less fit drug-resistant variants, the relative fitness of the virus decreases. In 

other words, the enzymatic efficiency of a drug-resistant HIV-1 RT variant is impaired relative to the 

wild-type enzyme (see review [131]). If the enzymatic efficiency of a drug-resistant viral variant is 

sufficiently impaired, the replication of the variant virus would be significantly decreased. Thus, an 

antiviral drug will be useful not because it would completely stop the growth of HIV-1 but because it 

selects viral variants whose replication is significantly impaired. Positive clinical benefit results from the 

fact that the viral load is decreased owing to reduced replication of the variant virus. As predicted by this 

model, some HIV-1 RT and protease inhibitors seem to select for relatively less fit drug-resistant 

variants. For example, treatment of HIV-1 infection with HBY 097, a quinoxaline inhibitor, induces 

development of an HIV-1 RT variant containing the Gly190Glu mutation that appears to have 

substantially decreased polymerase activity and replicates relatively slowly [22,84]. Replacement of the 

hydrogen atom of Gly190 with an acidic side chain of Glu190 in the hydrophobic NNIBP apparently 

interferes with the stability of the enzyme as well as the ability of the NNIBP to bind a hydrophobic 

inhibitor. The relative inefficiency of HIV-1 variant containing the Gly190Glu mutation in RT can be 

viewed as a positive outcome of the selection pressure provided by this particular inhibitor. However, 

most of the HIV-1 variants selected by 


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currently available antiviral agents are not significantly less fit than the wild-type virus and the clinical 

benefits are not obvious. In this regard, new drugs should be designed that would be intended to select 

HIV-1 RT variants that are significantly less fit and do not replicate efficiently. 

XV. Combination Therapy Using Multiple ANTI-HIV-1 Drugs 

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Monotherapy using either NRTIs or NNRTIs has led to the emergence of drug-resistant viral strains of 

HIV-1. Though many drug-resistance mutations confer cross-resistance to other inhibitors belonging to 

the same class, there are indications that some mutations conferring resistance to certain inhibitors are 

incompatible (see reviews [5,11,131]). A multidrug clinical trial with HIV-1 infected patients has shown 

that AZT resistance can be reversed by mutations that confer resistance to ddI [8]. The Leu74Val 

mutation appears to suppress the effects of the Thr215Tyr mutation that confers resistance to AZT 

[8,27]. The Met184Val mutation, which causes resistance to 3TC or other oxathiolane-cytosine analogs, 

also appears to reverse the effects of the AZT-resistance mutations [27]. Recent clinical studies have 

shown that a combination of AZT and 3TC led to a considerable decrease in viral load and a substantial 

increase of CD4 cells when compared with monotherapy using AZT alone, even after emergence of the 

Met184Val mutation [132]. Another example is the Pro236Leu mutation that confers resistance to 

BHAP. The sensitivity of this HIV-1 RT variant to TIBO, nevirapine, and pyridinone is increased ten 

fold [133]. Although the NRTIs and NNRTIs target two distinct binding sites of HIV-1 RT and lead to 

different sets of resistance mutations, some of the NRTI- and NNRTI-resistance mutations also appear 

to be incompatible. For example, the NNRTI-resistance mutations Leu100Ile and Tyr181Cys have been 

shown to suppress the effects of some AZT-resistance mutations [11,134]. This has led to the suggestion 

that a combination of anti-HIV-1 drugs would be more effective in inhibiting HIV-1 replication than 

using individual drugs alone. In fact, both clinical and in vitro studies have shown that combination 

therapy has considerable advantages over monotherapy. At least in some cases, the effectiveness of the 

therapy increases with an increase in the number of drugs in the combination [5,135]. Combination 

therapy may, in addition to increasing antiviral activity, also slow emergence of drug-resistant variants 

and may have the added benefit that reducing the dosage of individual drugs can reduce toxicity. It is 

generally believed that synergistic drug interactions arise from the fact that certain combinations of drugresistance 

mutations are particularly detrimental for the enzyme (and, by extension, the virus). This has 

focused attention on 


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determining the mechanism(s) underlying drug resistance and, from this understanding, to devise ways 

for identifying combinations of drugs which might provoke drug-resistance mutations incompatible with 

viral survival. 

Several protocols have been designed for combination therapy using a variety of anti-HIV-1 drugs (see 

reviews [5,11]). Combinations of different drugs that interact with the same binding site of the same 

viral protein but lead to mutually antagonistic or suppressive resistance mutations have been studied 

extensively, especially for the combined uses of different but structurally related NRTIs (see for 

example [136–138]) or NNRTIs [139–141]. Combinations of drugs or inhibitors that target different 

sites of the same viral protein, primarily the combination of NRTIs and NNRTIs of HIV-1 RT, show 

enhanced inhibition of HIV-1 RT polymerase activity and suppression of the emergence of drugresistance 

mutations (for example [142–146]). Experiments have also been conducted with combinations 

of drugs that target different viral proteins, e.g., inhibitors of virus adsorption, virus-cell fusion, and/or 

uncoating proteins have been tested in combination with protease inhibitors and/or RT inhibitors. 

Combinations of AZT with the glycosylation inhibitor castanospermine [147], or with the Tat inhibitor 

Ro 24-7429 [148], or with the protease inhibitor Ro 31-8959 [149] have been shown to potently inhibit 

HIV-1 viral replication in vitro. 

Combination therapy can increase the effectiveness of inhibition and significantly impair efficiency of 

viral replication. However, both NRTI- and NNRTI-resistance mutations can affect the positioning of 

the nucleic acid and/or the overall structure of HIV-1 RT [23]. These two sets of resistance mutations 

can communicate with each other and can result in cross resistance. Moreover, new drug-resistance 

mutations that confer cross-resistance to both NRTIs and NNRTIs can be selected, which reduce the 

effectiveness of some drug combinations (see reviews [5,11,26]). Biochemical studies showed that both 

HIV-1 RT mutants [150] and viral variants [151] could be obtained that are resistant to the combination 

of AZT, ddI, and nevirapine. In clinical trials, treatment with AZT and ddI or AZT and ddC led to a 

different spectrum of NRTI-resistance mutations [152,153]. The most notable of these new mutations is 

Gln151Met, which is located at a position close to the dNTP-binding site. Structural analysis of the HIV- 

1 RT/DNA/Fab complex suggests that the side chain of Gln151 in the wild-type enzyme may interact 

with the first unpaired template nucleotide. The side chain of this residue may play a role in selecting the 

correct base for the incoming nucleotide [72]. Since the RT mutant containing only the Gln151Met 

mutation can confer high-level resistance to a number of NRTIs, including AZT, ddI, and ddC, it is not 

clear why this mutation did not emerge in monotherapy of these NRTIs. However, Gln151 is relatively 

well conserved and mutations at this position may have an unfavorable impact on HIV-1 RT. 


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XVI. Perspective 

Page 70 

Substantial progress has been made in understanding the structure and function of HIV-1 RT and in the 

development of anti-HIV-1 inhibitors. However, the genetic flexibility of HIV-1 will continue to make 

development of a truly effective antiviral therapy for AIDS an exceptionally difficult task. We are 

beginning to understand how to circumvent drug resistance. The accumulated evidence has shown that 

the ability of the virus to develop drug resistance is limited and that the drug-resistant viral variants are 

less efficient than the wild-type virus. If the selection pressure provided by antiviral drugs makes the 

virus pay a sufficiently high price, then the viral load can be decreased and there will be a measurable 

clinical benefit. Based on a better understanding of the structure-function relationships of HIV-1 RT, we 

are now coming to grips with the mechanisms of polymerization, drug inhibition, and drug resistance. 

This information should make it possible to develop new or improved HIV-1 RT inhibitors that have 

different properties and provoke different patterns of drug-resistance mutations. Though it is likely that 

there will be no single drug which would be effective against all HIV-1 variants, we have reasons to 

believe that new or improved drugs or, more likely, new drug combinations, will be designed that are 

broadly effective against all of the HIV-1 variants that can grow efficiently. Detailed analysis of the 

conformational changes among the various HIV-1 RT structures may reveal additional sites (in addition 

to the currently known NRTI- and NNRTI-binding sites) for binding new inhibitors able to interfere 

with the polymerization and/or the flexibility of the enzyme required for its activity. The considerable 

physical and genetic flexibility of HIV-1 RT suggests that more effective anti-RT drugs should be 

designed to target the conserved portions of HIV-1 RT that the virus cannot easily afford to change. 

Such conserved elements can be identified by comparing the sequences of RTs from different 

retroviruses; the functions and relative importance of these conserved elements can be determined by 

mutagenesis and biochemical and structural analyses. It is our hope that application of structure-based 

drug design strategies may aid in the development of novel HIV-1 RT inhibitors for a more effective 

treatment of HIV-1 infection. 


We thank the other members of the Arnold and Hughes laboratories and our collaborators for their 

helpful discussions and assistance, including Koen Andries, Gail Ferstandig Arnold, Paul Boyer, Arthur 

Clark, Jr., Paul Janssen, Jörg-Peter Kleim, Luc Koymans, Tack Kuntz, Karen Lentz, Chris Michejda, 

Henri Moereels, Manfred Roesner, Marilyn Kroeger Smith, Rick Smith, Jr., and 


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Chris Tantillo. The work in Edward Arnold's laboratory has been supported by Janssen Research 

Foundation and NIH grants (AI 27690 and AI 36144). Research in Stephen Hughes' laboratory is 

sponsored in part by the National Cancer Institute, DHHS, under contract with ABL, and by NIGMS. 

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3 

Retroviral Integrase: Structure as a Foundation for Drug Design 

Alison B. Hickman and Fred Dyda 

National Institutes of Health, Bethesda, Maryland 


A. Retroviral Lifecycle 

Page 83 

The human immunodeficiency virus (HIV) is one of only a few retroviruses known to infect humans. It 

is estimated that approximately twenty-two million people are now infected worldwide [1]. With only a 

tiny number of exceptions, infection ultimately leads to the development of the lethal condition of 

acquired immunodeficiency syndrome, or AIDS. To date, only a handful of drugs have been shown to 

have any effect on the course of the disease. These are, in general, relatively ineffective at significantly 

prolonging life, and drug resistance develops rapidly. Equally discouraging, vaccines have not yet been 

developed to prevent infection. 

The retroviral lifecycle presents several steps that can be targeted as possible sites of intervention by 

inhibitors. As shown in Figure 1, when a retrovirus encounters a host cell, specific recognition between 

proteins on the surface of the virus and receptors on the host cell surface leads to membrane fusion. The 

viral core then enters the cell cytoplasm where the process of reverse transcription begins. The 

requirement of the conversion of viral RNA to double-stranded DNA is a feature unique to retroviruses. 

With the recent exception of the protease inhibitor saquinavir, ritonavir, and indinavir, the drugs 

approved to date by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection 

have been nucleoside analogs targeted against the viral enzyme that carries out this conversion, reverse 

transcriptase. 


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Figure 1 

Retroviral lifecycle as summarized in Reference 67. Reprinted by 

permission of Springer-Verlag Publishing Co., New York, NY. 

Page 84 

Although details of the timing of reverse transcription, nuclear localization, and integration are not yet 

clear, it is generally recognized that the movement of double-stranded viral DNA across the nuclear 

membrane is followed by insertion, or integration, of the viral genome into a host-cell chromosome. The 

viral DNA moves as part of a larger “preintegration complex,” a high-molecular-weight aggregate 

whose composition has not yet been completely defined. 

The end result of integration is the incorporation of the viral DNA into the DNA of the host cell. Once 

there, the provirus can serve as a template for the production of mRNA, allowing for the synthesis of 

viral proteins. These are assembled at the cell membrane to produce new viral particles, which then bud 

off to seek out new cells to infect. The integrated viral DNA is also necessarily copied whenever the 

host cell undergoes cell division. The insidious nature of 


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the virus arises because, once integrated, the viral DNA can no longer be distinguished from host cell 

DNA and has become a permanent fixture of the host cell genome. 

B. Rationale for Drug Design Against Integrase to Fight HIV and AIDS 

Page 85 

It has been demonstrated that the chemical steps that comprise DNA integration are carried out by the 

viral protein, integrase (IN). Integrase is encoded by the 3' end of the viral pol gene, which also codes 

for two other viral enzymes, the protease and reverse transcriptase. These three enzymes are initially 

synthesized as part of a larger polyprotein that is subsequently cleaved by the protease to the individual 

proteins. 

Why is integrase a good target for drug-design efforts to prevent infection by halting the viral replication 

cycle? First, integration is required for replication. In the absence of integration, the virus is unable to 

continue to make copies of itself. Secondly, the enzyme that carries out integration is virally encoded, 

and when the viral genome is disrupted so that functional integrase is no longer made, sustained viral 

replication does not occur [2]. This demonstrates that if viral integrase can be effectively inhibited, there 

is no protein encoded by the host cell that can replace it and carry out viral integration. Finally, since 

mammalian cells do not have enzymes capable of integrating HIV DNA, there are no vital host cell 

analogs of integrase carrying out essential reactions whose function would be blocked by integrase 

inhibitors. 

Effective inhibition of HIV integrase would add to the number of sites at which the virus replication 

cycle can be halted. One can imagine treatment protocols in which a mixture of inhibitors, each aimed at 

a different viral protein, could be administered. This is known as divergent combination therapy. As 

structural details are a necessary starting point for rational drug design, we present here our recent 

results on the high-resolution three-dimensional structure of the catalytic core domain of HIV-1 

integrase [3]. We also review the current literature discussing integrase inhibitors and present thoughts 

on ways in which knowledge of the chemical reactions carried out by integrase and its structure might 

direct the development of effective inhibitors. 

II. Biochemical Reactions Catalyzed By HIV Integrase 

A. In Vivo Integration 

Details of the initial chemical reactions that occur during HIV integration are now well understood (for 

reviews, see References 4,5). Once linear double-stranded DNA is available for integration, (Figure 2a) 

integrase then removes 


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Figure 2 

In vivo reactions carried out by HIV integrase. 

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two nucleotides from each 3' end of the viral DNA (Figure 2b). The two nucleotides are removed as a 

dinucleotide rather than in two individual steps. The specificity for this reaction is conferred by the third 

and fourth nucleotides from each 3' end, a -CA sequence that is absolutely conserved. Once two 

nucleotides have been removed, leaving recessed 3' hydroxyl groups, the next step is the joining of the 3' 

ends to target DNA (Figure 2c, d). This process, known as double-ended integration, occurs on opposite 

strands such that the joining sites on each of the target DNA strands are separated by five base pairs. The 

final step in integration is the repair of the single-stranded gaps generated by the staggered insertion of 

the viral 3' ends on opposite strands; this regenerates an intact double-stranded DNA molecule (Figure 

2e and f). Gap repair is probably carried out by host cell DNA repair systems. 

One necessary consequence of retroviral integration is the duplication of five base pairs of host cell 

DNA on either side of the integrated provirus. 


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Another is the loss from the ends of the viral DNA of the original two base pairs that preceded the 

conserved 3'-CA. 

B. In Vitro Assays to Monitor Integrase Activity 

Page 87 

In contrast to the in vivo reaction, concerted integration in vitro of two HIV DNA ends into a target 

DNA molecule separated by a 5 base-pair stagger occurs very inefficiently. However, in vitro systems 

have been developed [6,7] using recombinant HIV integrase that have allowed the chemistry of the 

single-ended integration event to be studied in fine detail. It is possible and routine to use short, doublestranded 

synthetic oligonucleotides that mimic the viral ends to monitor the removal of two nucleotides 

from 3' ends (denoted 3' processing or cutting) and the subsequent insertion of one 3' processed DNA 

molecule into another (known as strand transfer or joining). Typical reactions are depicted in Figure 3. 

The stereochemical mechanism of 3' processing and strand transfer has been investigated using DNA 

substrates that incorporate phosphorothioate link-ages [8]. For both reactions, the introduced chiral 

centers are inverted in the products, implying that the reactions occur via a one-step in-line displacement 

mechanism rather than via a covalent intermediate. 

A third assay of integrase activity, termed disintegration, has more recently been developed [9] that 

monitors the apparent reversal of strand- 

Figure 3 

Reactions carried out by integrase in vitro, using short oligonucleotide substrates. 


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transfer (Figure 3). While disintegration probably has no physiological significance, it has been useful in 

defining aspects of integrase biochemistry. 

The three in vitro activities of integrase require divalent metal ions as cofactors. The only two metals 

that support these activities are Mn 2+ and Mg 2+. Since quite high metal concentrations must be added to 

assays (1–10 mM for optimal activity), it has been presumed that Mg 2+ is the ion used in vivo. 

C. Evidence for a Multimer as the Active Unit of Integrase 

Several lines of evidence demonstrate that the active unit of integrase is a multimer. It is clear, as an 

isolated protein in solution, that integrase forms dimers [6,10–12], and it has been shown by 

sedimentation equilibrium studies that Rous sarcoma virus (RSV) integrase exists in reversible 

equilibrium between monomeric, dimeric, and tetrameric forms [13]. Protein-protein cross-linking 

studies of HIV-1 [14] and RSV [15] integrases confirm the existence of protein dimers and tetramers in 

solution, and in vivo, the yeast GAL4 two-hybrid system has demonstrated that HIV-1 integrase can 

interact with itself [16]. 

Complementation studies using mutant proteins in vitro provide compelling evidence that the active 

form of integrase must be at least a dimer [14, 17]. This can be inferred from the result that when certain 

inactive forms of integrase—generated either by truncation or point mutation—are mixed, robust 

activity can be reconstituted. This indicates that different monomers in a multimer are capable of 

providing different essential functions in the context of an active complex. 

Collectively, these studies suggest that integrase acts as a multimer. This would also seem the most 

straight-forward model to explain the observation that viral integration requires two coordinated cutting 

and joining reactions on the target DNA during strand transfer. However, physical studies have not yet 

addressed what form of integrase actually binds to DNA and carries out the chemical reactions of 

integration. 

III. Properties of HIV-1 Integrase 

A. Domain Structure of Retroviral Integrases 

A consistent view of the domain structure of retroviral integrases has emerged by combining the results 

from biochemical studies using deletion and site-specific mutants, limited proteolysis experiments, and 

sequence comparisons among the family of retroviral integrases. The organization of the domains of 

integrase is shown schematically in Figure 4. 

The central domain of HIV-1 integrase, consisting approximately of residues 50 to 200, is largely 

conserved among retroviral integrases, and forms 


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Figure 4 

Schematic domain structure of HIV-1 integrase as adapted from 

Engelman et al. [19]. Structures of two domains, the catalytic core 

extending from residues 50 to 212 [3] and the nonspecific 

DNA-binding domain from residues 220 to 270 [28,29], have 

recently been determined by x-ray crystallography and NMR 

spectroscopy, respectively. 

Page 89 

the protease-resistant core of the protein [18,19]. Within this domain are three invariant residues that 

comprise the “D,D-35-E motif” (see alignment in Figure 5). These are residues Asp64, Asp116, and 

Glu152. Even conservative substitution of any of these residues leads to loss of all three in vitro 

activities of integrase in parallel [19–21]. The D,D-35-E motif is also observed in retrotransposons and 

some prokaryotic transposases. A truncated form of HIV-1 integrase consisting of residues 50 to 212 is 

capable of disintegration [22], implying that the catalytic site is contained within this domain. These 

observations and the absolute requirement for metals for in vitro activity have led to the proposal that 

the three acidic residues constitute a divalent metal-binding site capable of binding one or two Mg 2+ or 

Mn 2+ ions to form a catalytically active enzyme. As will be seen in later sections, the three-dimensional 

structure of the core domain of HIV-1 integrase is consistent with this hypothesis. The catalytic 

mechanism may be, therefore, similar to the one proposed by Beese and Steitz for the 3'–5' exonuclease 

of E. coli DNA polymerase I [23]. It is proposed that for phosphate bond cleavage, one metal ion helps 

form the attacking hydroxide ion while the other stabilizes a pentacovalent intermediate around the 

phosphorus. 

The C-terminus of HIV-1 integrase, consisting approximately of residues 210 to 288, includes the 

dominant nonspecific DNA binding domain [24, 25], which has been more finely mapped to residues 

220–270 [26]. The C-terminus is the least conserved region of retroviral integrases; only one residue, 

Trp235, is absolutely invariant. However, it has been reported that removal of only five amino acids 

from the C-terminus of HIV-1 integrase is enough to severely reduce its 3' processing and strand transfer 

activities [27]. One notable feature of the C-terminus is its high proportion of positively charged 

residues. As discussed in Section IV.E, the structure of part of this region has recently been determined 

using NMR spectroscopic methods [28,29]. 


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Figure 5 

Alignment of amino acid sequences of retroviruses. See Engelman et al. [19] for details. 


Page 91

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Page 92 

The role of the N-terminus of integrase, residues 1 to 50, is still unclear. Within this region are four 

strictly conserved amino acids: two His and two Cys residues. In HIV-1 integrase, the spacing is His-X 3- 

His-X 23-Cys-X 2-Cys. This cluster of His and Cys residues is reminiscent of a zinc-binding motif, and it 

has been demonstrated that the full-length protein binds Zn 2+ [22,30], and that the separately expressed 

domain consisting only of residues 1 to 55 also binds Zn 2+ stoichiometrically [31]. However, it has not 

been shown that either the structural integrity or the enzymatic activities of integrase require Zn 2+. While 

truncation of residues from the N-terminus of HIV integrase results in loss of 3' processing and strand 

transfer activities [22,25], in the case of RSV integrase, the N-terminal region can be replaced by 

unrelated sequences, and the enzyme is still capable of all three in vitro activities [32]. 

B. Biophysical Properties of Full-Length Recombinant HIV-1 Integrase 

It has been known for some time that recombinant HIV-1 integrase is a particularly poorly behaved 

protein in solution. Its solubility in most usual buffers is limited to approximately 1 mg/mL, and even 

then only in the presence of high concentrations of NaCl. At ~1 mg/mL, HIV-1 integrase slowly 

precipitates out of solution, revealing one of its characteristic features, a tendency towards aggregation. 

These properties of the protein are not unreasonable, since in its viral environment integrase is probably 

never required to be a soluble protein. To maintain the integrity of preintegration complexes, it may 

even be advantageous for the protein to have the properties of being rather insoluble and sticking to 

itself, nucleic acid, and perhaps other proteins. 

C. Properties of Truncated Versions of HIV-1 Integrase 

It has been our approach to protein structure determination by x-ray crystallography that it is imperative 

to begin with well-characterized and well-behaved protein. In particular, it is important that the protein 

be reasonably soluble and monodisperse in solution. Unfortunately, as discussed above, recombinant 

HIV-1 integrase satisfies neither of these conditions. One approach we and others have taken to 

circumvent these problems has been to examine truncated versions of HIV-1 integrase to determine if 

removal of amino acids from either terminus or both affects solubility and aggregation properties. 

Although we observed that two proteins we constructed, IN 213–288 and IN 50–288, were more soluble than 

the full-length HIV-1 integrase, IN 1–288 [33, and unpublished observations], our first target protein for 

crystallization efforts was the core domain of HIV-1 integrase consisting of residues 50 to 212, IN 50–212. 

We reasoned that this protein domain was likely to be compact and 


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well folded since it is relatively resistant to proteolysis. As it is also active for disintegration, we 

concluded that it contained the enzyme active site. 

Page 93 

We exploited the convenience of histidine-tag (HT) technology to develop methods to purify large 

quantities of IN 50–212 [12]. A 20-amino-acid histidine-containing tag was added to the N-terminus of 

HIV-1 IN 50–212 [22] to allow rapid purification on nickel affinity columns. It was subsequently removed 

by thrombin cleavage. Biophysical studies showed that although buffer conditions could be identified 

where the protein was soluble to ~ 4 mg/mL, under these conditions the protein was highly aggregated 

(unpublished observations). Although the aggregation problem could be largely avoided by the addition 

of high concentrations of the zwitterionic detergent CHAPS, conditions could not be identified under 

which protein crystals formed in the presence of CHAPS. 

D. Systematic Mutation of Hydrophobic Residues to Improve Protein Solubility 

As it became clear that IN 50–212 was crystallographically challenged, a condition readily understood in 

terms of its aggregation problems and low solubility, a more radical approach was undertaken to try and 

improve its biophysical properties. Hydrophobic residues in the catalytic core were targeted for sitespecific 

mutation according to the following criteria: where two or more hydrophobic residues were 

encountered close together in the primary amino acid sequence, they were each changed to an alanine 

residue. When a hydrophobic residue stood alone, it was mutated to lysine. In this way, 29 different 

mutant proteins of IN 50–212 were rapidly generated using the overlapping polymerase chain reaction 

(PCR) and screened for improved solubility properties [34]. Three mutated proteins were identified that 

were more soluble at lower NaCl concentration than the unmutated core (V165K, F185K, and the 

double mutation of W131A/W132A). However, one of these in which Phe185 was mutated to Lys had 

dramatically improved solubility and was ultimately crystallized and its three-dimensional structure 

determined [3]. The remarkable biophysical properties of this single point mutant of IN 50–212 have 

recently been described [34]. 

IV. Structure Of The Catalytic Core Domain Of HIV-1 Integrase 

A. Description of the Structure 

The Overall Protein Fold 

The three-dimensional structure of the catalytic core domain of HIV-1 integrase is centered on a mixed 

five stranded β sheet flanked by several helices forming 


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Figure 6 

Molscript stereo figure of the three-dimensional structure of the catalytic core of 

HIV-1 integrase. The two catalytically essential aspartic acid residues (D64 and D116) 

visible in the x-ray structure are highlighted. 

Page 94 

an α-β meander sandwich (see Figure 6) [35]. In the crystal structure, interpretable electron density 

starts at Cys56, leading to a short loop. The first β strand starts at Gly59 and runs until Val68. The two 

central residues of a type I' reverse β turn, Glu69 and Gly70, change the polypeptide chain direction to 

form the second β strand between residues Lys71 and His78, which runs antiparallel with the first 

strand. A type I'β turn follows, with Val79 and Ala80 changing the chain direction again to form the 

third β strand between Ser81 and Ile89, which runs antiparallel with the second strand. A short loop 

between Pro90 and Glu92 leads to the first α helix (helix A) between Thr93 and Trp108. This helix 

packs against the bottom face of the sheet formed by the first three antiparallel strands by several 

hydrophobic interactions. A short loop (Pro109 and Val110) leads to the fourth β strand between Lys111 

and His114. This strand is parallel with the first. A short loop starting at Thr115 leads to helix B, a one 

turn helix between Gly118 and Thr122, followed by helix C between Ser123 and Ala133. This helix 

runs parallel to and packs against helix A. The residues Gly134 and Ile135 form a short loop prior to the 

fifth and last β strand of the structure between Lys136 and Ala138. This short strand is parallel with the 

first and the fourth. There is no interpretable electron density due to disorder between Gly140 and 

Met154. At Met154, the fourth α helix (helix D) starts and runs until Ala169 on the top face of the sheet 

formed by the first three β strands. The residue Glu170 leads into the next helix (helix E) running 

between His171 and Lys186, the first residue of a short basic sequence (Lys186, Arg187, and Lys188). 

Together with 


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Gly193 and Tyr194, Lys188 and Gly189 form two short antiparallel β strands separated by a turn of 

three residues (Gly190, Ile191, and Gly192) not involved in main chain hydrogen bonds. The first 

residue of the last helix, (helix F), which runs until Asp212, is Ser194. 

Three Conserved Acidic Residues at the Enzyme Active Site 

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There are four amino acids in the core domain sequence that are absolutely conserved among retroviral 

integrases: Asp64, Asp116, Glu152, and Lys159. The three acidic residues form the conserved D,D-35- 

E motif and have been shown to be essential for catalysis (see Section III.A). The role of Lys159 in 

retroviral integrases is not obvious; its replacement with Val does not abolish catalytic activity, although 

there is a decrease in strand transfer activity [20]. 

The first essential acidic residue, Asp64, is located in the middle of the first β strand, while Asp116 is in 

a loop region right after the fourth β strand. These two residues define the active-site area and they are 

right next to each other three-dimensionally with their α-carbons separated by only 6.7 Å. The closest 

approach is 3.4 Å between Oδ1 of Asp64 and Cβ of Asp116. These residues are on the surface of the 

molecule, not part of any obvious substrate-binding cleft. The third catalytically essential acidic residue, 

Glu152, is in the disordered and hence crystallographically invisible region between Gly140 and 

Met154. Its location therefore must be inferred from other parts of the structure and from available threedimensional 

structures of related proteins. The location of Met154, the residue only two positions 

upstream from Glu152, is known because of interpretable electron density. The distance between the αcarbons 

of Glu152 and Met154 cannot be larger than about 7.3 Å, which constrains Glu152 to the 

neighborhood of the two other essential carboxylates, allowing it to contribute to the formation of a 

divalent metal-binding site. 

More recently, a crystal structure of the avian sarcoma virus (ASV) integrase core domain was solved 

[36]. Within this domain, ASV integrase has 24% sequence identity to the HIV-1 integrase core and, as 

expected, its three-dimensional structure is remarkably similar. The ASV integrase core in its native 

form has much better solution properties than the HIV-1 integrase core, and did not require any point 

mutations to render it crystallizable. Due to this fact and perhaps also due to its different crystal packing 

interactions, the crystal lattice of the ASV integrase core domain is somewhat more ordered than that of 

HIV-1. The two three-dimensional structures can be aligned quite well, using 74 α-carbons, with an rms 

deviation of only 1.4 Å in these α-carbon positions. The most remarkable difference between the two 

structures is that in the ASV structure the electron density is interpretable in all parts of the molecule. 

This is not to say, however, that serious disorder is not present. For example, in one particular loop, the 

temperature factors are above 70 Å 2 for the α carbons, indicating larger than 1 Å mean displacement 

value for these atoms. The corresponding 


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Page 96 

region in the HIV-1 structure is the uninterpretable stretch from residues 140 to 153, showing that 

beyond the three-dimensional similarity, the molecules also share a similar disorder pattern despite their 

different crystal packing interactions. It is clear that in the apoenzyme (metal-free) form of the HIV-1 

integrase core, disorder is present in parts of the active site. However, in the holo form, structural 

stability must be necessary to form a metal-binding site. 

Position of the Third Essential Carboxylate 

Does the structure of ASV integrase give us a hint about the likely conformation of the polypeptide 

chain around Glu152 in the holoenzyme form of the HIV-1 integrase core? The answer is probably yes, 

considering the overall similarity of the structures. The first residue after the disordered part in the HIV- 

1 integrase core is Met154, which is also the first residue in helix C. The corresponding helix in the 

ASV core is longer, running between Gln153 and Gly175. The residue analogous to Glu152 is Glu157 

in the ASV integrase core structure, located a half-turn upstream from Ala159, which corresponds to 

Met154 in the HIV-1 integrase structure. It is plausible to assume, therefore, that the polypeptide chain 

in the holoenzyme form of HIV-1 integrase would also be in a helical conformation around Glu152, and 

its location would be very close to the one that can be inferred from the location of Glu157 in the ASV 

integrase core. Secondary structure prediction also supports this assumption, assigning α-helical 

structure around Glu152. Why does this helical turn show significant disorder in the HIV-1 integrase 

structure? The answer might be found in the amino acid sequence: Pro145 is a highly conserved residue 

among retroviral integrases, the only exception being ASV integrase where it is substituted with a Ser. 

Since the main chain nitrogen of a proline is not capable of participating in hydrogen bonding, it is very 

rarely found in helices. It is likely that if the polypeptide chain around Glu152 were helical in the 

holoenzyme form of the HIV-1 integrase core, then this helix would start after Pro145. There is no such 

restriction in the ASV integrase core, and it is possible that this is why helix C is longer in ASV than in 

HIV. This may also explain the disorder around Glu152 in the HIV-1 integrase core, since it is closer to 

the end of the helix and more susceptible to disordering effects. A longer helix and therefore a more 

ordered active site in the apo form may be a unique feature of the ASV integrase core. 

B. Similarity to Other Polynucleotidyl Transferases 

Overall Protein Folds 

The catalytic core domain of HIV-1 integrase has a topologically identical fold with the RNase H 

domain of HIV-1 reverse transcriptase [37], the RuvC Holli- 


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Figure 7 

Structures of the catalytic core of HIV-1 integrase, HIV-1 RNase 

H, RuvC, and the core domain of MuA transposase demonstrating 

similarities in folding topology. The catalytically essential residues 

are highlighted. 


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day junction resolving enzyme [38], and also the core domain of the phage MuA transposase [39] (see 

Figure 7). In the case of HIV-1 integrase, RNase H, and RuvC, definite three-dimensional similarity 

extends only to the ends of the last β strand; from this point, the structures diverge. In HIV-1 RNase H, 

there is only one more α helix corresponding to helix D in the HIV-1 integrase core but in a 40° 

different orientation. In RuvC there are three more helices, with the last one running parallel to helix D 

of HIV-1 integrase, but in the opposite direction and also 4.6 Å closer to the β sheet. In contrast, the 

homology between HIV-1 inte- 


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grase and the MuA transposase extends until the carboxyl termini of their respective catalytic domains 

with three very similarly positioned and oriented α helices. 

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Limited three-dimensional alignment between the four molecules can be accomplished by identifying 

structurally homologous stretches along the polypeptide chains if this search is restricted to between the 

first well-ordered amino terminal residue and the end of the last β strand. For the core domain of HIV-1 

integrase, this corresponds to the region between Ile60 and Gln137. Using the corresponding region in 

the MuA transposase, the two structures can be aligned with an rms deviation of 1.7 Å over 69 α-carbon 

positions. The main differences between these structures are two insertions in the transposase core: an 

11-residue β-stranded extension replacing the turn between the first and the second strand in the HIV-1 

integrase core, and a 15-residue extension before helix B with no secondary structure. Both of these 

extensions interact with the downstream nonspecific DNA-binding domain of the transposase. For HIV- 

1 RNase H, the alignment results in a rms deviation of 2.0 Å over 48 alignable α-carbon positions. The 

position of helix A is significantly different in RNase H, as it shifts more than 5 Å toward the β sheet. 

There is also an additional 2.5 turn helix following the fourth β strand and a 5-residue loop after this 

helix. For RuvC the alignment yields an rms deviation of 2.0 Å over 50 alignable α-carbon positions. In 

this case, the differences are mostly the result of longer secondary structure elements in RuvC. Of all the 

molecules compared, the HIV-1 integrase core is the smallest, with the most compact design in the 

region where these alignments were performed. For comparison, let us mention again that the 

homologous ASV integrase core can be aligned with an rms deviation of 1.4 Å over 74 α-carbon 

positions in this region. 

Both topological similarity and three-dimensional homology with the MuA transposase was expected 

based on the similarity of the reactions the enzymes catalyze, but the relationship with RNase H and 

RuvC was a surprise. This discovery led to the proposal of a new polynucleotidyl transferase 

superfamily. All the members of the superfamily are divalent metal ion-dependent endonucleases, and 

they all leave 3'-OH and 5'-phosphate groups at the site of cleavage. All the members of the superfamily 

display their catalytically essential acidic residues at the same general location. There are three such 

residues in HIV-1 integrase, RNase H, and the MuA transposase, while there are four in RuvC. Two of 

these residues are always located on the same three-dimensional structural elements, while the location 

of the third varies. The Asp64 residue HIV-1 integrase corresponds to Asp443 in HIV-1 RNaseH, Asp7 

in RuvC, and Asp269 in the MuA transposase. Based on the three-dimensionally aligned structures, the 

α-carbon positions of these residues cluster quite well around that of HIV-1 integrase, with an rms 

deviation of 0.84 Å. All these residues are located in the middle of first β strand. The side chain torsion 

angle, Chi 1, is 


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-62° for HIV-1 integrase, a frequently observed rotamer. For the other three molecules this Chi 1 value 

varies between -142° and -156°, a common range for rotamer angles. The preference for the first 

rotamer of the HIV side chain is probably due to the 2.73 Å-long hydrogen bond between Oο°2 of 

Asp64 and Nε2 of Gln62. There is no such interaction in the other molecules. The different rotamer 

causes a 2.46 Å rms separation of the Cγ positions around the HIV-1 Cγ, compared to only 1.63 Å 

around the Cγ of Asp443 in HIV-1 RNase H, indicting the carboxylate of Asp64 in HIV-1 integrase as 

the outlier. 

Position of the Second Carboxylate Residue 

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In all the analogous structures, the second essential carboxylate resides just after the end of the fourth β 

strand. The main-chain atoms are not involved in strand-forming direct hydrogen bonds, therefore the 

chain diverts from running parallel with the first strand, forming a small cleft. The equivalent residues 

are Asp116 in HIV-1, Asp498 in HIV-1 RNase H, and Glu66 in RuvC. The clustering is weaker than for 

Asp64; the rms deviation is 1.77 Å in α-carbon position around the HIV-1 integrase residue. 

Interestingly, by including the structurally otherwise highly homologous ASV integrase core, the rms 

deviation increases to 2.15 Å due to the 3 Å distance between the Cα of Asp116 of HIV-1 integrase and 

that of the corresponding residue, Asp121 of ASV integrase. The rms separation between the ASV 

position and the rest of the cluster (now excluding HIV-1 integrase) is 2.2 Å, which is rather high, 

identifying the ASV residue as the outlier. For the Chi 1 torsion angles, all three preferred rotamers are 

present: Chi 1 is -86° for HIV-1 integrase, 73° for HIV-1 RNase H, and -173° for the MuA transposase. 

The RuvC Chi 1 value is not included in this comparison because it has a Glu in this position. The 

different Chi 1 values combined with the variation in α-carbon positions leads to a somewhat more 

scattered Cγ (or Cδ for Glu66 in RuvC) position with an rms deviation of 2.71 Å around Cγ of Asp116 

of HIV-1 integrase. By including the ASV molecule, the scatter increases to 3.82 Å due to the 6 Å 

distance between Cγ of Asp116 in HIV-1 integrase and Cγ of Asp121 of ASV integrase. 

The Third Essential Carboxylate 

The location of the third essential catalytic carboxylate varies between different members of the 

superfamily. For HIV-1 integrase, Glu152 is in a disordered region with no interpretable electron 

density. Based on the location of the equivalent residue in the ASV integrase, its position is assumed to 

be on helix D, as discussed above. For RNase H, Glu478 is located on helix A, with its side chain 

pointing toward the other two carboxylates to complete the divalentmetal-binding site. Such metal 

binding has been observed crystallographically 


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[37]. For RuvC, four residues have been shown to be essential for catalysis [40]. The third of these, 

Asp138, is at the amino end of the last helix of the structure. In the three-dimensionally aligned 

structures, this helix is parallel with the helix D in HIV-1 integrase, although it is running in the opposite 

direction. The position of helix D in RuvC is also significantly different, mainly due to a 13 Å shift 

along its axis toward the active site, placing Asp138 close to the other two carboxylates. The fourth 

essential residue, Asp141, is on the first turn of the same helix, very close in the aligned structures to the 

essential Glu157 of ASV integrase, their α carbons separated by only 1.6 Å. For the MuA transposase, 

its third essential carboxylate, Glu392, is in a loop region, just one residue upstream from the amino end 

of a helix, the topological equivalent of helix D. Unexpectedly, this residue turns away from the region 

defined by the two other carboxylates to a position where it clearly cannot contribute to the formation of 

a metal-binding site. It is likely, therefore, that the conformation of the polypeptide chain around Glu392 

in the transposase core observed in the crystal structure belongs to an inactive form. In this case, a 

conformational change upon transposase tetramer assembly or perhaps upon substrate binding is 

required for activity. 

Significance of the Disordered Region 

From the point of view of HIV-1 integrase, it is interesting to note that the apparently flexible part of the 

MuA transposase structure is topologically equivalent with the disordered and uninterpretable part of the 

integrase. Similarly, in the crystal structure of the isolated RNase H domain of HIV-1 reverse 

transcriptase, a five-residue loop in a topologically equivalent location is disordered and therefore 

uninterpretable. In the ASV integrase core, the corresponding loop is visible but with rather high 

mobility. It seems that some kind of disorder or flexibility in this region is a common feature of the 

superfamily. Crystal structures of enzyme-substrate or enzyme-inhibitor complexes will tell us the 

functional significance of this flexibility as well as the exact configuration of the active site. 

C. The Dimer Interface 

HIV-1 integrase is active as a multimer, and the catalytic core domain alone forms dimers in solution, 

even at low protein concentration (see Section II.C). In the crystal structure, a roughly spherical dimer of 

about 45 Å diameter was observed, formed by a crystallographic two-fold axis. The dimer has a large 

solvent-excluded surface of 1300 Å 2 per monomer. This area is close to what is expected for dimers in 

this molecular weight range [41]. Therefore, we are convinced that in the crystal structure the authentic 

dimer is present. This was subsequently confirmed by the structure of the ASV integrase core. Although 


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crystallized under different conditions, forming crystals that are in a different space group with different 

crystal-packing interactions, the dimer observed for the ASV core is essentially identical with that of 

HIV-1 integrase, although the solvent-excluded surface is smaller (only 740 Å 2). This difference is 

largely due to the absence of helix F in the ASV structure. 

The core domain dimer of HIV-1 integrase is held together by several hydrophobic and polar 

interactions. Hydrophobic interactions dominate the interface between helix E from one monomer and 

helices A and B from the other. There is a buried salt bridge between Glu87 of the third β strand and 

Lys103 on helix A. There are also some water-mediated polar interactions between these two secondary 

structure elements. There are direct hydrogen bonds between residues on helix A and residues on helix E 

across the interface including one between Lys185 (the substitution responsible for the improved 

solubility and therefore crystallizability) and the main-chain carbonyl on Ala105. In the ASV core, 

His198 is in this position, forming a very similar hydrogen bond with the carbonyl oxygen of Ala110. 

There are about 10 water molecules buried in the interface, all involved in hydrogen bonds. The part of 

the solvent-accessible surface of the monomer which becomes buried upon dimer formation displays a 

high degree of shape compatibility with itself: by rotating it 180° around the crystallographic two-fold 

axis, the resulting surface will fit the original one without forming large pockets. It is possible that the 

core domain of HIV-1 integrase has evolved to optimize this compatibility in order to increase its 

stability. It would be interesting to see the effect on protein activity of site-directed mutations aimed at 

disrupting this interface and hence the dimer (or possibly the higher order multimers in the context of 

the full-length protein). 

D. Implications of Crystallographic Dimer for the Chemistry of Catalysis 

The nearly spherical nature of the dimer formed by two monomers of the integrase catalytic core places 

active sites on respective monomers on opposite sides of the dimer: approximately 35 Å separates the 

carboxylate oxygens of Asp64 of each monomer. While we are convinced that the observed dimer is not 

an artifact or consequence of crystallization, it would seem difficult to reconcile this distance with the 

observation that, during in vivo strand transfer, cuts on the target DNA occur with a separation of five 

base pairs, corresponding to 15–20 Å in B-form DNA. How can a single dimer accomplish this? One 

possibility is that the cuts do not occur simultaneously. One end of the viral DNA could be joined by a 

reaction at one active site, followed by carefully controlled movement of DNA and protein relative to 

one another such that the second active site is now positioned five base pairs away from the initial site of 

strand transfer. It has been proposed, in a variation on this theme, that the first strand-transfer 


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reaction is followed by DNA relaxation (unwinding, rotation, etc.), resulting in the site on the target 

DNA for the second transfer reaction site now being located close to the active site of the second 

monomer [42]. An alternate possibility is that a multimer larger than a dimer is responsible for the 

coordinated cutting and strand-transfer reactions. For example, two contacting dimers can be modeled 

such that two active sites of the resulting tetramer are located 15–20 Å apart. It is also possible that even 

higher order multimers are involved. 

There is, as yet, no convincing evidence in support of any one model. The observation that RSV 

integrase cuts target DNA with a six-base-pair stagger rather than the five observed for HIV correlates 

intriguingly with the apparently longer distance (~ 38 Å vs. 35 Å) between active sites in the RSV 

dimer. However, understanding the coordinated cutting and joining reaction awaits three-dimensional 

information on the arrangement of monomers within an integrase multimer binding to DNA. 

E. Three-Dimensional Structures of Other Domains of HIV-1 Integrase 

Three-dimensional structural information has not yet been obtained for a full-length integrase protein. In 

its absence, attempts have been made to determine the structure of the smaller domains consisting of the 

separately expressed N- and C-termini that flank the core whose structure is now known. 

The Amino Terminus of Integrase 

While the N-terminus of HIV-1 integrase, consisting of residues 1 to 55, has been separately expressed, 

purified, and biophysically characterized [31], structural data has not yet been obtained. This protein 

domain binds metal ions such as Zn 2+, Co 2+, and Cd 2+ stoichiometrically, and is monomeric at low 

protein concentrations. Dramatic changes in helix content (from 14% to 32%) are observed in the 

circular dichroism (CD) spectrum upon addition of metal. Analysis of CD spectral features led 

researchers to conclude that it is highly probably that integrase contains a zinc finger that folds in much 

the same way as the TFIIIA-like DNA binding proteins, with two His residues located on an α helix and 

two cysteines part of a β sheet [31]. However, confirmation of such a model awaits structure 

determination by x-ray crystallography or NMR spectroscopy. 

The Carboxy Terminus of Integrase 

When the C-terminal domain is expressed as a separate polypeptide, IN 213–288 can be purified from the 

initial soluble fraction from cell lysates [33]. This small protein fragment, therefore, was an attractive 

target for structure determination. 


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Figure 8 

Molscript stereo figure of the structure of the nonspecific DNA-binding domain 

of HIV-1 integrase, IN 220–270 , determined by heteronuclear NMR spectroscopy [28]. 

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The structure of this domain is of particular interest as it represents the dominant nonspecific DNAbinding 

region of integrase. Gel filtration and sedimentation equilibrium results indicated that purified 

IN 213–288 partitioned between dimers and highly aggregated material (unpublished observations). 

However, a smaller domain consisting of residues 220 to 270 maintains the DNA-binding properties of 

the longer C-terminal domain and is better behaved in solution. Recently, two groups have reported the 

structure of this smaller fragment, IN 220–270, determined using multidimensional heteronuclear NMR 

spectroscopic methods [28,29]. 

As shown in Figure 8, the overall structure of IN 220–270, is that of a β sandwich formed by two threestranded 

β sheets. As anticipated by biophysical studies, the polypeptide is a dimer in solution. The 

interface between monomers is formed by the antiparallel interaction of three β strands from each 

subunit and is stabilized predominantly by hydrophobic interactions. There is a long loop between 

strands β1 and β2 which, in the dimer, defines the sides of a cleft that is of the appropriate dimensions 

(about 24 × 24 × 12 Å) to accommodate double-stranded DNA. The folding topology is very similar to 

that of SH3 domains that are found in several proteins involved in signal transduction, despite the lack 

of significant sequence homology. This is rather unusual since SH3 domains are generally involved in 

protein binding rather than interactions with DNA. 

V. Prospects for Inhibitors 

A. Overview of Inhibitor Studies to Date 

The investigation of HIV integrase inhibitors has been largely restricted to testing available compounds 

that inhibit other enzymes with similar substrates or 


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Table 1 Representative Inhibitors of HIV-1 Integrase and IC 50 values of compounds that inhibit 3' processing and 

strand transfer activities of HIV-1 integrase 

Compound 

I aurintricarboxylic acid 

monomer 

IC 50 of 3' processing 

(μM) 

IC 50 of strand 

transfer (μM) 

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Active against 

IN 50–212 ? Reference 

10–50 n.d. n.d. 44 

II cosalane n.d. 25 n.d. 45 

III DHNQ 5.7 2.5 yes 47 

IV primaquine 15 3.6 n.d. 46 

V CAPE 220 19 yes * 46 

VI quercetin 24 14 n.d. 47 

VII quercetagetin 0.8 0.1 yes 47 

VIII AG1717 0.4 0.16 yes 50 

IX β-conidendrol 0.5 0.5 n.d. 51 

X suramin 0.25 0.11 n.d. 53 

XI curcumin 95 40 yes 55 

XII (neocuproine) 2-Cu + 3 3 yes 56 

XIII AZT-monoP i 100–150 100–150 yes 57 

XIV GT 17-mer 0.092 0.046 n.d. 59 

XV HCKFWW 2 2 yes 43 

The third column indicates if these compounds also inhibit disintegration by IN 50–212 . n.d. = not determined. * = only if 

pre-incubated. 


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proposed mechanisms. For example, as will be seen, many topoisomerase II inhibitors also inhibit HIV 

integrase. While screening of chemical databases is likely underway at several pharmaceutical 

companies, results have either not been made available or are discouraging (see below). One foray into 

integrase inhibitor design—rather than discovery—has recently been described using a peptide 

combinatorial library approach [43]. We summarize below published reports to date (March 1996) in 

which compounds have been identified that inhibit integrase in in vitro assays with IC 50 values of 100 

μM or less (IC 50 is the concentration at which the measured activity is inhibited by 50%). In vitro 

inhibition data is compiled in Table 1; structures of selected compounds are shown in Figure 9. 

An Effective Pharmacophore: Multiple Hydroxyl Groups on Aromatic Rings 

The first report of a class of compounds that inhibits HIV integrase appeared in 1992 [44]. 

Aurintricarboxylic acid (I) and its derivatives, known to inhibit other enzymes that process nucleic acids, 

were determined to inhibit 3' processing with moderate IC 50 values of 10–50 μM. As shown in Figure 9, 

a recurring structural theme was established early on in which integrase inhibitors often possess 

aromatic rings with multiple hydroxy substituents that are either located 


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Figure 9 

Chemical structures of reported inhibitors of HIV-1 integrase. (I) aurintricarboxylic 

acid monomer; (II) cosalane; (III) dihydronaphthoquinone or DHNQ; (IV) 

primaquine; (V) caffeic acid phenethyl ester or CAPE; (VI) quercetin; (VII) 

quercetagetin; (VIII) AG1717; (IX) β-conidendrol; (X) suramin; (XI) curcumin. 


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on the same ring or can potentially be positioned close together in three-dimensional space if rings stack 

on top of each other. More recently it was shown that cosalane (II), a steroid-substituted derivative of 

(I), was no better in inhibiting integrase in vitro than the parent compound, but showed promise in HIV 

cytopathicity cell-culture assays [45]. Although cosalane and a number of related analogues inhibit both 

HIV protease and integrase in vitro, the primary site of action is believed to be inhibition of gp120 

binding to CD4 receptors. 

In 1993, the effects of selected topoisomerase II inhibitors, antimalarial agents, DNA binders, 

naphthoquinones, and various other agents on integrase activity in vitro were investigated [46]. 

Although certain effective topoisomerase inhibitors are also good HIV integrase inhibitors, this is not a 

generalizable correlation. Since many topoisomerase inhibitors also bind DNA, it is difficult to assess 

whether the observed in vitro effects result from specific interaction with integrase or from the 

sequestering or distorting of the DNA substrate. However, several compounds were identified that are 

not known to be DNA binders but that inhibited integrase with reasonable IC 50 values. These included 

dihydroxynaphthoquinone (III), primaquine (IV), caffeic acid phenethyl ester (CAPE, V), and quercetin 

(VI). 

Motivated by the structural similarities between compounds III–VI, a more intensive structure-function 

study of flavones was undertaken in which approximately 50 related compounds were screened for 

inhibition of in vitro integrase activity [47]. Flavones are planar compounds containing three aromatic 

rings substituted with various polar groups such as hydroxy substituents. General trends were observed 

relating structure to inhibitory effectiveness; for example, inhibition required at least three hydroxy 

groups, the most favorable arrangement being when they were located ortho to one another. The most 

effective compound, quercetagetin (VII), is a potent topoisomerase II inhibitor and a known DNA 

intercalator. It was noted by the authors that many flavones are not integrase-specific; rather, they inhibit 

a broad range of enzymes including DNA polymerases, ATPases, and NAPDH-monooxygenases. They 

are also, in general, capable of DNA intercalation. It has not been established that their inhibitory effects 

are due to direct interactions with integrase. 

A subsequent detailed structure-activity relationship study of CAPE (V) revealed that while the ortho 

hydroxys were important for in vitro integrase inhibition, both the caffeic acid and phenethyl moieties 

could be substantially modified [48]. Ortho hydroxyl groups in the context of other classes of 

compounds also confer anti-integrase potency. For example, several semisynthetic compounds derived 

from arctigenin, a topoisomerase I inhibitor that itself is not active against integrase, have been shown to 

inhibit HIV integrase [49]. More compelling evidence of the importance of ortho hydroxyl groups was 

provided by the tyrphostins, a group of synthetic compounds that are tyrosine kinase inhibitors. Several 

of these also inhibit integrase in the submicromolar range 


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(for example, compound VIII, aka AG1717). In cell-based screening assays, AG1717 demonstrated 

some antiviral activity [50]. 

Page 107 

Another study that demonstrated a role for ortho hydroxyl groups in in vitro integrase inhibition 

identified β-conidendrol (IX) via random screening as an inhibitor with an IC 50 value of less than 1 μM 

[51]. Although β-conidendrol did not inhibit several other nucleic-acid processing enzymes, indicating 

some specificity for integrase, it was not active in cell-based antiviral assays at concentrations as high as 

100 μM. 

Other Classes of Integrase Inhibitors 

Several compounds and their derivatives that do not contain adjacent hydroxy groups on a phenyl ring 

have recently been identified as HIV integrase inhibitors. These include suramin (X), curcumin (XI), 

phenanthroline-Cu + complexes (XII), and 3'-azido-3'-deoxythymidylate (AZT) monophosphate (XIII). 

Suramin (X) is a known inhibitor of DNA and RNA polymerases, retroviral reverse transcriptases, and 

topoisomerase II. It has also been shown to prevent the infection of T lymphocytes by HIV in vitro [52]. 

Its six sulfonic acid groups confer a strong negative charge, and it was reasoned that there might be an 

inhibitory electrostatic interaction with the positive residues of the HIV C-terminus domain. Suramin 

was shown to be an effective inhibitor of 3' processing and strand transfer, with IC 50 values of 0.25 μM 

and 0.11 μM, respectively [53]. It was not demonstrated, however, that the mechanism of inhibition does 

involve binding to the C-terminus of integrase, although this could be readily addressed using Cterminal 

truncated mutants active for disintegration. 

Curcumin (XI), the coloring dye in the spice turmeric, is structurally related to CAPE (V). It has also 

been shown to inhibit HIV replication by inhibiting p 24 antigen production and tat-mediated transcription 

[54]. As shown in Table 1, it also has moderate integrase inhibitory properties [55]. Although its two- 

OH groups are neither adjacent to each other nor on the same phenyl ring, its conformations can be 

modeled to bring the hydroxy groups into close proximity by stacking the two phenyl rings on each 

other. 

Several tetrahedral cuprous phenanthroline complexes, known inhibitors of transcription, were tested 

against integrase and shown to be reasonably effective inhibitors [56]: IC 50 values in the range of 1–10 

μM were determined (for example, the neocuproine-Cu + complex, XII). Analyses of the mode of 

inhibition demonstrated that these compounds act noncompetitively, and that inhibition does not 

correlate with inhibition of DNA binding. Thus, it has been proposed that these metal chelates may act 

at a site distant from the active site, or perhaps in the context of an enzyme-DNA complex. 

3'-Azido-3'-deoxythymidine, or AZT, is a nucleoside analog approved for use to treat AIDS. Its 

metabolites, the mono-, di- and triphosphate forms, accu- 


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mulate during treatment; in particular, AZT-monoP i (XIII) accumulates in cells to millimolar levels. 

These metabolites were investigated as possible integrase inhibitors and it was shown that all three 

phosphate derivatives inhibit with IC 50 values of 100–150 μM, although AZT itself is not inhibitory [57]. 

These results suggest that despite the weak inhibition by these particular compounds, nucleoside analogs 

may serve as lead compounds for the development of integrase inhibitors. 

It was recently observed that oligonucleotides that form guanosine quartet structures inhibit HIV 

replication [58]. In light of the AZT results that suggested that there may be a nucleotide binding site on 

integrase—and because integrase binds DNA—these oligonucleotides (for example, 5'- 

GTGGTGGGTGGGTGGGT-3', XIV) were investigated as possible inhibitors [59]. These compounds 

have the lowest inhibition constants reported to date (see Table 1), and suggest an exciting new avenue 

for integrase inhibitor development. 

In a completely different approach to integrase inhibitors, a synthetic peptide combinatorial library was 

used to select a hexapeptide capable of inhibiting integrase proteins [43]. The first two amino acids were 

selected using a library of 400 dipeptides, and the remaining amino acids selected one-by-one in an 

iterative process. The optimal hexapeptide, HCKFWW (XV), inhibits HIV-1 integrase with an IC 50 of 2 

μM. The peptide does not compete for DNA binding to viral DNA, nor does it represent a sequence 

present in integrase itself. Although it is not expected that a peptide consisting of L-amino acids would 

be a suitable drug in itself, the use of D-amino acids or peptidomimetic backbones may be fruitful 

directions to pursue. 

Summary 

Many of the compounds identified to date that inhibit HIV integrase in vitro have common structural 

features as illustrated in Figure 9. Most notably, these include hydroxy-substitued phenyl rings. 

However, these compounds may inhibit in vitro activity for reasons unrelated to enzyme binding. For 

example, it is difficult to know what to make of inhibition studies where the compounds added to the 

assays are known to bind DNA. Do the compounds affect in vitro activity because they bind and 

sequester the substrate? It is possible that they distort the DNA or intercalate between base pairs, 

preventing appropriate binding to the enzyme. While this is itself is a valid basis for the design of 

inhibitors against HIV infection, particularly if it targets DNA specific to the virus such as the LTR 

sequences [60], it is not an approach that builds on knowledge of the three-dimensional structure of the 

protein. To this end, valuable information could be obtained from studies in which direct binding of 

compounds to integrase is measured. This is also true for those inhibitors that do not contain the hydroxysubstituted 

phenyl pharmacophore. Binding studies could be carried 


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out, for example, using radiolabeled inhibitors or possibly by monitoring any UV-spectral shifts in the 

case of aromatic compounds. 

B. What We Need to Know before We can Start Structure-Based Drug Design 

The review of known integrase inhibitors presented in the previous section demonstrates the paucity of 

effective inhibitors reported in the literature. A limited number of structural types have been 

investigated, focused heavily on compounds with aromatic ring systems and hydroxy substituents. Most 

inhibitors reported to date are only moderately effective in in vitro assays, with IC 50 values residing in 

the low μM range (see Table 1). 

To build on this knowledge base, and to use known molecules as lead compounds for the development 

of more effective inhibitors, it would be extremely valuable to obtain a high-resolution crystal structure 

of an integrase-inhibitor complex. We and others are vigorously pursuing this goal. It is not clear if the 

lack of success to date is because the inhibitors identified so far manifest their effects in in vitro assays 

predominantly at the level of the DNA, or if some aspect of the structure of the HIV-1 integrase core 

itself—for example, its mobility in certain regoins—prevents the formation of a tight complex. It is also 

possible that binding is weaker to the HIV-1 core domain than to the full-length protein because of 

missing enzyme-inhibitor contacts. Co-crystallization attempts would benefit from in vitro studies to 

determine relative binding constants as a guide in selecting the most tightly bound inhibitors. It would 

also be useful to obtain information on the effect of variables, such as Mn 2+ or Mg 2+, on binding 

constants. 

It may be futile at this stage to attempt to model the binding of known inhibitors to the catalytic core 

domain of HIV-1 integrase in the absence of more complete information. It cannot a priori be assumed 

that the site of action of all these inhibitors is the enzyme active site identified by the constellation of 

conserved acidic residues. For example, certain very effective nonnucleoside inhibitors of HIV reverse 

transcriptase bind not to the enzyme active site, but rather to a small pocket adjacent to it. There are no 

obvious structural features of the integrase core—such as the deep trough surrounding active site 

residues in the case of HIV protease [61,62]—that can be readily identified as a potential inhibitor 

binding site. Furthermore, since part of the region defining the active site of HIV-1 integrase is 

disordered in our crystal structure, this prevents a surface rendering of the region around the conserved 

acidic residues. It also seems unlikely, given the known differences in active-site geometries between 

the HIV and ASV integrase (see Section IV.B), that the structure of the related ASV integrase core 

domain by itself would be particularly useful in this regard. As the active form of the enzyme 

presumably binds divalent metal ions, it will be important to determine how or if the structure of HIV 

integrase changes 


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when metals are bound. Finally, there is no evidence to rule out the possibility that the two termini 

contribute to part of the active site, occlude some of it, or restrict access to it in as yet undetermined 

ways. For this and other obvious reasons, three-dimensional structures of larger versions of HIV-1 

integrase, such as IN 1–212, IN 50–288, and the full-length protein, IN 1–288, will be required. 

Page 110 

We also lack a clear picture of how the enzyme substrates, the viral DNA ends and the target DNA, bind 

to integrase. The DNA must at some point approach the region defined by the three conserved acidic 

residues so that bond cleavage and joining can occur. However, the dominant DNA binding domain is 

defined by residues in the C-terminus. It would be extremely valuable to determine the relative 

orientation of these domains in the context of a larger version of integrase. Even more revealing would 

be the structure of the full-length protein with bound DNA. Once we possess this information, it should 

be possible to rapidly progress with structure-based drug design. 

C. Possible Approaches to the Design of Effective Integrase Inhibitors 

There are a variety of approaches to the design of integrase inhibitors that are obvious and do not depend 

on knowing its three-dimensional structure. However, the rational implementation and refinement of 

these approaches will require high-resolutional structural data, much of which, as indicated above, is not 

yet available. It still may be useful to discuss here different classes of inhibitors that can be envisioned. 

Preventing DNA Binding 

One approach to the inhibition of integrase would be to prevent binding of the DNA substrate. 

Unfortunately, we do not yet know how or where DNA binds. There are likely to be several sites on the 

enzyme that contact DNA, including the C-terminus and the region around the active site. In the absence 

of the structure of an integrase-DNA complex, structures of related enzymes (RNase H, the MuA 

transposase, and RuvC) with their DNA substrates would be useful guides in suggesting ways in which 

DNA could interact with integrase. However, there is no guarantee that modes of DNA binding are 

conserved among members of this polynucleotidyl transferase family. The overall structure of the Cterminus 

fragment suggests that it should be possible to develop compounds that bind specifically in the 

cleft formed by dimers of IN 220–270 (see Section IV.E) which may prevent DNA binding. 

Inhibition at the Active Site 

It may be possible to inhibit integrase by preventing the binding of the required metal cofactor(s) to the 

active site acidic residues that are presumed to provide 


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coordinating ligands to the metal(s). This could be accomplished by sterically blocking access to the 

active site or by specifically binding the acidic residues themselves. Such a mechanism has been 

suggested to explain the inhibition of integrase by curcumin [55], which could bind to the active site 

aspartates or glutamates via its hydroxy groups. Compounds could also be devised that chelate the 

metal(s) once bound, preventing access of the active site to the substrate or distorting the active site 

geometry preventing phosphate bond cleavage. An intriguing approach to disrupting metal binding has 

recently been reported for the Zn 2+-binding HIV nucleocapsid (NC) protein [63]. In this case, 

compounds were developed that specifically eject Zn 2+ from the zinc-finger region of NC, interfering 

with viral replication. Such an approach might be applied to Mn 2+ or Mg 2+ binding at the active site, 

although the Zn 2+-binding domain at the N terminus of integrase also suggests itself as a target. 

It is curious that approaches have not been devised for mechanism-based inhibitors, particularly since 

the stereochemical mechanism of integration has been understood for some time now [8]. 

Interfering with Multimerization 

The structures of the domains of HIV-1 integrase determined to date both reveal dimers [3,28,29,36]. It 

may be possible to develop compounds that bind specifically to the dimer interfaces, preventing 

interactions between monomers that may be necessary for activity. The success of this approach 

presumes that during the retroviral lifecycle there is a point where the monomer surfaces are accessible. 

It is not clear that integrase in the context of preintegration complexes is in equilibrium between the 

monomeric and higher order forms. This approach need not be restricted to preventing dimer formation 

if higher order interactions (e.g. formation of a tetramer) are also mechanistically relevant. To this end, 

the structure of an integrase tetramer, such as that formed by the full-length protein, would be useful in 

identifying dimer-dimer interface(s). 

Other Ways to Confound Integrase 

There are other parts of the retroviral life cycle involving integrase that could be targeted for inhibition. 

For example, integrase can bind other proteins such as human Ini1 [64], and most likely interacts with 

the viral proteins that are part of the preintegration complex [65]. Although it is not known if these 

interactions are essential for viral replication, preventing protein-protein binding would provide another 

site at which to attack integrase. It is possible that interactions between integrase and other proteins in 

the preintegration complex are crucial for maintaining the integrity of this complex and its ability to 

migrate to the cell nucleus. Interfering with these protein-protein or protein-nucleic acid interactions 

may be another approach to halting viral replication. For example, prein- 


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cubation of phosphotyrosine with integrase has been shown to inhibit interaction with the MA protein 

[65]. Therefore, phosphotyrosine analogs could be a unique approach to antiviral development. 

D. Concluding Statement 

Page 112 

It is known from drug design studies with HIV reverse transcriptase and protease that the virus is able to 

escape from the pressures of inhibitors by mutation of the drug targets [66]. Although integrase is, a 

priori, a reasonable target for drug-design efforts, it must be anticipated that integrase will also be able 

to rapidly mutate and thereby avoid inhibition. 

By analogy with recent approaches to reverse transcriptase inhibitors, it may be possible to design a set 

of integrase inhibitors that act at slightly different binding sites and from which the virus cannot 

simultaneously escape. That is, the combination of mutations required to avoid inhibition may be severe 

enough to prevent integrase from carrying out its required chemistry. (In the absence of direct structural 

information on the sites of inhibitor binding to integrase, the generation of escape mutants in vitro and 

their subsequent sequencing may be an indirect way to identify inhibitor binding sites.) It will also be 

important to determine if the virus will be able to simultaneously mutate reverse transcriptase, integrase, 

and the protease in response to a combination of inhibitors targeted against all three pol gene products. 

The answers to these questions will require the development of suitable inhibitors and the beginning of 

in vitro testing. To this end—while large-scale screening and the development of combinatorial 

chemistry methods should continue—the structure of the catalytic core domain of HIV-1 integrase is a 

starting point for the rational design of integrase inhibitors. There is much more structural information 

that must be obtained for the full-length protein and its complexes with metals, inhibitors, and 

substrates. We and others are aggressively pursuing results on these fronts. 

E. Recent Developments 

Several studies published since March 1996 have expanded the list of in vitro integrase inhibitors 

effective at IC 50 values below 100 μM. These include two dicaffeoylquinic acids obtained from 

medicinal plants and a synthetic analog, L-chicoric acid [68], the HIV protease inhibitors NSC 117027 

and NSC 158393 [69], certain anthraquinone derivatives [70], coumermycin, and pyridoxal phosphate 

[71]. In addition to exhibiting in vitro inhibition, the dicaffeoylquinic acids effectively inhibited HIV-1 

replication in T-lymphoblastoid cell lines [68]. 


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Follow-up studies were also reported for two previously identified classes of integrase inhibitors. 

Several nucleotides that were more effective inhibitors than the originally tested AZT nucleotides were 

identified [71]. For example, the L-enantiomers of 5-fluoro-2',3'-dideoxycytidine monophosphate and 

triphosphate inhibit 3' processing and strand transfer with IC 50 values of ~40 μM. A structure-function 

study on GT-containing oligonucleotides showed that both the number of quartets formed and the loop 

sequences between the quartets are important for activity, and that inhibitors of this type may function 

by interacting with the N-terminus of integrase [72]. 

A particularly important contribution was the demonstration that preintegration complexes isolated from 

HIV-infected lymphoid cells can be used in assays to screen for inhibition of integration [70]. 

Intriguingly, many compounds previously identified as in vitro inhibitors of 3' processing and strand 

transfer had no effect on integration carried out by either crude or partially purified preintegration 

complexes. Thus, such an assay may be a valuable method of screening out “false positives” identified 

using in vitro oligonucleotide assays, or corroborating the evidence that a particular compound is indeed 

active against integrase. 


Our work described here was carried out in the laboratories of R. Craigie and D. R. Davies. We express 

our gratitude to our co-workers who, over the years, participated in the effort to determine the structure 

of HIV integrase. In particular, we acknowledge the contributions of F. D.Bushman, M. Carmichael, A. 

Engelman, S. Hosseini, T. Jenkins, K. Mizuuchi, I. Palmer, P. Rice, P. Sun, and P. Wingfield. We would 

also like to thank D. R. Davies and T. Jenkins for their comments on the manuscript and A. Mazumder 

for alerting us to the most recent work on integrase inhibitors and for his contributions to Section V.A. 

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72. Mazumder A, Neamati N, Ojwang JO, Sunder S, Rando RF, Pommier Y. Inhibition of the human 

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4 

Bradykinin Receptor Antagonists 

Donald J. Kyle 

Scios Nova Inc., Sunnyvale, California 


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The term “kinins” is generally made in reference to either the nonapeptide bradykinin (Arg1-Pro2-Pro3- Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) or the decapeptide kallidin (Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8- Phe9-Arg10). In rats another kinin, Ile1-Ser2-Arg3-Pro4-Pro5-Gly6-Phe7-Ser8-Pro9-Phe10-Arg11 (T-kinin) is 

produced under certain circumstances and binds to the same receptors as bradykinin [1,2]. A schematic 

of the human kinin-kallikrein system is shown in Figure 1. 

The release of kinins from precursor proteins (known as kininogens) is mediated by enzymes called 

kininogenases [3–5]. The predominant enzymes responsible are kallikreins, but others, which include 

trypsin, plasmin, and some snake venoms, also release kinins. Kininogens are primarily synthesized in 

the liver and represent an abundant source of the precursors that are required for kinin generation. These 

proteins are produced from alternative splicing of a single gene product and there are two forms: high 

molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) [6]. Unlike 

HMWK, which exists in the circulation as a complex with plasma pre-kallikrein, LMWK circulates 

freely. 

During immunological reaction, charged surfaces—which may be derived from bacterial 

lipopolysaccharide, oligosaccharides, connective tissue proteoglycans, or damaged basement 

membranes—facilitate the conversion of factor XII to factor XIIa. Once factor XIIa is present, prekallikrein 

can be cleaved to its active form, known as plasma kallikrein. This enzyme acts upon its 

preferred substrate, HMWK, to release bradykinin. Plasma kallikrein is further able to convert inactive 

factor XII to active XIIa, thereby participating in a positive 


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Figure 1 

Diagram of the human kinin-kallikrein system including the native ligands for B1 and B2 

receptor subtypes. 

feedback loop. The cleavage of bradykinin from HMWK is highly localized since pre-kallikrein and 

substrate (HMWK) circulate as a complex. 

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Another kinin, Lys-bradykinin (also known as kallidin), is produced via the action of the tissuekallikrein 

enzyme on LMWK. This enzyme is found in many tissues, either in the form of a precursor 

requiring activation or as an active enzyme. In contrast to plasma kallikrein, which preferentially acts 

upon HMWK, tissue kallikrein can release kallidin from either HMWK or LMWK. Through the action 

of aminopeptidases, kallidin can subsequently be converted directly into bradykinin. This enzyme is 

present in both the plasma and on the surface of epithelial cells. 

Both bradykinin and kallidin can be degraded by a variety of plasma and cell surface enzymes 

(kininases) [7]. The most widely recognized of these enzymes are kininase I, kininase II (angiotensin 

converting enzyme, ACE), and carboxypeptidase N. In plasma, kininase I cleaves the C-terminal 

arginine from both bradykinin and kallidin to form [des-Arg 9] kinins. These [des-Arg 9] kinins are known 

to act as agonists of B1 receptors that are present in some species and have been implicated in the 

pathophysiology associated with prolonged inflammation [8–10]. 


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Nearly all cells express kinin receptors that mediate the activities of both bradykinin and kallidin. The 

activation of these G-protein coupled receptors causes relaxation of venular smooth muscle and 

hypotension, increased vascular permeability, contraction of smooth muscle of the gut and airway 

leading to increased airway resistance, stimulation of sensory neurons, alteration of ion secretion of 

epithelial cells, production of nitric oxide, release of cytokines from leukocytes, and the production of 

eicosanoids from various cell types [11,12]. Because of this broad spectrum of activity, kinins have been 

implicated as an important mediator in many pathophysiologies including pain, sepsis, asthma, 

rheumatoid arthritis, pancreatitis, and a wide variety of other inflammatory diseases. Moreover, a recent 

report demonstrated that bradykinin B2 receptors on the surface of human fibroblasts were upregulated 

three-fold beyond normal in patients with Alzheimer's disease, implicating bradykinin as a participant in 

the peripheral inflammatory processes associated with that disease [13]. 

In contrast to the adverse physiologies associated with bradykinin release, there is a growing body of 

literature that implicates bradykinin as a protective agent during periods of cardiac or renal stress 

[14–16]. In this regard there is substantial evidence that the cardioprotective effects afforded by ACEinhibitor 

treatment are a result of metabolically preserving bradykinin and are therefore mediated by 

bradykinin B2 (and possibly B1) receptors [17–18]. These results point to a possible therapeutic role for 

a kinin receptor agonist. 

Overall, the kinins are an important part of a well-organized physiological system. The various aspects 

and interdependencies of the kinin system have been, and continue to be, the focus of intensive research 

efforts in many laboratories. Many pharmaceutical companies have identified this system as an ideal site 

for therapeutic intervention in many inflammatory diseases. Hence, there have been many diverse 

approaches taken toward the discovery of antagonists (peptide and nonpeptide) of B2 and B1 receptors. 

This review focuses on the structure-based design strategies pursued in our laboratories during the past 

several years. 

II. Ligand-Based Investigations 

A. The Solution Conformation of Bradykinin 

In the late 1980s when we began the pursuit of bradykinin receptor antagonists, information of relevance 

to medicinal chemists was scarce. For example, not one nonpeptide antagonist of this receptor was 

known, nor were any series upon which to base a structure-activity relationship. Moreover, all 

publications described bradykinin as being highly flexible in an aqueous environment, such that no 

structural mimetics could be rationalized. Of course the receptors had not been cloned at that time so 

nothing was known about the primary sequence of the receptor or the three-dimensional structure. 


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Initially our approach was to complete a detailed examination of bradykinin using two-dimensional 

NMR methods in combination with empirical energy calculations [19]. Our strategy was derived on the 

basis of spectral data, biological results from conformationally restricted analogs, as well as the 

relationship between ordering in bradykinin and the dielectric environment of the solvent. Our guiding 

hypothesis was that, although in aqueous solution bradykinin is conformationally random, the 

biologically active form of the peptide is likely ordered and stabilized within the lipid bilayer of the cell 

membrane prior to binding with its receptor. Alternatively, the receptor binding environment might also 

be hydrophobic and thereby lead to similar conformational biases in the ligand. We presumed that an 

appropriate solvent environment should be able to stimulate, at least in terms of hydrophobicity and 

dielectric constant, the nature of a cell membrane, and a 90:10 d 8-dioxane-H 2O mixture was selected for 

NMR experiments. It was anticipated that under these nonsolvating conditions the conformational 

diversity of bradykinin might be severely restricted. The ultimate analysis of the two-dimensional NMR 

data collected at 500 MHz supported a single major conformational species. There were five HN-CαH 

connectivities, one for each amide. This was confirmed in the 13C NMR spectrum where only nine 

carbonyl resonances, one for each amino acid, were present. 

In order to provide a starting point for subsequent molecular dynamics simulations the assumption was 

made, based on multiple observed long-range amide-to-amide nuclear overhauser effects (NOEs), that it 

was indeed a single major conformational species. Although bradykinin contains three proline residues, 

the absence of any strong CαH i-CαH j+, cross peaks in the nuclear overhauser enhancement 

spectroscopy (NOESY) spectrum was taken as proof that all peptide bonds were trans. In total, 35 

interproton distances were extracted from the NOESY spectrum and, whenever possible, stereospecific 

assignments for pro-R and pro-S hydrogens were made explicitly. A temperature-dependent study of the 

chemical shifts of the amide protons resulted in a near-linear dependence suggesting no major 

conformational changes were coinciding with the temperature change and thereby allowing a 

comparison of slopes (Δδ/Δt). The lowest values obtained for these slopes corresponded to Phe 8 and 

Arg 9 suggesting solvent sequestering for these amides. 

Given the high Chou and Fasman probability of β-turns in the sequences Pro 2-Pro 3-Gly 4-Phe 5 and Ser 6- 

Pro 7-Phe 8-Arg 9 (3.79 × 10 -4 and 1.99 × 10 -4, respectively), the computational strategy employed was to 

begin from two initial structures: (a) an extended β strand, and (b) a structure containing these two 

predicted β turns. Utilizing custom routines written using the program CHARMm, version 21 [21], the 

interproton distances were incorporated into the potential-energy expression in the form of an additional 

potential-energy term. During the 3-ps heating step of the molecular dynamics, the temperature was 

raised from 0K to 300K in steps of 20K every 0.2 ps. Since the target distances 


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were poorly satisfied in the starting structures, the potential-energy term corresponding to the imposed 

NOE data (E NOE) was applied gradually by increasing the scale factor in a nonlinear fashion such that it 

was 0.0 after 0.2 ps, 0.1 after 1.2 ps, and 1.0 after the full 3.0 ps. Following 15 ps of equilibration, 7 ps 

of incremental production dynamics was completed. During this stage the NOE scale factor was raised 

from 1.0 to 4.5. By slowly raising the force constants for the NOE restraints as the target distances 

became better satisfied, no dramatic increase in temperature was observed. Finally the NOE scale factor 

was set to 5.0, 10 ps of production dynamics were completed, and an average structure was extracted 

from the last 5 ps of the coordinate trajectory. 

Analysis of the two average structures obtained from the two unique starting points demonstrated 

convergence to a similar conformational species. In each, the sum of the NOE restraint energy was less 

than 4.7 kcal/mol and the RMS deviation from the target distances was below 0.25 Å. Similar results 

were obtained for each simulation when they were repeated without the electrostatic term being included 

in the total potential-energy function. This important data lends credence to the hypothesis that the final 

structures are derived from the NOE restraints and not by poorly represented electrostatic interactions. 

The average dynamic structures are characterized as having all trans peptide bonds and hydrophobic side 

chain groups oriented outward into solution, perhaps ready to interact with the receptor. There is a 

possible 1–3 hydrogen-bonded γ turn bridging Phe 5, although it is not explicitly defined by the NOE 

data set. If present, then the preferred overall geometry would be U shaped and, if absent, an S shaped 

geometry is possible based on coincident conformational analyses. According to the dihedral angle 

values for Pro 7 and Phe 8, a type-II β turn extending from Ser 6 to Arg 9 also exists. A variety of reports 

have subsequently appeared that are in agreement with the conformation we described in this work. 

A similar C-terminal turn structure was observed in an analogous NMR study of a first-generation kinin 

antagonist, NPC 567 (DArg 0-Arg 1-Pro 2-Hyp 3-Gly 4-Phe 5-Ser 6-DPhe 7-Phe 8-Arg 9), although the type of 

turn was not the same. Our initial speculation was that this slight structural difference might partially 

account for the functional differences of bradykinin and NPC 567. These solution conformations, one of 

an agonist and the other of an antagonist, were subsequently used to focus the design and synthesis of 

conformationally constrained peptide analogues of NPC 567. 

B. Conformationally Constrained Bradykinin Antagonist Peptides 

The ligand-based approach of conformationally constrained peptides has been widely used. The process 

involves the incorporation of conformational constraints into known peptides, either agonist or 

antagonist, which enforce a 


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predictable geometry. A series of peptides containing these types of constraints can be useful for 

extrapolating the steric and electronic environment of a given binding site. This structural information 

can be derived regardless of whether or not the constrained peptide binds to the target receptor. Since 

peptides can be prepared rapidly, it is typical to establish a structure-activity relationship using them and 

then at some later time transpose that information onto a nonpeptide lead molecule in an attempt to 

improve its potency. 

As part of an expansion upon the hypothesis that a C-terminal β turn was a structural prerequisite to highaffinity 

antagonist binding, a novel series of constrained decapeptides was prepared [22–24]. These 

peptides are of the sequence DArg 0-Arg 1-Pro 2-Hyp 3-Gly 4-Phe 5-Ser 6-DHype 7-Y 8-Arg 9, where Y is either 

tetrahydroisoquinoline-3-carboxylic acid (Tic), or octahydroindole-2-carboxylic acid (Oic). DHype 

denotes an organic ether of D-4-hydroxyproline in either the cis or trans geometric form. The C-terminal 

portion of a representative member of this class of peptides was shown—first by empirical calculation 

[22], then by NMR at 600 MHz—to adopt a β turn nearly unambiguously (figure 2) [25,26]. Moreover, 

it was shown by calculation that the turn was adopted regardless of the nature of the ether group (alkyl, 

aryl, etc.) or its geometry (cis or trans). Hence, a diverse series of these peptides was initially used as a 

tool to probe the steric and electrostatic topology of an antagonist 

Figure 2 

Lowest 5 kcal mol -1 of the calculated overall potential energy surface for a model 

peptide of Ser-DHype(trans propyl)-Oic-Arg. The contour interval is 0.5 Kcalmol -1 

and the highest (outermost) and lowest contour energy values are labeled. 

Superimposed on the contour plots are values for ψ i+1 and ψ i+2 from each of the 

thirty structures generated from the NMR data corresponding to the tetrapeptide 

Ser-DHype(trans propyl)-Oic-Arg. 


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binding site on the bradykinin B2 receptor in the guinea pig ileum. The cis ethers, in all cases, bound to 

the receptor with significantly lower affinity than did the trans. A more complete listing of the peptides 

used in the study is shown in Table 1. These results support the hypothesis that the domain of the 

receptor that binds these antagonist ligands is partly made up of a hydrophobic cavity about one side of 

the C-terminal turn. However, adjacent to the other side of the 


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Figure 3 

Receptor binding curves for the binding of NPC 17410 and NPC 17643 to B2 receptors 

from the guinea pig ileum and cloned rat and human B2 receptors. Legends are noted on 

the figure. 

turn, there appears to be some type of steric interference (or lack of a pocket) that might otherwise 

accommodate the ethers of the cis configuration. 

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More recently, bradykinin B2 receptors have been cloned from both rat and human sources [27,28]. In 

receptor-binding experiments using these new receptors, selected members of the DHype-containing 

decapeptides were used to probe these receptors [24], a representative sample of the data is shown in 

Figure 3. Specifically, NPC 17643 (a trans propyl ether of D-4-hydroxyproline at position 7) and NPC 

17410 (a cis propyl ether of D-4-hydroxyproline at position 7) were used. Although the trans ethercontaining 

decapeptide behaved similarly in binding assays directed toward the bradykinin B2 receptors 

in guinea pig, rat, and human, the cis ether-containing decapeptide, NPC 17410, displayed an interesting 

pharmacology. In particular, NPC 17410 bound with similar affinity to both the guinea pig and rat 

bradykinin B2 receptors, but had an appreciably higher affinity for the human B2 receptor. This result 

strongly suggests that there are slight structural differences in the antagonist binding sites of the rat and 

human B2 receptors. With clones available for the rat and human bradykinin B2 receptors, this 

prompted a systematic search using NPC 17410 binding to rat/human bradykinin B2 receptor chimeras 

and point mutations in an attempt to discover residues on the receptor that comprise this antagonist site. 

The details and results of the subsequent application of these novel receptor-probing ligands is fully 

described later in this chapter. 


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The des-Arg 9 forms of these peptides have also been shown to have high affinity for the recently cloned 

human B1 receptor [24]. An extension of the work described herein would be to use a more complete 

series of des-Arg 9 DHype-containing nonapeptides to probe the binding site of this new receptor where 

other interesting pharmacological differences are likely to exist since the B1 receptor is only 33% 

homologous to the human B2 [29]. 

In summary, the DHype-containing decapeptides have been useful in many regards. First, they 

incorporate a novel β-turn mimetic that was alternatively functionalized and used to probe the unknown 

topology of the guinea pig, rat, and human bradykinin B2 receptors. In this role, one of these tools 

showed differential pharmacology between rat and human forms of the receptor. This tool was used in a 

synergistic fashion with subsequent molecular biological and computational procedures in the 

elucidation of an antagonist binding site. Second, these peptides, together with another potent 

decapeptide antagonist with similar conformational constraints [30,31], provide the first strong 

experimental evidence that high-affinity decapeptide bradykinin receptor antagonists adopt a C-terminal 

β turn in the receptor-bound conformation. Third, certain members of this series of decapeptides contain 

alkyl ethers of D-4-hydroxyproline at position seven. In this regard, they are the very first examples of 

decapeptide bradykinin receptor antagonists that do not contain a D-aromatic amino acid at the seventh 

position as had been previously deemed to be essential. Commercially, this renders the series patentably 

distinct from all other known bradykinin receptor antagonists. Finally, several members of the series 

(i.e., NPC 17731, NPC 17761, NPC 17974) are among the most potent antagonists for this receptor yet 

reported. Hence, there may be applications for these compounds as human therapeutics. 

Several “second generation” decapeptide antagonists have been reported, but the prototype from the 

class, which was first to be reported, is HOE 140 (DArg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-DTic7-Oic8- Arg9) [30,31]. This decapeptide has also been shown to preferentially adopt a C-terminal β turn, 

consistent with the previous discussion [26,32,33]. The side chain of DTic at position seven is, however, 

flexible. While side-chain rotational movement is not allowed, the saturated six-membered ring easily 

undergoes an endo/exo ring-flipping motion. Hence, the β turn predominates about the backbone 

dihedral angles, but the side chain of DTic could be either endo or exo ring flipped in the receptor-bound 

conformation. In the absence of an appropriate x-ray crystallographic structure, there is no definitive 

means of establishing which possibility is correct. This type of ring-flipping conformational change 

serves to orient the bulky hydrophobic side chain of the DTic residue to either one side of the β turn, or 

the other. The data collected from the decapeptides containing either cis or trans ethers of D-4hydroxyproline 

at the analogous position in the 


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sequence (discussed above) support a hypothesis that the DTic is exo ring flipped in the receptor-bound 

state. 

There are two factors that must be considered when applying structure-activity-relationship (SAR) 

information from a series of peptides toward the design of nonpeptide mimetics and putative library 

scaffolds. One is in regard to the backbone conformation that primarily serves as a structural scaffold 

upon which the various functionalities (side chains) are attached. The other factor is the side chains 

themselves whose spatial positions are primarily dictated by the backbone structure. Usually, the threedimensional 

arrangement of these differing chemical groups are responsible for affinity and triggering of 

the receptor. Knowledge of the relative importance of the individual side chains and amide bonds for 

receptor affinity is therefore a critical aspect of small molecule design from a peptidic structure-activity 

relationship. 

Conformationally constrained derivatives of HOE 140 have been prepared in continuing efforts to 

elucidate the ideal backbone conformation peptide antagonists must adopt for bradykinin B2 receptor 

interaction. One such series made use of Cα- or N-methyl substituted amino acids, incorporated at 

position(s) Gly4, Phe5, or both, in the peptide D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Tic7-Oic8-Arg9 (NPC 18545) [34]. An N-methyl substitution in the backbone of an L-amino acid is known to disfavor 

helical, or twisted, backbone conformations while favoring an extended backbone. The contrasting Cα methyl modification tends to favor a helical (twisted), rather than extended, conformation [35,36]. These 

conformational preferences apply only to the backbone φ, ψ angles (where φi and ψi correspond to 

backbone dihedral angles for residue i, defined by the four adjacent amino acid backbone atoms C i-1-N i- 

Cαi-Ci band Ni-Cαi-Ci-Ni+1, respectively) of the amino-acid residues bearing the modification. Receptor 

binding assays were performed in membrane preparations of the guinea pig ileum, a source of B2 

receptors, wherein these constrained peptides were evaluated for their abilities to compete with 

bradykinin binding. 

With the exception of the C α-methyl-Phe 5-containing peptide (NPC 18540), each conformational 

constraint caused a significant, at least 1000-fold, loss in binding affinity with respect to the 

unconstrained parent peptide, NPC 18545. There are several factors that could contribute to the poor 

receptor affinities measured for these peptides. In addition to the possible induction of an adverse 

conformation via the N-methyl substitution, this modification also eliminates an amide proton that might 

be an important hydrogen-bond donor during ligand-receptor interaction. Furthermore, the N-methyl 

substitution enhances the likelihood of trans-cis amide bond isomerization, which could also disrupt an 

optimal ligand-receptor interaction by altering the spatial display of the local side chains. The C α-methyl- 

Phe 5 substitution of NPC 18540 is well 


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tolerated by the receptor as evidenced by only a seven-fold loss in receptor affinity (K i=0.54 nM) with 

respect to the parent of the series, NPC 18545. This implies that the φ, ψ backbone dihedral angles about 

Phe 5 are on the order of -60°, -60° in the biologically active conformation. This combination of dihedral 

angles represents a helical twist or “kink” in the midsection of the peptide. 

Since the original submission of the manuscript describing these constrained linear and cyclic peptides, 

bradykinin B2 receptors have been cloned from other species including human [27,28]. Toward the goal 

of designing a nonpeptide antagonist as a human therapeutic agent, it would be interesting to evaluate 

these analogues against these newly reported receptor homologues. This would likely be fruitful and 

valuable given that there is evidence, including that presented in this chapter, showing that guinea pig 

and rat B2 receptors differ structurally from the human B2 receptor at the antagonist binding site. Hence, 

a structure-activity relationship established against the guinea pig or rat receptors could be misleading in 

the context of potential human therapeutics. 

A systematic study of the relative importances of amides and side chains in a prototypical second 

generation antagonist, NPC 18545 (DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9) has 

recently been described [37,38]. The D-Arg0 and Ser6-DTic7-Oic8-Arg9 segments were left intact in all 

peptides on the assumption that N-terminal positive charge(s) and a hydrophobic C-terminal β turn are 

minimally required for binding. In a systematic fashion, the amino acids in the core of the peptide (Arg1- Pro2-Hyp3-Gly4-Phe5) were substituted with glycine, an amino acid bearing no chirality or side chain. 

Binding assays, either in membranes from the guinea pig ileum or in membranes from a stable cell line 

expressing the human B2 receptor, were performed on each peptide and the results compared with the 

parent, NPC 18545, which has a Ki against [ 3H]-bradykinin of 0.08 nM. The elimination of all chirality 

and sidechain moieties in the segment Arg1-Pro2-Hyp3-Gly4-Phe5 via replacement by Gly1-Gly2-Gly3- Gly4-Gly5 (NPC 18152), led to a peptide that no longer binds the receptor. This demonstrated that one or 

more of the side chains in this segment are critical during ligand-receptor interaction. Incorporation of 

either the Arg1 or Phe5 side chains led to improved potency (285 nM and 483 nM, respectively). 

Constructing a peptide with side chains of both Arg1 and Phe5 in place (NPC 18149) yielded a peptide 

with good affinity, Ki of 13.7 nM. Overall, this study shows that to maintain potency in the low 

picomolar range, peptides in this series require Arg1, Phe5, and either Pro2 or Hyp3 (but not both). The Nterminal 

charged moieties and the hydrophobic C-terminal β turn are also required. Potencies in the low 

nanomolar range are attainable without including the side chains or chirality associated with Pro2 and 

Pro3. These data have 


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subsequently been successfully applied toward the design and synthesis of several nopeptide scaffolds 

and mimetics. Ultimately these mimetics were assembled in a combinatorial fashion as discussed in 

Section IV. 

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In a related study, wherein NPC 18149 (DArg 0-Arg 1-Gly 2-Gly 3-Gly 4-Phe 5-Ser 6-DTic 7-Oic 8-Arg 9; K i = 

13.7 nM; Guinea pig ileum) was taken as the lead peptide, the relative contributions to binding affinity 

from each amide bond in the segment Arg 1-Gly 2-Gly 3-Gly 4-Phe 5 were examined. Aminovaleric acid was 

used in a systematic fashion as a surrogate for any pair of adjacent Gly-Gly residues in the peptide. 

Aminovaleric acid is atomically identical to Gly-Gly with the exception that the amide bond linking the 

two glycines is replaced by two methylenes. The synthesis of Gly 4-Phe 5 required a special Gly-Phe 

mimic that has since been reported [39]. Since this substitution introduces flexibility into the peptide, it 

is a means of probing the structural role a given amide bond plays during receptor interaction. Potential 

hydrogen-bond donor and acceptor groups in the amide bond are removed via this substitution, which 

yields additional insights into potential electrostatic interactions that may also be important during 

ligand-receptor interactions. 

The conclusions drawn from the data are that in terms of structural or electrostatic interactions with this 

antagonist site on the receptor, the amide bond linking residues two and three may not be as critical as 

those linking residues three to four and four to five. 

Each of these investigations was aimed toward an understanding of either the backbone conformation of 

this prototypical decapeptide or the relative importance of the functional groups in the side chains that 

make significant contributions to receptor affinity. From the former, nonpeptide frameworks and 

scaffolds can be imagined. From the latter, insights into which functionality is required for high-affinity 

binding is derived. The remaining challenge is to reassemble these fragments onto synthetically feasible 

nonpeptide frameworks as potential new lead compounds. Our approach toward addressing this 

challenging problem is described later in this chapter. 

III. Receptor Structure-Based Investigations 

A. Elucidation of an Agonist Binding Site on the B2 Receptor 

In addition to the deductions one might make about a receptor binding site on the basis of receptor 

binding data from conformationally constrained ligands as previously described, models of bradykinin 

and bradykinin antagonists bound to their respective sites on the receptor as complimentary aspects of 

the overall strategy are also valuable. Unfortunately, due to the nature of the bradykinin 


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receptor, it has not yet been obtained in crystalline form, nor is it likely to be in the near future. 

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The bradykinin receptor is a member of a family of receptors for which an intracellular interaction with 

a G-protein is a critical part of the signal transduction pathway following agonist binding. Structurally, 

these G-protein-coupled receptors extend from beyond the extracellular boundary of the cell membrane 

into the cytoplasm. The tertiary structure is such that the protein crosses the bilayer of the cell membrane 

seven times, thus forming three intracellular loops, three extracellular loops, and giving rise to 

cytoplasmic C-terminal and extra-cellular N-terminal strands. It is generally presumed that the 

transmembrane domains of these receptors exist as a bundle of helical strands. This assumption is 

derived primarily from the known structure of the trans-membrane portions of a structurally related 

protein, bacteriorhodopsin [40]. 

G-Protein-coupled receptors do not lend themselves to analysis by either NMR or x-ray crystallography 

due to their structural dependence on an intact cell membrane. In our laboratories we pursued this 

valuable structural information by utilizing a combination of structural homology modeling, molecular 

dynamics, systematic conformational searching methods, and mutagenesis experiments. The 

combination of these techniques led to a proposed model of bradykinin bound to the agonist site on its 

receptor [41]. 

A hydrophobicity (Kyte-Doolittle) calculation [42] on the amino acid sequence of the rat bradykinin 

receptor yielded seven segments, each of which were 21 to 25 contiguous residues with predominantly 

hydrophobic side chains. These were presumed to be the seven transmembrane portions of the receptor. 

Cartesian coordinates of the backbone atoms within each of these seven segments were built by 

structural homology from the cryomicroscopic structure of the analogous segments of 

bacteriorhodopsin. Subsequently, side chains were added to these seven segments as appropriate for the 

rat bradykinin receptor, and the resulting geometry was optimized via constrained energy minimization 

to alleviate bad contacts. Extracellular and intracellular loops were extracted from the Protein Data Bank 

library, following a geometric search based upon a vector defined by terminal alpha carbons in adjacent 

helices. The model was subsequently subjected to a series of constrained and unconstrained energy 

minimizations as well as molecular dynamics simulations. The resulting structure of the receptor was 

used in a novel two-step docking procedure. 

Following our hypothesis that bradykinin adopts a C-terminal β turn upon complexation with the 

receptor, the φ, ψ backbone dihedral angles in the tetrapeptide corresponding to the C-terminus of 

bradykinin (Ser-Pro-Phe-Agr) were constrained in a harmonic fashion (force constant = 15 Kcal Å -1 mol - 

1) to values that define a type II' β-turn [43]. This tetrapeptide probe was then systematically translated 

about the interior of a theoretical box inscribing the rat 


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receptor model. The translations were such that the tetrapeptide probe molecule was incrementally 

repositioned within the receptor by following a 3 Å × 3 Å × 3 Å grid pattern. At each new position, both 

the probe and receptor were reset to their initial conformations, then the geometry of the complex was 

optimized using 200 steps of steepest descent followed by 500 steps of Adopted-Basis Newton-Raphson 

energy minimization. Subsequently, the sum of the steric and electrostatic contributions to the overall 

potential energy (interaction energy)—as measured only between the tetrapeptide probe molecule and 

the atoms of the receptor—were calculated. Slices through the receptor illustrating the energy of 

interaction as grayscale contour lines (darker gray = lower interaction energy) for that portion of 

receptor that was sampled by the tetrapeptide probe molecule is shown as an edge-on, frontal view in 

Figure 4. In this Figure it is qualitatively clear where the transmembrane domains are located (white), as 

well as where the most favorable sites of probe interaction are located (black). 

Figure 4 

Complete group of contour plots showing energy of interaction between probe 

and receptor. Each contour plot corresponds to a different horizontal slice as part 

of the first stage in the conformational search. Darker gray indicates most favorable 

interaction and the light shades represent least favorable interactions. 


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This initial stage of the docking process was used to reduce the computational difficulties that would be 

inherent in “tumbling” a complete bradykinin molecule (which has great flexibility) about the receptor 

in a similar fashion. However, following this initial stage, insight into those regions of the receptor 

capable of accommodating the C-terminal portion of the bradykinin molecule was obtained. On the basis 

of energetics, and as qualitatively shown in Figure 4, those particular regions are clustered in the central 

part of the receptor near to the extracellular domain. Using this information as a steering device to limit 

the size of the problem, an exhaustive conformational search was performed using the entire nineresidue 

sequence of bradykinin as a probe molecule, again enforcing a C-terminal β turn via dihedral 

angle constraints. Specifically, 24 unique geometric orientations (eight on each of three axes) of the 

bradykinin molecule were sampled at each of 100 grid points identified during the initial stage as likely 

zones to bind the tetrapeptide probe molecule. Bradykinin-receptor complexes within the lowest 150 

kcal mol -1 interaction energy with respect to the lowest found (17 complexes out of 2400) were grouped 

into sets of related conformational families, of which there were five. Computationally, each of the five 

complexes were presumed to be equally likely. All of these simulations were accomplished using 

custom routines written using the program CHARMm [21]. 

To guide the selection of which of the five bradykinin-receptor complexes to consider a “lead” model, 

supporting experimental evidence was sought from site-directed mutagenesis experiments. This support 

was taken primarily from work describing bradykinin binding assays performed of mutant rat B2 

receptors [44,45]. The underlying strategy of the mutation studies was based on the hypothesis that, 

since bradykinin has positive charges at either end of its sequence (Arg 1 and Arg 9), separated by a group 

of rather hydrophobic amino acids (Pro 2-Pro 3-Gly 4-Phe 5-Ser 6-Pro 7-Phe 8), it was likely that some acidic 

residues in the receptor participated during ligand binding. Several mutant receptors were made such 

that each contained either a point mutation or a small cluster of point mutations, wherein native residues, 

having negatively charged side chains (Asp, Glu), were replaced by alanine(s). Table 2 lists the initial 

cluster mutations (rat) that were prepared as well as the follow-up single point mutations (rat). 

Figure 5 shows a stereoview of the selected ligand-receptor complex chosen on the basis of best 

agreement with the results of these mutagenesis studies. None of the other four putative complexes were 

in agreement with this experimental data and were not considered further. Of particular significance in 

this work was that the trans-membrane residue Glu 49, when mutated to alanine, showed no adverse 

effect on bradykinin receptor affinity with respect to rat wild type. A similar result was reported for the 

Glu 196 rarrow.gif Ala 196 mutation. These residues are remotely situated with respect to the proposed site 

of bradykinin 


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binding and are colored light gray in Figure 5. In contrast, the [Asp175, Glu178,179] rarrow.gif 

Ala175,178,179 cluster mutation showed a 12-fold loss in bradykinin binding affinity, and the [Glu282, Asp 286] rarrow.gif Ala 282,286 cluster mutation lost 17-fold with respect to the wild type receptor. The 

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Asp 268 rarrow.gif Ala 268 and Asp 286 rarrow.gif Ala 286 point mutations caused 19-and 28-fold 

respective losses in affinity for bradykinin. Close inspection of the bradykinin Arg 1 side chain location 

and surrounding receptor interactions led to the suspicion that Asp 286 and Asp 268 

Figure 5 

Proposed model of bradykinin bound to the rat B2 receptor at the agonist binding site. 

Only the upper portion of the receptor is shown as gray helical ribbons. Bradykinin 

backbone and side chain atoms are shown as thick white licorice. Positions of point 

mutations having no significant adverse effects on bradykinin binding are shown as 

light gray spheres. Positions of mutations affecting bradykinin binding are shown as 

dark gray spheres. 


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might be jointly interacting either with the guanidino group in the side chain of Arg1 or the N-terminal 

amino group in bradykinin. Therefore a receptor containing a double mutation (Asp268,286 rarrow.gif 

Ala268,286) would be expected to show a much more dramatic loss in affinity for bradykinin than would 

receptors containing the individual point mutations. The appropriate double mutation experiment 

confirmed this by causing a 500-fold loss in affinity for bradykinin, as predicted (Table 2). The 

mutagenized residues of this double mutant B2 receptor are colored dark gray in Figure 5. This type of 

an ionic interaction is also precedented by the body of literature that exists supporting the requirement of 

an N-terminal arginine residue and a free N-terminal amino group in both bradykinin peptide agonists 

and antagonists for high affinity binding. All of these mutant receptors were demonstrated to be 

functional receptors on the basis of bradykinin-induced membrane depolarization in a Xenopus oocyte 

expression system [44,45]. 

The selected agonist site model is characterized by an overall twisted S-shape ligand, similar to the 

conformation of bradykinin determined previously in a hydrophobic environment by NMR [19,46]. 

Overall, the model suggested that the N-terminal amino and guanidine groups of Arg 1 interact directly 

with negatively charged amino acids in extracellular loop three, and the C-terminal end is in a β-turn 

conformation buried just below the extracellular boundary of the trans-membrane domain of the 

receptor. Noteworthy is the presence of a hydrophobic cavity in our receptor model located adjacent to 

Pro 7 of the bradykinin ligand. This cavity is made up, in part, by the residues Phe 261, Leu 104, Val 108, and 

Ile 112. Given the historical significance of position seven in peptide bradykinin-like ligands, these 

residues represent interesting targets for further mutagenesis experiments. One such result, the mutation 

of Phe 261 to Ala 261, has already been described, and the results were supportive of this proposed model 

[47]. Antibodies to the extracellular loops two and three have also been shown to compete with 

bradykinin binding, lending further experimental support for an extracellular domain on this agonist 

binding site. 

More recently, chemical crosslinking combined with site-directed mutagenesis was used to analyze the 

bradykinin binding site in the human B2 bradykinin receptor [48]. Previous studies using the bovine B2 

receptor showed that heterobifiunctional reagents reactive to amines and free sulfhydryls crosslink the 

bound bradykinin N-terminus to a sulfhydryl(s) in the receptor [49]. To identify this sulfhydryl(s), two 

conserved candidate residues in the human B2 receptor—Cys 20 in the N-terminal domain and Cys 277 in 

extracellular loop 3—were mutated to serine residues. Single and double mutants were expressed in Cos 

7 cells. All mutants bound [ 3H]bradykinin with typical B2 receptor specificity. The heterobifunctional 

reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester crosslinked bradykinin to wild-type and 

mutants with maximum efficiencies of 35% (wild type), 40% (Ser 20), 20% (Ser 277), and 0% 


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(Ser 20, Ser 277). This clearly demonstrated that Cys 20 and Cys 277 are the only sulfhydryls available for 

crosslinking receptor-bound bradykinin. These results provided direct biochemical evidence that the Nterminus 

of bradykinin, when bound to the B2 receptor, is adjacent to extracellular loop 3 and the Nterminal 

domain in the receptor. 

Further consideration of the model led to the hypothesis that agonist peptides may minimally require an 

intact C-terminal β-turn structure with appropriate side chains in place and N-terminal amino and 

guanidine groups for primary electrostatic interaction(s) with Asp286 and Asp268 in extracellular loop 3. 

As a test of this hypothesis, the prototypical second generation antagonist, NPC 18545, (DArg0-Arg1- Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9) was modified such that residues 2–5 were replaced by a 

simple twelve carbon chain spacer (12-aminotridecanoic acid). The resulting compound, NPC 18325, 

contains only the appropriately charged moieties at the N-terminus, separated by a simple organic spacer 

moiety from a known β turn forming tetrapeptide [25,26]. This pseudopeptide was tested in the human 

bradykinin B2 receptor binding assay and found to have a Ki of 44 nM against [ 3H]bradykinin binding 

[50]. Functionally, this pseudopeptide was an agonist as measured by its ability to stimulate IP 

production in a stable CHO cell line expressing the human B2 receptor and in WI-38 cells. Since it was 

designed on the basis of an agonist site on the receptor, this result was not completely surprising despite 

the incorporation of the DTic-Oic pair at the C-terminus that previously had been shown to be critical in 

high affinity antagonists. 

Subsequently the length of the linear carbon chain was varied to further explore the hypothesis that the 

agonist site on the receptor had two domains, a hydrophilic site in the extracellular loop area and a 

hydrophobic domain in the transmembrane area [50]. Presumably, the two terminal portions of NPC 

18325 can only simultaneously interact with each putative domain of the receptor binding site when the 

carbon chain is 12–13 methylenes long. But if the carbon chain length is shortened too far, this ligand 

might be unable to simultaneously interact with both domains, resulting in an affinity loss. This series of 

pseudopeptides and their respective human bradykinin B2 receptor affinities are presented in Table 3. 

The data are consistent with the hypotyhesis since the receptor affinity decreases as the carbon chain 

length is shortened. An alternative explanation of the data is that a certain hydrophobicity profile is 

required of the compounds in this series for good receptor affinity. These results indicated that there 

may be additional hydrophobic or flexibility prerequisites to binding in this series of pseudopeptides. 

One noteworthy observation was that NPC 18325 showed divergent behavior when evaluated against 

different species homologues of the bradykinin B2 receptor. Notably, in the guinea pig ileal membrane 

preparation assay, the affinity for the receptor was approximately 10-fold less than what had been 


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observed for the human B2 [37]. Furthermore, in contrast to the functional activity of NPC 18325 at the 

human B2 receptor, the compound is a functional antagonist as measured against bradykinin-induced 

contraction of the isolated guinea pig ileum (pA 2 = 5.5). These findings are in agreement with the 

concept that as a ligand is made smaller (i.e., fewer contact points possible with the receptor), the subtle 

structural differences in the binding sites on species variants of the same receptor become amplified. 

This observation further supports a cautionary posture toward developing nonpeptide antagonists for use 

in human diseases on the basis of results obtained in some animals including the guinea pig. Taking this 

new molecule as a lead structure, together with the receptor model and structure-activity relationship 

associated with related peptides including cyclic antagonists, the pursuit of several related 

pseudopeptides was undertaken. 

B. Elucidation of an Antagonist Site on the B2 Receptor 

There have been a variety of single alanine point mutations experimentally introduced into both rat and 

human bradykinin B2 receptors. Several of these have been shown to decrease the affinity of bradykinin 

to the receptor and have been implicated structurally near the agonist binding site. In contrast, at the 

time of this manuscript, there have been no mutations reported that adversely affect the ability of any 

peptide antagonists to bind to the receptor. Furthermore, antibodies raised against the certain 

extracellular domains of the kinin receptor compete with bradykinin for binding to the receptor but have 

no inhibitory 


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action on the binding of antagonist peptides. In addition, it has been shown that bradykinin can be 

covalently crosslinked to the B2 receptor while antagonists cannot. These observations have fostered the 

belief that the agonist and antagonist binding sites of the receptor are not the same. At best, they may be 

partially overlapping, although there is no direct evidence for this. The ultimate identification of the 

amino acid residues that make up the antagonist site would be another important step toward the goal of 

structure-based design of novel nonpeptide antagonists. 

As described in a previous section of this chapter, characterizations of the bradykinin B2 receptors from 

rat and human using NPC 17410 (Figure 3) revealed different pharmacologies. Specifically, it showed a 

higher affinity for the human B2 receptor than it did for the rat B2 (human IC 50 = 0.95 nM, rat IC 50 = 

48.0). This ligand “tool” provided a means for evaluating a series of bradykinin rat/human B2 receptor 

chimeras [51–53]. Several different chimeras were prepared in a systematic fashion and the affinity of 

NPC 17410 was determined for each. The chimeras are depicted schematically in Figure 6 together with 

the IC 50 values determined for NPC 17410. Chimeras I through III sample the N-and C-terminal sections 

of the receptor for any contributions to an antagonist binding site. The remaining chimeras sample the 

core transmembrane domains of the receptor. Each chimera was shown to induce a membrane 

depolarization similar to wild type receptor in response to bradykinin when expressed in Xenopus 

oocytes. For each NPC 17410 assay, [ 3H]-NPC 17731 was used as the radioligand. 

From this systematic approach, specific groups of contiguous residues within the receptor were 

identified as possible contributors to an antagonist binding site. The NPC 17410 binding to chimeras III, 

IV, and VIII showed rat-like pharmacology (low NPC 17410 affinity). The NPC 17410 binding to 

chimeras I, II, VI, and VII showed human-like NPC 17410 pharmacology (high receptor affinity). 

Binding to chimeras V and VIII, however, was similar to rat-like NPC 17410 pharmacology, but the 

affinity of the compound was slightly shifted back toward human-like results. Comparisons of rat and 

human receptor sequences in the regions sampled by the chimeras reveals that only two clusters of 

residues differ between rat and human B2 receptors. Specifically, TM2 has the same sequence in rat and 

human receptors so it is unlikely that the differential pharmacology associated with NPC 17410 binding 

can be attributed to residues there. However, TM3 has a cluster of 3 residues that differ (rat rarrow.gif 

human: Thr110 rarrow.gif Ala108, Met111 rarrow.gif Ile109, Tyr113 rarrow.gif Ser111) and TM6 has a 

cluster of 5 residues that differ (rat rarrow.gif human: Phe259 rarrow.gif Leu257, Leu256 rarrow.gif 

Ile254, Val255 rarrow.gif Ile253, Gly252 rarrow.gif 

Leu 250, Ala 249 rarrow.gif Val 247) in rat and human receptors. These differences represent important 

targets for follow-up point (and cluster) mutation experiments. Our current thinking is that the largest 

effects on NPC 17410 pharmacology, if any, might be derived from the TM3 mutants since 


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Figure 6 

Rat and human B2 receptor chimera constructs and affinity data 

for binding NPC 17410. Also shown is the affinity NPC 17410 to 

rat and human wild type receptors. 

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between rat and human, these are quite diverse. However, it is also possible that the cluster of residues 

identified in TM6, while not radically dissimilar, may as a group create different hydrophobic 

environments between these species homologues. The most significant individual difference within the 

TM6 zone is the Phe 259 (rat) rarrow.gif Leu 257 (human) swap and might therefore be most significant 


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Figure 7 

Schematic of the primary amino sequence of the human B2 receptor. Shown 

in black are residues experimentally identified as contributing to an agonist binding site. 

The dark gray residues are suspect positions for contributing to an antagonist site. The 

residues colored light gray have been mutagenized only in the rat B2 receptor, but they 

are conserved in the human. The Thr 263 rarrow.gif Ala mutation interferes with agonist binding 

only, while Gln 260 partially interferes with agonist and first generation antagonist 

binding. 

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within the context of these TM6 residues. Currently, we have prepared these mutant receptors, but at this 

time binding to NPC 17410 remains unfinished. 

A summary of the amino acids in the human B2 receptor implicated in comprising either agonist or 

antagonist sites are highlighted in Figure 7. Marked in dark black are the residues of extracellular loop 3, 

TM 6, and the extracellular N-terminal segment that have been shown to participate in agonist binding. 

Marked in dark gray in TM 6 and TM 3 are residues likely to partially comprise an antagonist binding 

site based primarily on the chimeric receptor studies described previously, although there is no explicit 

experimental evidence as yet. Shown in light gray are two residues that are conserved between rat and 

human B2 receptors. Mutagenesis experiments have been done on this pair in the rat B2 


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receptor with interesting results [54]. Mutations in Thr 263 only affect agonist binding, not antagonist. 

Mutations in Gln 260 affect binding of bradykinin and first generation antagonist peptides. As depicted in 

the figure, it is possible that the agonist and antagonist binding sites have domains on opposite sides of 

the helix that makes up TM 6, with Gln 260 being situated partly in both. 

IV. Design and Combinatorial Synthesis of Nonpeptidic Antagonists 

As was previously described, a significant body of information was generated that provides insights into 

the key structural features of bradykinin receptor binding sites and the residues that participate in ligand 

binding. In addition, from the ligand-based studies, knowledge about relevant structure-activity 

relationships was acquired. Our modular synthetic strategy was based primarily upon the recognition 

that high-affinity ligands appear to be comprised of three domains. These domains are (1) a positively 

charged N-terminal segment, (2) a midsection containing a bend or twist with some hydrophobic 

substituent attached and, (3) a C-terminal segment of appropriate hydrophobicity and structurally 

simulating a type II' β turn. Models of potent cyclic and linear peptide bradykinin receptor antagonists 

(described previously) were used in a comparative fashion to select nonpeptide ring systems from a 

database of chemical structures fine chemicals database. For each, some degree of chemical diversity 

was achieved by altering one of several parameters including, o, m, or p substitution of an aromatic ring 

or nature of alkyl substituent(s) as well as point(s) of synthetic attachment [55,56]. 

Each nonpeptide fragment was designed within the framework of several criterion. First, a given 

scaffold must closely match the known SAR and be compatible with the putative ligand binding site 

structure. Second, each scaffold must be a relatively simple synthetic target, having readily available 

starting material, no chiral centers and having a total synthesis of not more than 4–5 steps. Finally, each 

template must have a “C-terminal” carboxylate and an “N-terminal” amino group with no interfering 

functionality such that it could be readily used in a solid phase synthetic strategy. Given that each 

nonpeptide we identified was a viable surrogate for either the second or third domain of high-affinity 

ligands (as described above) our goal was to rapidly explore the receptor affinities of all possible 

combinations of these nonpeptide templates at position X and Y of the sequence DArg-Arg-X-Y-Arg, 

hence a combinatorial synthetic approach was taken. 

In this study, there were four linear aminoalkanoic acids [50], four different cinnamic acids, three 

different carbolines, three different phenanthridinones, and five different spirocyclics. The variability in 

the phenanthridinione series 


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was that the central ring could be opened or cleaved and the amino group could be meta or para 

substituted on the latter. In the carboline series, the cyclic amino group was either at the β or γ position 

of the cyclohexenyl ring and the methylene chain bearing the “C-terminal” carboxylate could be of 

variable length. The spirocyclic series was varied by alkyl, cycloalkyl, and aryl substitution on the fivemembered 

ring amine nitrogen. The cinnamic acids had two carbon chains that could be of varying 

length, one of which had the further possibility of containing a double bond(s). 

Rather than perform individual syntheses of all possible combinations of these nonpeptide units, 

members of each ring type or scaffold family were pooled in equimolar amounts prior to incorporation 

into the sequence DArg-Arg-X-Y-Arg. Since each individual member of a given pool was constructed 

on a similar carbocyclic scaffold, the chemical environment of the N-terminal amino group and Cterminal 

carboxylate groups were expected to follow similar kinetic and thermodynamic controls during 

the attachment of the nonpeptide residue to the growing peptide chain. The use of these smaller, directed 

libraries made it readily practical to obtain HPLC and mass spectral data for each and therefore confirm 

the composition of the library. 

Ultimately, 10 libraries of novel nonpeptidic structures were synthesized following typical solid-phase 

methodologies. Each library contained from nine to thirty-six different compounds in approximately 

equimolar amounts. Unpurified libraries were tested in a receptor binding assay utilizing membrane 

preparations from a stable CHO cell line expressing the human B2 receptor. Each library was tested at 

concentrations between 10 nM and 1μM. The ability of each library to inhibit[ 3H]-bradykinin binding 

was assessed and the results are presented in Figure 8a. Although this type of screening is highly 

qualitative, certain libraries appear in Figure 8a that show higher affinity to the receptor than other 

libraries. Library one (of the series DArg-Arg-PH-CN-Arg) was ultimately selected for further 

deconvolution. This library was further broken down (decoded) in order to determine which 

compound(s) were responsible for the apparent activity. It is important to note that breaking these 

libraries down to elucidate the structure of the hit(s) was feasible due to the inherently small size of each 

library. 

Library one contained 12 different structures (recall that there were originally three different 

phenanthridinones and four different cinnamic acids). The first deconvolution step of the approach is 

shown in Figure 9. Here, only the CN position was randomized, and the PH moieties were specific. This 

led to the preparation of three new libraries of 4 compounds each. Receptor binding was again 

performed as before and only one of these three new sublibraries showed affinity for the receptor at 10 

μM (Figure 8b). The final step in the process required to elucidate the active component(s) was to 

synthesize and purify each of the 4 members of this library as shown in Figure 9. Receptor binding on 

these 


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Figure 8 

(a) Binding assay results for 10 original nonpeptidic libraries 

of the sequence DArg-Arg-X-Y-Arg, where X and Y are defined 

as PH = phenanthridinone, CB = carboline, SP = spirocycle, 

SC = straight chain, CN = cinnamic acid. Each library was tested at 

1 μnM and 10 nM. Results were compared to cold bradykinin 

binding, which was tested at two lower concentrations, 0.1 nM 

and 1 nM. Panels (b) and (c) correspond to the receptor binding 

results obtained using the two breakdown steps from original 

library number 1, as shown in Figure 9. 


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Figure 9 

Composition of ten original nonpeptidic libraries of the sequence DArg-Arg-X-Y-Arg. 

X and Y were selected from the set of scaffolds shown in Table 1. Also shown 

are the subsequent breakdown libraries from original library number 1. Two-letter codes 

used in the figure correspond to the different nonpeptide moieties described in Table 1. 

Specifically, PH = phenanthridinone, CB = carboline, SP = spirocycle, SC = Straight 

chain, CN = cinnamic acid. 

Page 144 

four novel nonpeptidic structures showed that only one of the four had affinity to the receptor (Figure 

8c). This new compound, I, was subsequently shown to be an antagonist in a cellular assay measuring 

bradykinin-stimulated IP turnover [18]. Overall, there were 285 possible structures to survey due to the 

number of structure-based scaffolds that were prepared. This was rapidly accomplished via 19 synthetic 

couplings, 19 assays, and 4 purifications. 

Not surprisingly, compound I showed divergent potency when assayed in different species homologues 

of the bradykinin B2 receptor. In particular, in a model of bradykinin-induced hypotension in rats and 

rabbits, it showed no activity. Likewise, it did not block bradykinin-induced contraction of the isolated 

guinea pig ileum. Since compound I is considerably smaller than previously reported decapeptide 

antagonists, subtle structural differences (which are 


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known to exist in species homologs of the bradykinin B2 receptor) are likely amplified. A more 

comprehensive pharmacological analysis of compound I is currently underway. 

A. Lead Optimization 

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We have previously reported that the C-terminal guanidinyl moiety of Arg [9] in prototypical peptide 

bradykinin antagonists is likely to behave more as an aromatic functional group rather than a hydrogenbond 

donor/acceptor. This speculation was based on proposed models of the agonist and antagonist 

binding sites of this receptor that have been elucidated using molecular biological and computational 

procedures. On this premise, the newly discovered lead compound, I, was altered such that the Cterminal 

arginine was replaced by 3',5'-dimethylpyrimidylornithine in an attempt to increase potency. 

This known mimetic of arginine contains an aromatic 3',5'-dimethylpyrimidyl ring in the side chain 

rather than the guanidino group on naturally occurring arginine. 

The results of the receptor binding assay performed using this compound, IA, are shown in Table 4 

where it is clear that affinity to the human B2 receptor is improved with respect to compound I. This 

data is supportive of the notion that the C-terminal residue(s) in this new series of bradykinin antagonist 

compounds interact with a hydrophobic environment, perhaps within the transmembrane domain of the 

receptor as previously suggested. 

The discovery of I and IA is significant in many regards. First, they are highly nonpeptidic lead 

compounds that could be further modified to improve potency and/or reduce molecular weight. Such 

improvements might lead to novel therapeutic agents for the treatment of inflammatory diseases. Thus 

far in the kinin antagonist literature there is significant evidence showing that, for compounds containing 

a C-terminal arginine residue, removal of that arginine generally yields compounds that are antagonists 

of the B1 subtype of the bradykinin receptor. Following a similar strategy with compound I could lead to 

the discovery of a novel series of nonpeptidic B1 receptor antagonists, although this remains to be 

demonstrated. 

V. Conclusions 

There has been a significant effort invested toward the discovery of novel bradykinin receptor 

antagonists during the past decade. In that time, several generations of peptide antagonists have been 

developed and a few are in human clinical trials. The pursuit of nonpeptide antagonists of the human 

bradykinin B2 receptor continues and incorporates a wide range of strategic approaches. The approach 

described herein is an early and very good example of a combinatorial synthesis of nonpeptide building 

blocks that mimic peptide structure, 


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ultimately tested in a nontagged, solution-phase form. Perhaps more significant is that the success 

described here demonstrates a possible synergy between structure-based design and combinatorial 

methodology. This approach has many merits, but the most significant is the application of structurally 

directed libraries toward target binding-site structures which, for one reason or another, may not be fully 

characterized. This method serves to aim the combinatorial syntheses in a logical direction, rather than 

attempt to prepare libraries of vast diversify (and numbers). 

Finally, since the libraries of compounds that were prepared contained few members, it was possible to 

analytically characterize each of the pools to assess integrity of their composition. Overall, there are 

many important advantages in the paradigm we have adopted that make the strategy generally viable in 

the context of structure-based lead compound discovery. 

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31. Wirth K, Hock FJ, Albus U, Linz W, Alpermann HG, Anagnostopoulos H, Henke S, Breipohl G, 

Knoig W, Knolle J, Schölkens BA. Br J Pharmacol 1991; 102:774. 

32. Sawutz DG, Salvino JM, Seoane PR, Douty BD, Houck WT, Bobko MA, Doleman MS, Dolle RE, 

Wolfe HR. Biochem 1994; 33:2373. 

34. Chakravarty S, Wilkens D, Kyle DJ. J Med Chem 1993; 36:2569. 

33. HOE SDS MICELLES 

35. Momany FA. In: Metzger, RM, ed. Topics in Current Physics. Vol. 26; New York: Springer Verlag, 

1981:41–79. 

36. Momany FA, Chuman H. Meth Enz 1986; 124:3–17. 

37. Kyle DJ. Brazil J Med Bio Res 1994; 27:1757. 

38. Chakravarty S, Mavunkel BJ, Lu S, Goehring R, Wu JP, Connolly M, Valentine H, Liu YW, Tam C, 

Andy R, Kyle, DJ. 23rd European Peptide Symposium, Braga: Sept 4–10, 1994. 


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39. Mavunkel BJ, Lu Z, Kyle DJ. Tett Lett 1993; 34:2255. 

40. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH. J Mol Biol 1990; 

213:899. 

41. Kyle DJ, Chakravarty S, Sinsko JA, Stormann TM. J Med Chem 1994; 37:1347. 

42. Kyte J, Doolittle RF. J Mol Biol 1982; 157:105. 

43. Rose GD, Gierash LM, Smith JA. Adv Prot Chem 1985; 37:1. 

44. Novotny E, Bednar D, Connolly M, Connor J, Stormann T. In: Burch RM, ed. Molecular Biology 

and Pharmacology of Bradykinin Receptors. Austin, TX: R. G. Landes Company, 1993:19–30. 

45. Novotny EA, Bednar DL, Connolly MA, Connor JR, Stormann TM. BBRC 1994; 201:523. 

46. Lee SC, Russell AF, Laidig WD. Int. J Pept Prot Res 1990; 35(5):367. 

47. Freedman R, Jarnagin K. Cloning of a B2 Bradykinin Receptor: Recent Progress on Kinins. Basel: 

Birkhauser Verlag, 1992:487–496. 

48. Herzig MCS, Leeb-Lundberg F, Nash N, Connolly M, Kyle DJ. Kinin '95. International conference 

on kallikreins and kinins, Denver, CO, Sept, 1995. 

49. Herzig MCS, Leeb-Lundberg F. J Biol Chem 1995; in press. 


50. 

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51. Nash N, Connolly MA, Stormann TM, Kyle DJ. 14th Am Pep Sym, June 18, 1995. 

52. Nash N, Connolly MA, Stormann TM, Kyle DJ. Mol Pharm 1996; manuscript in preparation. 

53. Burch RM, Kyle DJ, Stormann TM. In: Molecular Biology and Pharmacology of Bradykinin 

Receptors. Austin, Texas: R. G. Landes Company, 1993:19–32. 

54. Nardone J, Hogan PG. PNAS 1994; 91:4417. 

55. Chakravarty S, Mavunkel B, Andy R, Kyle DJ. Network Science 1995; 1:1. 

56. Chakravarty S, Mavunkel BJ, Andy R, Kyle DJ. 14th American Peptide Symposium, Columbus, 

Ohio, June, 1995. 


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5 

Design of Purine Nucleoside Phosphorylase Inhibitors 

Y. Sudhakara Babu, John A. Montgomery, and Charles E. Bugg 

BioCryst Pharmaceuticals, Inc., Birmingham, Alabama 

W. Michael Carson, Sthanam V. L. Narayana, and William J. Cook 

The University of Alabama at Birmingham, Birmingham, Alabama 

Steven E. Ealick 

Cornell University, Ithaca, New York 

Wayne C. Guida and Mark D. Erion * 

Ciba-Geigy Corporation, Summit, New Jersey 

John A. Secrist, III 

Southern Research Institute, Birmingham, Alabama 


A. Enzymology 

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Purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) catalyzes the reversible phosphorylysis of 

ribonucleosides and 2'-deoxyribonucleosides of guanine, hypoxanthine, and related nucleoside analogs 

[1]. It normally acts in the phosphorolytic direction in intact cells, although the isolated enzyme 

catalyzes the nucleoside synthesis under equilibrium conditions. Figure 1 shows the chemical reaction. 

The enzyme has been isolated from both eukaryotic and prokaryotic organisms [2] and functions in the 

purine salvage pathway [1,3]. Purine nucleoside phosphorylase isolated from human erythrocytes is 

specific for the 6-oxypurines and many of their analogs [4] while PNPs from other organisms vary in 

their specificity [5]. The human enzyme is a trimer with identical subunits and a total molecular mass of 

about 97,000 daltons [6,7]. Each subunit contains 289 amino acid residues. 

* Current affiliation: Gensia, Inc., San Diego, California. 


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B. Pharmacology 

Figure 1 

The reaction catalyzed by PNP. 

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Interest in PNP as a drug target arises from its ability to rapidly metabolize purine nucleosides and from 

its role in the T-cell branch of the immune system. Unfortunately, PNP can also cleave certain 

anticancer and antiviral agents that are synthetic mimics of natural purine nucleosides, thus interfering 

with therapy. One such substance is ddI (2'3'-dideoxyinosine), which the Food and Drug Administration 

approved as a treatment for AIDS in 1991. Another is the potential anticancer agent 2'-deoxy-6thioguanosine 

[8]. Our goal was to develop a compound that when administered with the nucleoside 

analogs would inhibit PNP while the anticancer and antiviral agents accomplished their therapeutic 

missions. The combination of purine nucleoside analogs and a PNP inhibitor might prove to be a more 

effective treatment. 

The PNP inhibitors alone have potential therapeutic value based on the importance of PNP to the 

immune system. Patients lacking PNP activity exhibit severe T-cell immunodeficiency while 

maintaining normal or exaggerated B-cell function [9]. We, like other researchers, quickly recognized 

that PNP inhibitors might selectively suppress the T-cell proliferation associated with an array of 

autoimmune disorders such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus, multiple 

sclerosis, and insulin-dependent (juvenile-onset) diabetes [10]. This profile also suggests that PNP 

inhibitors might be useful in the treatment of T-cell proliferative diseases—such as T-cell leukemia or Tcell 

lymphoma—and in the prevention of organ transplant rejection. 

C. Drug Design Strategy 

Recent advances in biotechnology, macromolecular crystallography, computer graphics, and related 

fields have led to a new approach in drug discovery called structure-based drug design. Structure-based 

drug design requires a detailed structural knowledge of the target (enzyme or receptor) and the 

interaction of small molecules with it. 


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Figure 2 

Structure-based drug design strategy. 

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A tight fit is necessary for potency and specificity. A drug that binds to its target and inactivates it for a 

long time can be administered in lower doses than one that rapidly separates from its target. A substance 

designed to mesh perfectly with a particular binding site of one target is unlikely to interact well with 

any other molecule, minimizing unwanted interactions and side effects. 

Having chosen PNP as the target, we followed a systematic strategy for designing inhibitory 

compounds. Figure 2 outlines the overall strategy of this approach. To serve as a drug, an inhibitor has 

to readily cross cell membranes to the interior of cells, where PNP is located. 


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We determined the structure of human PNP by x-ray crystallography and used these results in 

combination with computer-assisted molecular modeling to design inhibitor candidates. We examined 

how well the shape and chemical 


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structure of a candidate would complement the active site of PNP. We used computational chemistry to 

estimate the strength of the attractive and repulsive forces between a candidate and the enzyme. 

We synthesized only those candidates suggested by chemical intuition and computer simulation to have 

high affinity for the target. Then we measured the inhibition of PNP and compared the proposed with the 

actual fit. Because modeling programs and expert opinion are imperfect, certain compounds did not 

meet expectations. After exploring the reasons for the successes and the failures, we returned to 

interactive computer graphics to propose modifications that might increase the effectiveness of drug 

candidates. 

The resulting compounds were evaluated by determination of their IC 50 values (the inhibitor 

concentration causing 50% inhibition of PNP) and by x-ray diffraction analysis using difference Fourier 

maps. This iterative strategy—modeling, synthesis, and structural analysis—led us to a number of highly 

potent compounds that tested well in whole cells and in animals. 

D. Previously Known Inhibitors 

At the beginning of our studies several PNP inhibitors had been reported with Ki values in the 10-6 to 10- 7 range, including 8-aminoguanine [11], 9-benzyl-8-aminoguanine [12], and 5'-iodo-9-deazainosine [13]. 

Acyclovir diphosphate had been shown to have a Ki near 10-8 if assayed at 1 mM phosphate rather than 

the more frequently used value of 50 mM phosphate [14]. During our studies, the synthesis of 8-amino- 

9(2-thienylmethyl)guanine was reported with a Ki of 6.7 × 10-8 M [15]. Figure 3 illustrates some of these 

structures. 

Despite the potential benefits of PNP inhibitors and the large number of PNP inhibitors that had been 

synthesized, no compound had reached clinical trials. None of these compounds were potent enough to 

be useful for therapy and also capable of crossing the cell membrane intact. Although potencies for the 

best compounds had affinities 10–100 fold higher than the natural substrate (K m = 20 μM), it is expected 

that T-cell immunotoxicity will only occur with very tight binding inhibitors (K i < 10 nM) due to the 

high level of in vivo PNP activity and competition with substrate. 

II. Crystallography 

At the present time, x-ray crystallography is the preferred technique for obtaining the required atomic 

resolution structural data. In the late 1970s when this project was first conceived, determining the 

structure of a protein was far from routine. The x-ray structural determination occupied a team of 

crystallographers led by Steven E. Ealick, then at the University of Alabama at Birmingham through 

most of the 1980s. 


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Figure 3 

Previously known inhibitors. 


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The stumbling block did not lie with obtaining pure PNP or converting the protein into crystals. Robert 

E. Parks, Jr. and Johanna D. Stoeckler of Brown University had already isolated the enzyme from 

human cells. They supplied quantities of protein to William J. Cook, who succeeded in preparing the 

well-ordered crystals required for x-ray studies [16]. We established that PNP crystals function normally 

as a catalyst. Thus crystalline PNP is essentially identical to PNP in the body. If it were profoundly 

different, one would have no justification for basing drug design on the crystal structure. 

In the early years we had to depend on a relatively low-intensity x-ray source. High-resolution data was 

obtained through collaboration with John R. Helliwell and his group at the Daresbury Laboratory 

Synchrotron Radiation Source in England. Today greatly improved equipment and more synchrotron 

facilities are available for protein crystallography. 

The three-dimensional structure was determined by multiple isomorphous replacement techniques using 

synchrotron radiation [17]. The native and guanine-PNP complex structures have been refined to 2.8 Å 

resolution [18,19]. 

A. Structure of the Enzyme 

Crystals of human PNP are grown from ammonium sulfate solution and stored in artificial mother liquor 

solution made of 60% ammonium sulfate in 0.05 M citrate buffer at pH 5.4. The space group is R32 with 

hexagonal cell parameters a=142.9(1) Å and c=165.2(1) Å. The PNP crystals contain about 76% solvent 

and diffract to around 2.8 Å resolution. 

The x-ray data established that PNP crystals contain a high percentage of water. This feature proved 

very useful; proposed drugs could easily be soaked into the active site without disrupting the crystal 

packing. Figure 4A shows the 


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Figure 4 

Structure of PNP as stereo drawings. (A) Crystal packing. The low-resolution 

surfaces of six trimers are shown. The top level is related to the bottom level by a 2-fold 

axis along X. The distance between the 3-fold axes is 143 Å. A drug molecule is shown 

in the solvent channel near the entrance to an active site. (B) Ribbon drawing. The 

lowermost trimer of Figure 4A is shown. This is the native structure; the guanine and 

phosphate are shown to mark the active site. (C) The swinging gate. The trimer of Figure 

4B is rotated about 30° counterclockwise in the plane, followed by a roughly 90° rotation 

about X to view the entrance to the active site. A model of the transition state is shown 

as a line drawing. Conformational changes of the protein on binding guanine are shown. 

Arrows are drawn from the C α positions in the native structure to their positions in the 

complex. (D) Active site of PNP. The orientation is approximately that of Figure 4C, but 

enlarged and clipped to focus on the substrate. Key side-chain residues are labeled. 

Residue 159'F, in the center of figure toward the viewer, is the only residue from the 

adjacent subunit. The guanosine and phosphate are shown with thicker bonds. Oxygen 

and sulfur atoms are shown as white spheres, nitrogen and phosphorus as black spheres. 

(E) Purine binding pocket. The style is the same as Figure 4D, but the figure is rotated 

slightly and enlarged. The key group interacting with bound guanine are highlighted. (F) 

The best inhibitor. The style is the same as Figure 4D, but the figure has been enlarged 

and rotated to place the phosphate binding site far from the viewer in the upper left. 


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Figure 4 

(Continued) Only one nitrogen atom from Arg 84 is visible, which—along 

with Ser 220 and His 86—interact with the acetyl group branching from the benzylic 

carbon. The chlorinated phenyl group is in the center of the figure, interacting with the 

aromatic groups in the sugar binding site. The guanine group interactions are the same as 

seen in Figure 4E. Figure prepared with ribbons (http://www.cmc.uab.edu/ribbons). 


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large solvent channels and the position of the active site. The x-ray analysis confirmed the trimeric 

nature of the enzyme, as the subunits are related by the crystallographic three-fold axis. 

Page 159 

A ribbon diagram of the trimer is shown in Figure 4B. Each monomer contains an eight-stranded β sheet 

and a five-stranded β sheet that join to form a distorted β barrel. Seven α helices surround this β sheet 

structure. 

The active site is an irregular indentation on the surface of the enzyme, located from the position of a 

tightly bound sulfate ion and various substrate analogs. These investigations revealed the identity of the 

exact amino acids constituting the active site region; such detail was a prerequisite to drug design. 

Information of greater import emerged from analyses of the complexes formed when synthetic 

nucleosides, including previously discovered inhibitors, were diffused into the active site. 

B. The Active Site 

The structural determinations also yielded a surprise. The shape of the enzyme changes when a purine is 

bound. The famous lock-and-key analogy [20] has a fallacy; the shape of the lock is not static, but 

flexible. Awareness of these conformational changes critically aided our modeling efforts, allowing 

prediction of which parts of PNP could change shape to interact with a proposed inhibitor. 

A “swinging gate” consisting of residues 241–260 controls access to the active site (Figure 4C). These 

residues in the native structure had poorly defined electron density with high thermal motion. The gate 

opens in the native enzyme to accommodate the substrate or inhibitor. The maximum movement caused 

by substrate or inhibitor binding occurs at His 257, which is displaced outwards by several angstroms. 

After binding, the electron density becomes well defined. The gate is anchored near the central β sheet 

at one end and near the C-terminal helix at the other end. The gate movement is complex and appears to 

involve a helical transformation near residues 257–261. 

Consequently, initial inhibitor modeling attempts using the native PNP structure were far less successful 

than subsequent analyses in which coordinates for the guanine-PNP complex were used. Because of the 

magnitude of the changes that occur during substrate binding, it is unlikely that modeling studies based 

on the native structure alone would have accurately predicted the structure of PNP/inhibitor complexes. 

The active site is located near the subunit-subunit boundary within the trimer and involves seven 

polypeptide segments from one subunit and a short loop from the adjacent subunit (Figure 4D). The 

purine binding site employs residues Glu 201, Lys 244, and Asn 243 to form hydrogen bonds with N1, 

O6, and N7 of purine. The remainder of the purine binding pocket is largely 


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hydrophobic, composed of residues Ala 116, Phe 200, and Val 217. The phosphate binding site uses 

residues Ser 33, Arg 84, His 86, and Ser 220 with the phosphate positioned for nucleophilic attack at C1' 

of the nucleoside. The sugar binding site is mostly hydrophobic consisting of residues Tyr 88, Phe 200, 

His 257 from one subunit and Phe 159 of the adjacent subunit. This hydrophobic pocket orients the 

sugar to facilitate nucleophilic attack by phosphate and subsequent inversion of C1'. 

C. Initial Inhibitor Complexes 

In order to understand the interaction of inhibitors with the active site residues, the previously known 

inhibitors were obtained and crystallographic analyses were carried out. The most important findings 

were (1) 8-amino substituents enhance binding of guanines by forming hydrogen bonds with Thr 242 

and possibly the carbonyl oxygen atom of Ala 116; (2) substitution by hydrophobic groups at the 9position 

of a purine enhances binding through interaction with the hydrophobic region of the ribose 

binding site; and (3) acyclovir diphosphate is a multisubstrate inhibitor with the acyclic spacer between 

the purine N9 and the phosphate of near optimal length to accommodate these two binding sites. Based 

on these results, a number of starting compounds were proposed that incorporated these and other 

features predicted to enhance inhibitor binding. 

III. Molecular Modeling 

Structural information in combination with graphical methods for displaying accessible volume, 

electrostatic potential, and hydrophobicity of the active site of the target macromolecule greatly 

facilitates the drug design process. Accurate prediction of binding affinities and protein conformational 

changes are currently not routinely possible, although significant advances are being made. 

Proposed compounds were screened by modeling the enzyme-inhibitor complex using interactive 

computer graphics. Macromodel [21] and AMBER [22] based molecular energetics were used along 

with Monte Carlo/energy minimization techniques [23] to sample the conformational space available to 

potential inhibitors docked into the PNP active site. Methods based on the work of Goodford [24] 

employing custom software were also used. Qualitative evaluation of the enzyme-inhibitor complexes 

by molecular graphics and semiquantitative evaluation of the interaction energies between the inhibitors 

and the enzyme aided in the prioritization of compounds for chemical synthesis. 


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IV. Drug Design Progression 

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We focused initially on filling the purine binding region of the active site. That done, we planned to fill 

the sugar binding region and, finally, the phosphate binding site. We expected that each successive step, 

moving the compound closer toward fully occupying the active site, would enhance the affinity of the 

drug candidate for the enzyme. 

A. The Purine Site 

From our crystallographic examinations, we knew that three amino acids in the purine binding pocket of 

PNP formed hydrogen bonds with purines and their mimics. Such linkages are among the strongest 

reversible chemical bonds that exist. In proposing inhibitor candidates, we concentrated on compounds 

that would at least form hydrogen bonds with the same three amino acids. Figure 4e shows a close-up of 

the purine site. 

We favored exchanging a carbon atom for the nitrogen atom that normally occupies position nine, since 

there was no interaction of this nitrogen with the active site and earlier studies showed such a change 

promotes binding to PNP. Guanine modified in this way is called 9-deazaguanine. The first structures 

selected for synthesis were 9-deazaguanines substituted by an arylmethyl group at the 9 position. These 

compounds were prepared by adaption of a literature procedure [25]. We further expected that attaching 

an amino group to the carbon atom in position eight on 9-deazaguanine would enhance affinity, since 8aminoguanine 

was the first significant inhibitor of PNP. 

Both 8-aminoguanine analogs and 9-deazaguanine analogs are good inhibitors of PNP. However, 

introduction of an 8-amino group into the 9-deazaguanine derivatives resulted in decreased potency. To 

understand this poor binding, we undertook crystallographic analysis of PNP complexes with four 

compounds having the 9-thienyl substituent attached to guanine (G), 8-aminoguanine (8AG), 9deazaguanine 

(DG), and 8-amino-9-deazaguanine (8ADG). The results of this analysis are summarized 

in Figure 5. These data show one mode of binding for compounds that accept a hydrogen bond from Asn 

243 at N7 (G and 8AG) and another for compounds that donate hydrogen to Asn 243 from N7 (DG and 

8ADG). The 8AG analogs make use of the Thr 242 side chain to form an additional hydrogen bond, 

which improves binding affinity. In the 9-deazaguanine series, where N7 has an attached hydrogen 

atom, Asn 243 undergoes a shift that is clearly seen in difference Fourier maps. This shift is caused by 

the formation of the N7-H…OD(243) hydrogen bond. A concomitant shift by Thr 242 prevents it from 

hydrogen bonding to the 8-amino group of 8ADG. Furthermore, the shift moves the methyl group of Thr 

242 towards the 8-amino 


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Figure 5 

Comparison of 8-amino and 9-deaza substitutions on guanine. Details are extensively 

discussed in the text. Cross-hatching schematically indicates the enzyme. Ball and 

stick diagrams show the inhibitor and key side-chain residues. Nitrogens are dark 

gray spheres, oxygens are light gray. Arrows indicate hydrogen bonding, with the 

arrow size showing relative strength. 

amino group, generating a hydrophobic environment for the group and decreasing binding affinity. 

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The carbon-for-nitrogen switch in the 9-deaza variant favors association with PNP by substituting a 

strong hydrogen bond for the relatively weak one occurring between Asn 243 and guanine. Formation of 

a simple 8-aminoguanine variant leads to tight binding by giving rise to an extra hydrogen bond between 

the purine derivative and Thr 242. 

The combination of the two “improvements”—the carbon-for-nitrogen substitution and the addition of 

the amino group to position eight—was counterproductive because the carbon in position nine prevented 

the amino group at 


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position eight from forming the extra bond with Thr 242. In fact, it set up an unfavorable, repulsive 

clash between the threonine and the added amino group. 

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In the absence of detailed structural information, it would have been extremely difficult to explain why 

affixing the amino group to the carbon in position eight proved unhelpful. But crystallography quickly 

provided the explanation. 9-Deazaguanine itself would be a better choice for the purine component of an 

inhibitor. This experience underscores the wonderful economy of the structure-based approach. Without 

crystallographic data, we might have pursued a logical but unproductive avenue of research much longer 

than we did. 

B. Ribose Site 

The next task was to fill the sugar binding site. The sugar in a nucleoside does not attach to PNP 

primarily by forming hydrogen bonds, but through hydrophobic attractions. The sugar binding pocket of 

PNP consists of three hydrophobic amino acids: Phe 200 and Tyr 88 from the same monomer that binds 

guanine and Phe 159 from the adjacent monomer. Several known inhibitors carried a benzene group 

attached to position 9 of the purine in place of the sugar in the nucleoside. An initial series of 

compounds was synthesized to exploit the hydrophobic region in the ribose binding site. 

A number of 9-substituted 9-deazapurine analogs were prepared with various aromatic, heteroaromatic, 

and cycloaliphatic substituents. The first 9-deazaguanine derivatives synthesized, such as 9-benzyl-9deazaguanine, 

were three to six times more potent than the most potent known inhibitor, 8-amino-9-(2thienylmethyl)guanine. 

The optimum spacer between the purine base and the aromatic substituent 

proved to be a single methylene group. Crystallographic data showed that generally the planes of the 

aromatic rings tend to orient in a reproducible conformation. The aromatic groups optimize their 

interaction with Phe 159 and Phe 200, which results in the classic “herringbone” arrangement reported 

in a variety of aromatic systems [26]. 

Inhibitors with cycloaliphatic substituents at N9 of deazaguanine were also as potent as the aromatic 

analogs. The cycloaliphatic substituents occupied the same general volume as the aromatic groups. As 

with the aromatic series, the optimum spacer between the 9-deazaguanine and the hydrophobic 

substituent is one carbon atom. X-ray analysis of the PNP complexes of 9-cyclohexyl-9-deazaguanine, a 

relatively poor inhibitor, and the complex of 9-cyclohexylmethyl-9-deazaguanine, a potent inhibitor, 

showed the two cyclohexyl groups occupy approximately the same space in the active site with the 

purine base pulled out of its optimal position in the former. 

The chemistry is more straightforward with the aromatic series. From modeling studies, we saw that the 

sugar binding pocket could be filled more 


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completely by adding any of several chemical groupings to the benzene ring. The best fit came from 

adding a chlorine atom to position 3 of the benzene ring. 

C. Phosphate Site 

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The final step added a group that would interact with the phosphate binding site either directly or via 

electrostatic interactions. We could not use phosphate itself, because phosphate-containing compounds 

are not metabolically stable and have difficulty passing through cell membranes intact. Acyclovir 

diphosphate, which is not membrane permeable and is subject to extracellular metabolism, is a good 

example. 

Our results suggested that an ideal PNP inhibitor in the 9-deazapurine series would contain an aromatic 

group and a substituent with affinity for the phosphate site interlinked by spacers with optimum lengths. 

Crystallographic and modeling studies suggested a two-to-four-atom spacer. Initial modeling studies 

encouraged us to prepare several structures, but they failed to improve the binding affinity of our twopart 

structure. 

Crystallographic analysis of a number of PNP inhibitor complexes revealed significant displacement of 

the inhibitors. These displacements appear to be the result of close contacts between the inhibitor and 

the ion in the phosphate binding site. Sulfate ions occupy the phosphate site in PNP crystals as they are 

grown from ammonium sulfate solution. These inhibitors were more potent when the binding was 

measured in 1 mM phosphate solution rather than in 50 mM phosphate. Kinetic studies showed that 

these inhibitors were competitive not only with inosine but also with phosphate, in keeping with the 

above observation. 

These results, summarized in Table 1, show that the IC 50 (50 mM) is equal to or larger than the IC 50 (1 

mM), in some cases by as much as 100-fold. The ratio and the dimension of the 9-substituent show some 

correlation. Compounds such as 8-aminoguanosine and 8-amino-9-(2-thienylmethyl)guanine show no 

difference. Since the concentration of phosphate in intact cells is 1 mM, we routinely used this assay 

condition for all PNP inhibitors. 

Starting with a model of the 9-benzyl-9-deazaguanine/PNP complex, we concluded that two of the 

positions on the 9-benzyl group, namely the 2-position of the phenyl ring and one of the benzylic sites, 

appeared to be oriented so that a group attached to either one could interact favorably with the phosphate 

binding site. The first compound made in this series, 9-[2-(3-phosphonopropoxy)benzyl]guanine, turned 

out to be a poor PNP inhibitor. Subsequent crystallographic analysis revealed that the plane of the 

aromatic ring had rotated approximately 90° from its optimum position in the hydrophobic pocket. This 

reorientation of the ring was presumably necessary to accommodate the four atom spacer between the 

phenyl ring and the phosphonate group. A compound 


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Table 1 Inhibition Data for Selected PNP Inhibitors by Increasing IC 50 Value 

R 2a 

R 2 

IC 50, μM Ratio d 

50 mM 

phosphate b 

1 mM 

phosphate c 

(S)-3-Chlorophenyl CH 2CO 2H 0.031 0.0059 5.3 

3-Chlorophenyl CH 2CN 1.8 0.010 180 

2-Tetrehydrothienyl H 0.22 0.011 20 

3,4-Dichlorophenyl H 0.25 0.012 21 

3-Thienyl H 0.08 0.020 4 

3-Trifluoromethylcyclohexyl H 0.74 0.020 37 

Cyclopentyl H 1.8 0.029 62 

Cycloheptyl H 0.86 0.030 29 

Pyridin-3-yl H 0.20 0.030 7.3 

2-(Phosphonoethyl)phenyl e H 0.45 0.035 13 

Cyclohexyl H 2.0 0.043 47 

2-Furanyl H 0.31 0.085 3.6 

(R)-3-Chlorophenyl CH 2CO 2H 0.90 0.16 5.6 

2-Phosphonopropoxyphenyl e H 42 1.0 42 

a Compounds with R2 not equal to H are racemic mixtures unless the R or S isomer is designated. 

b Calf spleen PNP assayed in 50 mM phosphate buffer. 

c Calf spleen PNP assayed in 1 mM phosphate buffer. 

d IC50 at 50 mM phosphate divided by IC 50 at 1 mM phosphate. 


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e Guanine base. 

Source: Ref. 27. 

with a two-carbon spacer was a much better PNP inhibitor; however, it was clear from x-ray analysis 

that the aromatic ring was unable to form the ideal “herringbone” packing interaction. 

Alternatively, compounds were modeled in which the spacer to the phosphate binding site branched 

from the benzylic carbon, thus placing no restrictions on the tilt of the aromatic ring. Examination of the 

9-benzyl-9-deazaguanine/PNP complex indicated that of the two benzylic positions, one (pro-R) pointed 

into a sterically crowded area within the active site, whereas the other (pro-S) pointed into a relatively 

empty space adjacent to the phosphate binding site. This analysis led to the synthesis of racemic 9-[1-(3chlorophenyl)-2-carboxyethyl]-9-deazaguanine. 

This compound adds an acetate group (CH 2COO-) to 

the methylene carbon atom that joined 9-deazaguanine to the chlorinated benzene ring. This compound 

was resolved into its (S) and (R) enantiomers. 


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As predicated, the (S) acid was a 30-fold more potent inhibitor of PNP than the (R) form. X-ray 

crystallographic analysis of the complexes revealed that the (S) acid was oriented properly for optimal 

interactions with all three subsites (Figure 4F), whereas the (R) acid was not. This series of compounds 

contains the most potent membrane-permeable inhibitors of PNP yet reported [27]. 

V. Summary 

Recently, scientists at BioCryst have successfully completed a project to design and synthesize potent 

inhibitors of the enzyme Purine Nucleoside Phosphorylase (PNP) using the three-dimensional structure 

of the active site. Crystallographic and modeling methods have been combined with organic synthesis to 

produce inhibitors. Our experience in creating a set of potential drugs—one of which (BCX-34) is now 

in human trials for treating psoriasis and a form of T-cell lymphoma—illustrates the process and the 

power of structure-based design. 

This structure-based inhibitor design approach led to a number of inhibitors more than 100 times more 

potent than any membrane-permeable inhibitor available at the beginning of this project. During the two 

and half years of this project, about 60 active compounds were synthesized. This is a remarkably small 

number compared with the extensive synthesis programs generally involved in drug discovery by trial 

and error techniques. The large number of active compounds and the enhancement of inhibitor potency 

stand as proof that crystallographic and modeling techniques are now capable of playing a critical role in 

the rapid discovery of novel therapeutic agents. The entire protocol, from choosing the target to creating 

a drug suitable for clinical trials, can probably be accomplished today in two or three years. 

A. Obstacles Encountered and Lessons Learned 

Crystallographic analysis was based primarily on the results of difference Fourier maps in which the 

interactions between residues in the active site and the inhibitor could be characterized. During these 

studies, about 35 inhibitor complexes were evaluated by x-ray crystallographic techniques. It is 

noteworthy that the resolution of the PNP model extends to only 2.8 Å and that all of the difference 

Fourier maps were calculated at 3.2 Å resolution, much lower than often considered essential for drug 

design. Crystallographic analysis was facilitated by the large solvent content that allowed for free 

diffusion of inhibitors into enzymatically active crystals. 

Initial inhibitor modeling attempts using the native PNP structure were far less successful than 

subsequent analyses in which coordinates for the guanine- 


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Page 167 

PNP complex were used, mainly because of the magnitude of the changes that occur during substrate 

binding. We found that computer modeling required significant tuning in order to provide useful results. 

Crystallographic results were useful in testing and modifying modeling parameters. The most useful 

modeling results were achieved after incorporation of the conformational searching techniques described 

earlier and when the coordinates for the PNP-guanine complex model were used. Visual inspection and 

chemical intuition were very important. 

B. Perspectives of Treating Targeted Disease 

One of the inhibitors designed during the drug discovery process, 9-(3-pyridylmethy)-9-deazaguanine 

(BCX-34), was selected for initial clinical development. Current clinical trials utilize both topical and 

oral formulations of the drug. 

Researchers at the University of Alabama at Birmingham and Washington University School of 

Medicine have recently completed small Phase II clinical trials of two indicated applications, cutaneous 

T-cell lymphoma (CTCL) and psoriasis, using a topical formulation of BCX-34. Although patients 

showed improvement in both trials, the duration of each was too short (six weeks) to adequately assess 

the efficacy of the drug. Subsequently 80% of the patients from the CTCL trial (24 patients) entered an 

open label trial for treatment of their disease for up to twelve months. At the end of the first six months 

of treatment, seven of the patients were in complete remission (verified by biopsy), two patients showed 

a clinical complete response, and nine patients had shown definite improvement. The other six patients 

had shown no change or progression of disease. No serious, drug-related adverse events were reported 

during the study. 

The process of structure-based drug design helped to ensure that the inhibitor would be highly selective 

for the PNP enzyme, and thus far no other targets for the drug have been identified. The mechanism of 

action of BCX-34 appears to be entirely related to its effect on the proliferation of human T-cells. This 

high degree of specificity probably also contributes to the high safety profile of the drug. Although longterm 

studies in more patients will be necessary to substantiate these results, it appears likely that BCX- 

34 will have a significant clinical effect on at least some T-cell mediated diseases. 

Based on the results from these three trials, BioCryst has initiated a multicenter Phase III trial for the 

treatment of CTCL, as well as a large, multicenter Phase II trial for psoriasis. In addition to the two 

clinical trials using the topical formulation, a Phase I clinical trial in CTCL and T-cell 

lymphoma/leukemia has begun using an oral formulation of BCX-34. In the future, a number of other Tcell 

mediated diseases or processes are possible targets for BCX-34, including rheumatoid arthritis, 

multiple sclerosis, inflammatory bowel disease, and organ transplant rejection. 


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References 

1. Parks RE Jr., Agarwal RP. In: Boyer PD, ed. The Enzymes. 3rd Ed., New York: Academic, 1972; 

7:483–514. 

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2. Stoeckler JD. In: Glazer, RE, ed. Developments in Cancer Chemotherapy. Florida: CRC, Baco Raton, 

1984; 35–60. 

3. Friedkin M, Kalckar H. In: Boyer PD, Lardy H, Myrback K, eds. The Enzymes. 2nd Ed. New York: 

Academic, 1961; 5:237–55. 

4. Agarwal KC, Agarwal RP, Stoeckler JD, Parks RE Jr. Purine nucleoside phosphorylase. 

Microheterogeneity and comparison of kinetic behavior of the enzyme from several tissues and species. 

Biochemistry 1975; 14:79–84. 

5. Bzowska A, Kulikowska E, Shugar D. Properties of purine nucleoside phosphorylase (PNP) of 

mammalian and bacterial origin. Z Naturforschung C Biosci 1990; 45:59–70. 

6. Stoeckler JD, Agarwal RP, Agarwal KC, Schmid K, Parks RE Jr. Purine nucleoside phosphorylase 

from human erythrocytes: physiocochemical properties of the crystalline enzyme. Biochemistry 1978; 

17:278–83. 

7. Williams SR, Goddard JM, Martin DW Jr. Human purine nucleoside phosphorylase cDNA sequence 

and genomic clone characterization. Nucleic Acids Res 1984; 12:5779–87. 

8. LePage GA, Junga IG, Bowman B. Biochemical and carcinostatic effects of α'-deoxythiguanosine 

Cancer Res 1964; 24:835–40. 

9. Giblett ER, Ammann AJ, Wara DW, Sandman R, Diamond LK. Nucleoside-phosphorylase deficiency 

in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet 1975; 1:1010–3. 

10. Otterness I, Bilven M. In: Rainsford K, Velo G, eds. New Developments in Antirheumatic Therapy. 

Inflammation and Drug Therapy Series. Norwell, MA: Kluwer Academic, 1989; 2:277–304. 

11. Stoeckler JD, Cambor C, Kuhns V, Chu SH, Parks RE Jr. Inhibitors of purine nucleoside 

phosphorylase, C(8) and C(5') substitutions. Biochemical Pharmacology 1982; 31:163–71. 

12. Shewach DS, Chern JW, Pillote KE, Townsend LB, Daddona PE. Potentiation of 2'-deoxyguanosine 

cytotoxicity by a novel inhibitor of purine nucleoside phosphorylase, 8-amino-9-benzylguanine. Cancer 

Res 1986; 46:519–23. 

13. Stoeckler JD, Ryden JB, Parks RE Jr, Chu MY, Lim MI, Ren WY, Klein RS. Inhibitors of purine 

nucleoside phosphorylase: effects of 9-deazapurine ribonucleosides and synthesis of 5'-deoxy-5'-iodo-9deazainosine. 

Cancer Res 1986; 46:1774–8. 


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14. Tuttle JV, Krenitsky TA. Effects of acyclovir and its metabolites on purine nucleoside 

phosphorylase. J Biol Chem 1984; 259:4065–9. 

15. Gilbertsen RB, Scott ME, Dong MK, Kossarek LM, Bennett MK, Schrier DJ, Sircar JC. Preliminary 

report on 8-amino-9-(2-thienylmethyl) guanine (PD 119,229), a novel and potent purine nucleoside 

phosphorylase inhibitor. Agents and Actions 1987; 21:272–4. 

16. Cook WJ, Ealick SE, Bugg CE, Stoeckler JD, Parks RE Jr. Crystallization and preliminary X-ray 

investigation of human erythrocytic purine nucleoside phosphorylase. J Biol Chem 1981; 256:4079–80. 


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17. Ealick SE, Rule SA, Carter DC, Greenhough TJ, Babu YS, Cook WJ, Habash J, Helliwell JR, 

Stoeckler JD, Parks RE Jr, Chen SF, Bugg CE. Three-dimensional structure of human erythrocytic 

purine nucleoside phosphorylase at 3.2 Å resolution. J Biol Chem 1990; 265:1812–20. 

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18. Narayana SVL, Bugg CE, Ealick SE. Refined structure of purine nucleoside phosphorylase at 2.75 Å 

resolution. Acta Cryst D 1996; accepted. 

19. Babu YS, Refined structure of guanine: purine nucleoside phosphorylase at 2.8 Å resolution. In 

preparation. 

20. Koshland DE Jr. The key-lock theory and the induced fit theory. Angew Chem Int Ed Engl 1994; 

33:2375–8. 

21. Mohamadi F, Richards NGJ, Guida WC, Liskamp R, Lipton M, Caufield C, Change G, Hendrickson 

T, Still WC. MacroModel—An integrated software system for modeling organic and bioorganic 

molecules using molecular mechanics. J Comput Chem 1990; 11:440–67. 

22. Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C, Alagona S, Profeta S, Weiner P. New force 

field for molecular mechanical calculations simulations of proteins and nucleic acids. J Am Chem Soc 

1984; 106:765–84. 

23. Chang G, Guida WC, Still WC. An internal coordinate monte carlo method for searching 

conformational space. J Am Chem Soc 1989; 111:4379–86. 

24. Goodford P. A computational procedure for determining energetically favorable binding sites on 

biologically important macromolecules. J Med Chem 1985; 28:849–57. 

25. Montgomery JA, Niwas S, Rose JD, Secrist JA 3d., Babu YS, Bugg CE, Erion MD, Guida WC, 

Ealick SE. Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(arylmethyl) 

derivatives of 9-deazaguinine. J Med Chem 1993; 36:55–69. 

26. Burley SK, Petsko GA. Aromatic-aromatic interaction: a mechanism of protein structure 

stabilization. Science 1985; 229:23–8. 

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of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors. 

PNAS USA 1991; 88:11540–4. 






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6 

Structural Implications in the Design of Matrix-Metalloproteinase 

Inhibitors 

John C. Spurlino 

3-Dimensional Pharmaceuticals, Inc., Exton, Pennsylvania 

I. Matrix-Metalloproteinases 

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The matrix metalloproteinases (MMPs) are a family of ubiquitous enzymes that are involved in 

extracellular matrix degradation and remodeling. They are critical for the processes of morphogenesis 

and wound healing, but are also implicated in many human diseases including arthritis, metastasis, and 

cancer tumor growth [1, 2]. This family includes matrilysin, fibroblast collagenase (HFC), neutrophil 

collagenase (HNC), stromelysin 1 (HFS), stromelysin-2, stromelysin-3, gelatinases A and B, collagenase- 

3, and the membrane type MMP. In addition to the destructive involvement in diseases, MMPs play a 

critical role in the remodeling of the extracellular matrix [3]. 

The MMP enzyme family is part of the superfamily of metzincins. The metzincin superfamily is 

distinguished by a conserved zinc binding motif for the catalytic zinc and a Met-turn region [4]. The 

MMPs are unique in that they also contain a second structural zinc, however this zinc may be absent in 

the intact full-length enzyme [5]. The presence of one to four structural calcium ions has been detected 

in the MMPs that have been characterized to date. The importance of the zinc ions and at least one of the 

structural calcium ions to enzymatic activity has been proven [6]. 

The MMPs are secreted as inactive proenzymes, which are activated by proteolytic cleavage. Once 

activated they are subject to control by tissue inhibitors of metalloproteinases (TIMPs). It is the 

imbalance between the active enzymes and the TIMPs that leads to destructive tissue degradation that 

potent directed pharmaceuticals can overcome. These enzymes have been the target of 


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drug design since the late 1970s [7]. Batimastat, [{4-N-hydroxyamino}-2R-isobutyl-3S-{thienylthiomethyl} 

succinyl]-L-phenyl-alanine-N-methylamide, a potent nonspecific MMP inhibitor from 

British Bio-Tech, is now in Phase III trials. The information gained from current studies indicate that 

there is some efficacy in the treatment of disease states by MMP inhibitors [8–10]. 

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There is still much debate about whether a broad spectrum or directed MMP inhibitor is the best course 

of treatment for a variety of diseases, partly because the exact role of individual MMPs is still unclear. 

Both collagenase-3 and HFC are suspected to cause osteoarthritis [11]. It is currently believed that 

gelatinase and collagenase-3 have a role in breast cancer [12]. Gelatinase A and B have been implicated 

in hemorrhagic brain injury [13]. Gelatinase A and B, HFC, and stromelysin may all be involved in 

gastric cancer [14]. Matrilysin may be implicated in colon cancer [15]. Increased gelatinase A and B 

activity has also been seen in response to beta-amyloid production [16]. The role that individual MMPs 

play in causing these diseases, however, remains unclear. This uncertainty underscores the need to 

develop selective inhibitors of individual MMPs to ferret out the roles each play in the development of 

specific disease states. 

The MMPs consist of one or more structural domains (Figure 1). The first domain, the propeptide 

domain, confers a self-inhibitory action on the full-length MMP. The second domain contains the active 

site residues and is referred to as the catalytic domain. The catalytic domain is characterized by the 

conserved zinc-binding sequence (HEXGHXXGXXHS), which also contains the glutamate residue that 

is essential for activity [17]. The MMPs are activated by cleavage of the prodomain. All MMPs contain 

these first two domains. Matrilysin, the simplest of the MMPs, is an example of a two-domain enzyme, 

where the active enzyme consists solely of the catalytic domain. 

The remainder of the MMPs also contain a hemopexin-like domain connected to the catalytic domain by 

a proline-rich linker. This domain is involved in the interaction of the collagenases and stromelysins 

with collagen and, in the case of the collagenases, is essential for activity against collagen [18]. The 

cleavage of the proline-rich linker region in HFC and HNC is another route to control collagenase 

activity. The hemopexin domain of the gelatinases is not necessary for collagen binding, but may be 

involved in receptor recognition [19]. 

The gelatinases also contain a fibronectin-like insert in the catalytic domain that is involved in binding 

collagen [20]. The fibronectin domain has also been shown to be essential for elastolytic activity [21]. A 

structural picture of these additional domains is essential for an understanding of the mode of action for 

these larger MMPs, but is not necessary for a structure-based drug design strategy. Differences in the 

catalytic domains of the MMPs can be used to drive a targeted drug discovery effort. 


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Figure 1 

A ribbon model of the full-length collagenase structure (1fbl.pdb). The 

prodomain would precede and include the portion labeled in the figure. The 

catalytic domain is shown, with a highlighted region where the fibronectin-like 

domain of the gelatinases is inserted. 

II. 3-Dimensional Structure of MMPs 

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Catalytic domain structures for fibroblast collagenase [22–25], neutrophil collagenase [26,27], 

matrilysin [28], and stomelysin [29, 30] have all been determined and deposited in the Protein Data 

Bank [31]. The catalytic domains of MMPs, as seen in the archetypal collagenase structure (shown as a 

ribbon drawing [32] in Figure 1), consist of an upper 5-stranded β sheet flanked by two α helices on one 

side of the active site cleft and a long loop that contains the Met-turn flanked by a single α helix on the 

other side of the cleft. 

The active-site groove as seen in the solvent-accessible surface [33] is an obvious structural feature 

(Figure 2). The top wall of the cleft (as seen in Figure 2) is formed by the top strand of the β sheet and 

the loop that contains the calcium binding site. The lower wall of the cleft is formed from the residues 

on either side of the Met-turn. These residues can be considered as an interrupted strand which, together 

with the substrate, complete the twisted β sheet of the catalytic domain. The bottom of the cleft is 

formed by the second helix, which 


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Figure 2 

The accessible surface of HFC with a modeled substrate from human collagen 

showing the binding sites. 

contains the HExxH motif, the catalytic zinc, and the S1' pocket. Substrates bind in an extended 

conformation that approximates an antiparallel strand. The cleft, however, is not large enough to 

accommodate a triple helix collagen molecule. 

Page 174 

A structure for the full-length active porcine synovial collagenase [34] has been determined. The 

structure of the catalytic domain of this full-length enzyme is equivalent to the structures of the isolated 

catalytic domains of HFC, HNC, and matrilysin. The flexible linker domain between the catalytic and 

hemopexin domains is disordered and the orientation of the hemopexin domain in the structure offers no 

real clue as to the mode of action for the full-length collagenases. Furthermore, the matrilysin structure 

of the full-length active enzyme has almost identical secondary structural features (a Ca overlap of 


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Figure 3 

The sequence alignment of MMPs with the catalytic domain region highlighted. 

The residues that line the subtrate pockets are marked: S3 (3), S2 (2), S1 (1), S1 

(*), S2' (@), and S3' (#). The highlighted catalytic domain alignment was dominated by 

the structural alignment of the determined structures of HFC, HNC, and matrilysin. 


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0.43 Å) as the catalytic-domain structure of HFC. This demonstrates that the absence or presence of the 

hemopexin domain does not affect the overall structure of the catalytic domain. 

The sequence homology of the catalytic domains of the collagenases is 62%. This can be extended to the 

other members of the MMP family as seen in Figure 3. An understanding of the structural features of the 

target enzyme is essential for structure-based drug design. In this example we will be looking at 

inhibiting MMPs by binding to the active site. The numbering system used throughout this chapter will 

be in regard to the HFC sequence used in 1hfc.pdb. 

III. Surface Features 

The surface features of matrilysin, fibroblast collagenase, and neutrophil collagenase are all similar 

(Figure 4). The active-site groove can be plainly seen on the surface: two main pockets punctuated by 

the active-site zinc. Modeling 

Figure 4 

The accessible surfaces of HFC (a), HNC (b), and matrilysin 

(c) are shown with a bound P'-side hydroxamate inhibitor. 


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studies show that there is not sufficient room in the S1'-S3' cleft to accommodate the native coiled triple 

collagen bundle. 

The active site consists of a series of subsites on either side of the catalytic zinc. These subsites are 

numbered starting at the catalytic zinc and preceding from N to C as S1', S2', etc., corresponding to the 

residues (P1', P2', etc.) of the substrate that is bound (Figure 2). Likewise the subsites are numbered S1, 

S2, etc. outward from the other side of the catalytic zinc. 

While the interaction of the substrate (and the inhibitor) with the catalytic zinc is the most important 

interaction, the remainder of the substrate (inhibitor) also forms hydrogen bonds with residues from the 

top strand of the β sheet and the loop region posterior to the Met-turn. These interactions with the 

substrate in the binding pockets of the MMPs are the prime targets for engineering specific MMP 

inhibitors. An in-depth understanding of the differences of the properties of these pockets in the different 

MMPs and the interactions of specific residues within these pockets is essential for structure-based 

design of inhibitors. 

IV. Main-Chain Substrate Interactions 

Most of the hydrogen bonds between the substrate and the MMP occur with the top strand of the β sheet. 

The P3 residue does not make any direct hydrogen bonds with the MMP. The P2 residue makes two 

hydrogen bonds with residue 184, which is a conserved alanine residue in all the aligned MMP 

sequences. Residue 183 is a conserved histidine, which is bound to the structural zinc, further stabilizing 

the conformation of the top strand. The carbonyl oxygen of P1 is liganded to the catalytic zinc. The lefthand 

side of the substrate is thus held in place by only two hydrogen bonds with the enzyme and one 

interaction with the catalytic zinc. Although the P3 residue does not make any hydrogenbond 

contributions to substrate binding, it is essential for catalytic activity [43]. 

The right-hand side of the substrate is held much tighter. The P1' residue's carbonyl oxygen makes a 

hydrogen bond with the amide nitrogen of residue 181, which is a conserved leucine residue. The amide 

nitrogen of the P1' residue is hydrogen bonded to the conserved alanine-182 carbonyl oxygen. The P2' 

substrate residue is held in place by hydrogen bonds to proline 238 and tyrosine 240, two more 

conserved residues. The amide nitrogen of P3' makes a hydrogen bond with the carbonyl oxygen of 

residue 179, the only nonconserved residue, making a main-chain interaction with the substrate. 

The use of conserved residues to maintain the main-chain interactions along the substrate backbone 

makes differentiation of the MMPs through these interactions difficult. Instead, differences in the 

regions where the side chains of the substrate interact can be used to drive the discovery of specific 

MMP inhibitors. The hydrogen-bonding pattern also indicates that right-hand side (P'- 


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side) inhibitors will bind with greater affinity. Indeed, most of the structures of MMPs were determined 

with right-hand side inhibitors, and most of the pharmaceuticals currently in development are also righthand 

side binders. A closer look at the binding pockets themselves also demonstrates the reasons for the 

preference of researchers for the right-hand side. 

V. Substrate-Binding Pockets 

The nonprimed or left-hand side of the cleft consists of a large shallow depression. The S1 pocket 

consists of a shallow ridge that complements the glycine residue of the collagen strands. Most of the 

interactions with the glycine residue are brought about due to its interaction with the catalytic zinc. 

Asparagine 180 approaches the P1 residue in HFC. Crystallographic evidence indicates an interaction of 

the thiophene ring of batimastat via electrostatic interactions of the p orbitals and the catalytic zinc and 

the possibility of a water-mediated hydrogen bond to the carbonyl O of residue 184 [35]. Larger 

substituents can be accommodated in regions adjacent to the P1 pocket, possibly in the large pocket 

above the S1 site (see Figure 2). Increased potency was noted for several compounds with cyclic imido 

P1 substituents that could bind here [2]. 

The S2 pocket is a large shallow depression offering no real binding cavities. One side of the pocket is 

made up from the conserved histidine at position 228 and the main chain from residue 227. The bottom 

of the pocket is formed by histidine 222. Both of these histidines are liganded to the catalytic zinc. The 

other side consists mostly of the residue 186 side chain with some hydrogen-bonding contacts possible 

from the tip of the glutamine side chain in the case of HFC and HNC. 

The S3 pocket offers a shallow cavity to bind the conserved proline. The proline residue of the substrate 

would lie between the side chains of His 183, Phe 185, and Ser172 [36]. Residues 183 and 185 are 

conserved among the MMPs with the minor exception of a tyrosine replacing phenylalanine 164 in 

stromelysin. Residue 172 shows some variability among the MMPs existing as a serine in HNC and 

HFC and a tyrosine in the remainder of the aligned MMPs. 

The primed or right-hand side of the active site exists as a narrow canyon with a large well at the 

beginning. The S1' pocket is a narrow, deep cavity providing an ideal binding site for inhibitor design. 

The S1' pocket is the most significant feature of the surface, extending as a tunnel completely through 

the enzyme in the case of neutrophil collagenase and stromelysin. This feature makes the S1' pocket an 

ideal candidate for use in designing an inhibitor with specificity for HNC, HFC, or HFS. 

The volumes of the S1' pockets vary greatly. Matrilysin has the smallest S1' pocket at 111 Å 3. The 

fibroblast collagenase pocket is not much larger at 


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Figure 5 

(a) A cut away view of the S1' pocket of HFC showing the termination of 

the pocket by Arg214. Matrilysin also has a truncated pocket. (b) A cut away 

view of the S1' pocket of HNC showing the clear path through to the other 

side of the enzyme. The gelatinases are also likely to have large extended S1' 

pockets. 

123 Å 3. The pocket of the neutrophil collagenase that travels through the enzyme has a volume of 305 

Å 3. Figure 5 demonstrates the differences in the relative sizes of the S1' pockets of HFC and HNC. 

Stromelysin has a pocket that should be of similar size as that of neutrophil collagenase [21]. The 

gelatinases and collagenase-3, based on sequence alignment, should also posses long tunnel-like S1' 

pockets. 

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The residues that line the S1' pocket are mostly hydrophobic residues (see Figure 3) and show a 

remarkable overall similarity. The specificity of a number of inhibitors of MMPs can be linked to 

differences in the S1' pockets. The S1' pocket of HFC is terminated by arginine 214, while matrilysin 

has a tyrosine residue that accomplishes the same thing. The remainder of the aligned MMPs have 

leucine residues at that position. In addition the conformation of the leucine residue is swung back, 

forming the tunnel. There are three additional residues that are significant in their differences within the 

S1' pocket: residues 239 and a two-residue insert, relative to HFC, after residue 242. These residues 


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form the lower end of the tunnel. The major effect of the different residues found here is on the diameter 

of the exit hole in the S1' tunnel. However, there are some residues that could be targeted for hydrogenbond 

formation. 

The S2' binding cavity is a narrow cleft that can easily accommodate a peptide backbone, but with no 

room for a side chain. The interaction of the P2' side chain is made with the exterior surface of the 

enzyme. The S2' site is exposed to solvent and presents two possible interaction sites for bound 

inhibitors that are related by a rotation about χ1. These sites consist of residues 179–180 on one side and 

residues 238–240 on the other. 

The S3' binding cavity opens up out of the canyon and consists almost entirely of surface interactions. 

The side chain of P3' interacts with residues 210 and 240, which are mostly conserved tyrosine residues. 

Additional interactions could be formed with some of the additional residues found in the insertions 

after residue 242. 

VI. Structure-Based Design 

Structure-based drug design is an iterative process that starts with a lead compound, a structural model 

of the target, and a structure-activity relationship 


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(SAR) model. The lead compound can come from compound screening, previously discovered 

inhibitors, or it can be based on a known substrate. The model can be obtained from x-ray 

crystallography, high-field nuclear magnetic resonance spectroscopy, or from homology-based model 

building. Inhibitor structures are developed and docked into the model of the binding site of interest, 

typically the active site of the enzyme. The interactions of the inhibitor-enzyme complex are evaluated 

and ranked. The most promising compounds are then synthesized and tested. Based on the results of the 

testing, additional enzyme-inhibitor structures are determined, the SAR model is updated, and the 

process beings again. 

As a model case of structure-based drug design for MMPs we will look at the design of a right-handed 

inhibitor based on the x-ray structures of HFC and HNC. 

VII. Zinc-Binding Group 

The design of active-site inhibitors based on the natural substrate of the collagenases has produced a 

variety of zinc-binding groups to anchor the inhibitor to the catalytic zinc. These group include 

hydroxamates, thiols, phosphorous acid derivatives (phosphinate, phosphonate, phosphoramidate), and 

carboxylates. The selection of a suitable zinc-binding group has been studied in depth [37–40]. The most 

potent zinc-binding group found for the collagenases to date is the hydroxamate. 

The structural comparison of hydroxamate, carboxylate, and sulfodiimine in matrilysin provided 

information on the contribution of the zinc ligand to the overall potency of the inhibitor [19]. The 

potency of the zinc-bind group can be directly related to the number of bonds in which it is involved for 

this instance. The hydroxamate is the perfect bidentate ligand to the zinc with both oxygens being within 

2.2 Å of the zinc. The hydroxamate group also is involved in hydrogen bonds with Glu219 and the 

carbonyl oxygen of Ala182. The carboxylate group is also a bidentate ligand to zinc, however the 

oxygens are not equidistant from the zinc. The carboxylate forms only one additional hydrogen bond 

with Glu219 of the enzyme. The sulfodiimine bound to matrilysin is a monodentate zinc ligand and the 

weakest of the zinc-binding groups. The comparison of several inhibitors with both carboxylate and 

hydroxamate zinc-binding groups demonstrates this property in fibroblast and neutrophil collagenases as 

well. While the potency of inhibitors with different zinc-binding groups maps directly to the number of 

bonds formed by the zinc-binding group, some of the increase in potency of the hydroxamate group over 

charged groups most likely is a result of the decreased energetic cost of the desolvation of the neutral 

hydroxamate. 


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VIII. S1' Interactions 

Matrix metalloproteinase structural studies of the P'-side inhibitors to date show a common set of 

inhibitor-enzyme interactions. This can be attributed primarily to the strong directional zinc-binding 

forces. Further stabilizing forces from the backbone hydrogen-bonding patterns common to a β sheet 

allow for minor adjustments due to the zinc interactions to be made while maintaining a common 

pharmacophore. 

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The fairly rigid constraints of binding based on the known hydroxamate inhibitors allows the use of 

computer-aided modeling to play a useful role in the design of specific MMP inhibitors. Exploration of 

the S1' pocket was carried out by docking the P1' group within the cavity followed by rounds of energy 

minimization. In order to maintain the integrity of the MMP structure several limitations were used. All 

MMP atoms that are greater than 8 Å from the docked inhibitor were frozen. The Cα atoms of all 

residues within 8 Å of the inhibitor were initially constrained to their original position by a 20 kcal/mol 

Å 2 force constant that was gradually relaxed to 1 kcal/mol Å 2. Strong initial constraints were also placed 

on the conserved hydrogen bonds and zinc-ligand interactions. 

This method has several advantages and disadvantages over the common static treatment of target 

structures. The advantages are that it more closely approximates the actual dynamic state of protein 

structure and is not computationally prohibitive. The disadvantages include the increased computational 

cost over a static enzyme target and the fact that gross structural rearrangements can still not be 

accounted for. 

The structural similarity of the active site of the MMP family allows structure-based drug design to 

effectively be used for those enyzmes whose structures have not been determined yet. Examination of 

the S1' cavities of HFC and HNC clearly indicates a path for designing inhibitors that bind preferentially 

(Figure 5). The cavity of HFC is mostly filled by the leucine side chain of the preferred substrate, while 

the S1' pocket of HNC [26] and HFS [30] remains unfilled. The sequence similarities of the gelatinases 

with HNC indicate that they too can accommodate a much larger P1' group. 

A series of compounds was designed to explore filling the long S1' tunnel of gelatinase B [41, 42]. The 

optimum length for binding to gelatinase B was found. There was an increase in affinity for the phenolic 

ethers versus the benzylic ethers of the same overall length for binding to gelatinase B, but there was no 

preference seen in binding to HFS. Surprisingly the phenolic ethers also showed potent binding to HFC. 

The size of the bottom of the S1' pocket and the differences in the preferred torsion angle for the bond to 

the aromatic ring both play a role in this differential binding. 


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IX. S2' Interactions 

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The interactions at the S2' site display an interesting structure-activity relationship. While the 

interactions do not include any hydrogen bonds, favorable stacking interactions do play a significant role 

in binding. A glycine residue at P2' results in a loss of three orders of magnitude in potency for 

otherwise identical inhibitors [41]. There is a preference for an aromatic ring at this position in natural 

peptide substrates [43]. Structural considerations also allow the placement of a t-butyl group here. The 

potency of a t-butyl glycine P2' is less than that of a phenylalanine (Table 1), but the expected gain in 

bioavailability brought about by shielding the amide bond from solvation effects should compensate for 

the loss in potency. 

X. Conclusions 

High resolution x-ray crystallographic structure determination is an essential step in structure-based drug 

design. The need for high resolution structural data 


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Figure 6 

(a) The pocket of HFC with a leucine residue in the S1' pocket. 

(b) The volume of the S1' pocket of HFC can change when there are 

favorable interactions. The binding of the (CH 2) 4OPh can cause 

Arg214 to move, thereby making room for the extended side chain. 


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to develop an appropriate SAR is demonstrated in the case of the inhibitors shown in Table 1. The 

unusual potency of a benzylic ether for HFC was unexpected and would not have been predicted with 

standard docking and minimization studies (Figure 6). 

Page 186 

The differences in the potency of the various 4-substituted analogs of inhibitor 10 against HFC suggest ππ 

interactions are the driving force for the displacement of arginine 214. The electron-withdrawing Cl 

substitution decreases the affinity for HFC while increasing the affinity for HFS. The leading 4-pentyl 

group can not effectively interact with arginine 214 in HFC; therefore, the rearrangement does not 

occur. The open channel that is present in HFS and gelatinase B presents no such impediment to binding 

and the affinity is essentially equal to the unsubstituted form. 

Not all structure-based design experiments are successful. Attempts to displace the arginine residue that 

caps the S1' pocket of HFC by forming a salt link with a P1' carboxylate or hydroxyl moiety were 

unsuccessful [42]. However, these failed attempts offer some redeeming features in the refinement of 

parameters that can be used to evaluate the energetic potentials for displacing buried water molecules as 

well as the inherent desolvation energies for polar compounds. 

The outlook for structure-based drug design is good. The advancement in both x-ray area detectors and 

computer hardware will make the determination of a series of compounds bound to a target enzyme for 

use in SAR development commonplace in drug-discovery efforts. The continued explosion of structural 

studies will lead to an increased understanding of the dynamics of protein interactions, which will, in 

turn, lead to better docking algorithms. The combination of structural information and greater 

computational power will also make more accurate predictions of protein—ligand interactions possible. 

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7 

Structure—Function Relationships in Hydroxysteroid Dehydrogenases 

Igor Tsigelny and Michael E. Baker 

University of California, San Diego, La Jolla, California 


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Steroid hormones regulate a multitude of physiological processes in humans. Androgens and estrogens 

regulate sexual development and reproduction; glucocorticoids are important in the response to stress; 

vitamin D is important in bone growth; progestins are important for a viable fetus during pregnancy; 

mineralocorticoids regulate sodium and potassium balance to maintain normal blood pressure. 

Moreover, the growth of some breast and prostate tumors depends on steroids. With this multitude of 

medically important steroid-dependent actions, much research has gone into understanding their mode 

of action, with most of the effort concerned with the receptors that mediate the actions of steriods. 

A. High Blood Pressure and 11β-Hydroxysteroid Dehydrogenase 

It is only in the last decade that the role of hydroxysteroid dehydrogenases (Figure 1) in regulating the 

actions of steroids has been appreciated [1–6]. This mechanism for regulating steroid hormone action 

was uncovered in several laboratories studying various aspects of high blood pressure. One source was 

the study in the 1970s that identified the syndrome, Apparent Mineralocorticoid Excess (AME), a 

genetic disease that results in high blood pressure in children [7–10]. Also important is the work from 

laboratories investigating paradoxes in the mechanism of action of aldosterone in the kidney [1–4,9,10]. 

These studies identified 11β-hydroxysteroid dehydrogenase (11β-HSD) as a key enzyme. 


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Figure 1 

Reactions catalyzed by 11β-hydroxysteroid and 17β-hydroxysteroid 

dehydrogenases. (a) 11 β-hydroxysteroid dehydrogenase type 1, an NADPH-dependent 

enzyme, catalyzes the conversion of the inactive steroid, cortisone, to cortisol, which is 

the biologically active glucocorticoid. 11β-hydroxysteroid dehydrogenase type 2, an 

NAD + -dependent enzyme, catalyzes the reverse direction. (b) 17β-hydroxysteroid 

dehy-drogenase type 1, an NADPH-dependent enzyme, catalyzes the reduction of 

estrone to estradiol. Type 2, an NAD + -dependent enzyme, catalyzes the oxidation of 

estradiol to estrone. Type 3, an NADPH-dependent enzyme, catalyzes the reduction of 

androstene dione to testosterone. Type 4, an NAD + -dependent enzyme, catalyzes the 

oxidation of estradiol to estrone, and androstenediol to dehydroepiandrosterone. 


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The enzyme 11β-HSD interconverts the active glucocorticoid cortisol and cortisone, an inactive 

metabolite (Figure 1a). By the oxidation of cortisol to cortisone, 11β-HSD prevents glucocorticoids 

from deleterious actions in certain cell types. For example, excess glucocorticoids in Leydig cells inhibit 

testosterone synthesis [3,11]. Expression of 11β-HSD in Leydig cells prevents this effect of 

glucocorticoids. In this way, 11β-HSD is important in androgen action. The enzyme 11β-HSD is also 

important in aldosterone action in the distal tubule of the kidney. Glucocorticoids have high affinity for 

the mineralocorticoid receptor [12] and can stimulate the mineralocorticoid response—uptake of sodium 

from urine—one effect of which is to increase blood pressure. Local expression of 11β-HSD in the 

distal tubule prevents this effect of glucocorticoids. The steroid aldosterone, which is not metabolized by 

11β-HSD, can bind to the mineralocorticoid receptor and regulate sodium and potassium balance. Thus, 

11β-HSD has an important role in regulating the biological actions of both glucocorticoids and 

mineralocorticoids. 

As would be expected, interference with 11β-HSD activity due to a mutation [13,14] or by an inhibitor 

such as licorice (Figure 2) [1–3,15] has a variety of physiological effects including high blood pressure 

due to mineralocorticoid actions of glucocorticoids in the kidney's distal tubule. Thus, studies to unravel 

genetic hypertension in children and the actions of aldosterone in the kidney yielded the general insight 

that, at specific times, altered expression of 11β-HSD in specific tissues is an important mechanism for 

regulating glucocorticoid, mineralocorticoid, and androgen action. 

A similar mechanism has been found for 17β-hydroxysteroid dehydrogenase (17β-HSD), the enzyme 

that regulates the concentrations of estradiol and testosterone in human [5,16,17] (Figure 1b). Genetics 

diseases associated with mutations in this enzyme lead to developmental abnormalities [18]. Enzymes 

that regulate the concentrations of retinoids [19] and prostaglandins [20] may also have a similar role 

[6]. 

B. Multiple Divergent 11β-Hydroxysteroid and 17β-Hydroxysteroid Dehydrogenases 

The cloning and sequencing of 11β-HSD [21–25] and 17β-HSD [16–18,26] revealed two 11β-HSDs and 

four 17β-HSDs with very different sequences 


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Figure 2 

Structure of licorice and carbenoxolone. Glycyrrhizic acid, a constituent of 

licorice extract, is found in the root of Glycyrrhiza glabra. The glycosidic group at C3 is 

cleaved by bacteria in the small intestine to form glycyrrhetinic acid, the compound that 

inhibits 11β-hydroxysteroid dehydrogenase. Carbenoxolone, a water soluble synthetic 

analog of glycyrrhetinic acid, is widely used to regulate 11β-HSD in vitro and in vivo. 

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(Figure 3, Table 1). This was surprising, as one would expect the two 11β-HSDs to be similar because 

they recognize the same substrates. Instead, the two 11β-HSDs have less than 20% sequence identity, 

after including gaps in the alignment (Table 1). The same degree of sequence divergence is found in the 

four 17β-HSDs [6]. This sequence divergence is reflected in differences in their catalytic properties. For 

example 11β-hydroxysteroid dehydrogenase-type 1 (11β-HSD-1) is an NADPH-dependent enzyme that 

converts cortisone to cortisol, and 11β-hydroxysteroid dehydrogenase-type 2 is an NAD +-dependent 

enzyme that oxidizes cortisol to cortisone. The enzyme 17βHSD-1 is an NADPH-dependent enzyme 

that converts estrone to estradiol, and 17βHSD-2 is an NAD +-dependent enzyme that oxidizes estradiol 

to estrone and testosterone to androstenedione. 


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Figure 3 

Alignment of 11β-and 17β-hydroxysteroid dehydrogenases. As seen in this 

Figure and Table 1, the sequences of the two 11β-HSDs and four 17β-HSDs are very 

divergent. Boxes denote sites where either 5 or 6 residues are conserved, which are 

likely to be functionally important. 

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There is considerable interest in understanding the structural bases for these differences because this 

information would be very useful in designing steroids and other compounds to selectively regulate the 

activity of one or more steroid dehydrogenases as a means of treating hormone-responsive diseases. 

There is precedent for this kind of treatment in the use of licorice extract from the root of the plant 

Glycyrrhiza glabra [15,27] to treat Addison's disease, 


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Figure 3 

(Continued) 

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which is characterized by insufficient levels of cortisol. Licorice inhibits 11β-HSD-2, which raises the 

circulating levels of cortisol and provides some relief from the symptoms of Addison's disease. This use 

of licorice is an example of a plant-derived compound having important uses in mammalian steroid 

hormone physiology and indicates another reason why elucidation of the structure of steroid 

dehydrogenases is of medical interest. Plants contain a wide variety of compounds, many of which have 

been purified and had their structures determined; however, we don't know which of these compounds 

inhibit steroid 


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Table 1 Percent Identity Between Hydroxysteroid Dehydrogenase Sequences Shown in Figure 3 

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11β-HSD-2 17β-HSD-2 17β-HSD-3 17β-HSD-1 11β-HSD-1 17β-HSD-4 

11β-HSD-2 0.00 46.1 20.3 28.6 20.6 18.1 

17β-HSD-2 0.0 21.1 21.2 17.7 18.9 

17β-HSD-3 0.0 19.1 19.6 17.5 

17β-HSD-1 0.0 21.1 20.4 

11β-HSD-1 0.0 17.1 

17β-HSD-4 0.0 

dehydrogenases. Knowledge of structure-activity relationships for the binding site on steroid 

dehydrogenases will be helpful in identifying novel compounds from plants and other sources that could 

be useful in regulating steroid dehydrogenases. 

At this time, we are just beginning to work on this ambitious goal. Structural information is limited. The 

3-D structure of 17β-HSD type 1 has been determined [28], but without the steroid or cofactor in the 

binding site. Fortunately, 11β-HSD and 17β-HSD belong to a large family of enzymes that are called 

short-chain alcohol dehydrogenases [29–31] or sec-alcohol dehydrogenases [32]. The structures of 

dehydrogenase homologs in bacteria, plants, and animals have been determined [33–37] and we used 

them as templates for modeling 11β-HSD and 17β-HSD [38,39]. There also is information about the 

effects of mutations on catalytic activity in 11β-HSD-1 [40] and 17β-HSD-1 [41] and in homologs, 

especially for Drosophila alcohol dehydrogenase (ADH) [42–48]. Together they enable us to begin to 

understand the relationship between structure and function in hydroxysteroid dehydrogenases, as we 

discuss in this chapter. 

II. Methods 

A. Molecular Modeling 


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Important for the validity of the models that we constructed is the evidence from models of other 

proteins indicating that two proteins can have as little as 20 to 25% sequence identity and still have very 

similar 3D structures, especially in α helices and β stands [49–52]. Variation is found in the loops and 

coiled structures. A relevant example for this chapter is the comparison of the tertiary structure of rat 

dihydropteridine reductase [33] and Streptomyces hydrogenans 20β-hydroxysteroid dehydrogenase [34]. 

As noted by Varughese et al. [33], despite less than 20% sequence identity between dihydropteridine 

reductase and 


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Figure 4 

Amino acids important in cofactor and catalysis in human 11b-hydroxysteroid 

dehydrogenase types 1 and 2. (a) 11b-HSD type 1. Preference of 11b-HSD type 1 

for NADPH resides in lysine-44 and arginine-66, which have positively charged side 

chains that stabilize the binding of the 2'-phosphate on NADPH. These residues also 

counteract the repulsive interaction between glutamic acid 69 and the phosphate group. 

(b) 11b-HSD type 2. Preference of 11b-HSD type 2 for NAD+ is due to favorable bonds 

with aspartic acid-91, serine-92, and threonine-112. Moreover there is a coulombic 

repulsion between aspartic acid-91 and NADP+, which destabilizes binding of NADP+ 

11b-HSD type 2 lacks a nearby amino acid with a positively charged side chain that 

could diminish the repulsive interaction between NADP+ and aspartic acid-91. Also 

shown are threonine residues that could hydrogen bond 

to nicotinamide's carboxamidemoiety. 


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20β-hydroxysteroid dehydrogenase, the root mean square deviation for the two tertiary structures is 2 Å 

over 160 C α carbon atoms. 

We aligned human 11β-HSD-1 [21] and 11β-HSD-2 [22] with S. hydrogenans 20β-hydroxysteroid 

dehydrogenase and Escherichia coli 7α-hydroxysteroid dehydrogenase [37] for 3D modeling. Human 

11β-HSD has extra segments at the amino terminus and carboxyl terminus. Previously reported 

alignments [30,31,53] were used to find the core structure consisting of about 225 residues that are 

structurally similar to the template. The first 190 residues of the 255 residues are reasonably well 

conserved among the hydroxysteroid dehydrogenases. Alignment of the C-terminal 65 residues is less 

certain as this part contains gaps and insertions. Fortunately, the core 190 residues contains the catalytic 

site and the cofactor binding site. We also superimposed the two 11β-HSD structures on mouse carbonyl 

reductase [37]. The 11β-HSD 3D structures superimpose nicely on α helices E and F and other helices 

and strands that are important in binding of cofactor and substrate. Then, we extracted NADPH from 

carbonyl reductase and NAD + from 7α-hydroxysteroid dehydrogenase and inserted the cofactors into 

the structures of the two 11β-HSDs. 

The α helix F in 17β-HSD-1 [16], 17β-HSD-2 [17], 17β-HSD-3 [18], and porcine 17β-HSD-4 [26] was 

constructed by modeling on α helix F in 20β-hydroxysteroid dehydrogenase. Comparisons with other 

3D structures [33–37] have demonstrated that this α helix is highly conserved. The modeled dimers 

were not minimized as a dimer complex to avoid the artifactual adjustment of α helix F side chains. 

The Homology program (Biosym Technologies, Inc., 1995) was used to model a 255-residue segment of 

11β-HSD and α helix F of 17β-HSD on the S. hydrogenans 20β-hydroxysteroid dehydrogenase 

template. To produce the final model (Figures 4 and 5) this program finds an optimal configuration of 

the residues when arranged in the template structure by minimizing unfavorable interactions between 

amino acid side chains. The side chains of each monomer were then minimized (1,000 iterations of the 

conjugate gradient) using the Discover program (Biosym Technologies Inc., 1995). 


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III. Results and Discussion 

Figure 5 

Structure of α helix F interface of human 11β-hydroxysteroid dehydrogenase types 1 

and 2. The α helix F part of the dimer interface on 11β-HSD-1 and -2 is shown along 

with side chains of the highly conserved tyrosine and lysine residues and other residues 

that are oriented into the cavity that binds substrate and nucleotide cofactor. 

A. NADPH Binding Site on 11β-Hydroxysteroid Dehydrogenase Types 1 and 2 

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Several lines of evidence—sequence analysis, mutagenesis studies, and the solved 3D structure of 

homologs of 11β-HSD—indicate that the nucleotide binding site in these enzymes has many similarities 

to that in other classes of dehydrogenases. For many dehydrogenases, the nucleotide binding domain 


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consists of a β strand α helix, β strand in a fold that provides a hydrophobic pocket for the adenosine 

monophosphate (AMP) part of the nucleotide cofactor [51,54,55]. In short-chain alcohol 

dehydrogenases, this βαβ fold is at the amino terminus. The turn between the first β strand and the α 

helix is a glycine-rich segment of the form Gly-X-X-X-Gly-X-Gly. This glycine-rich segment forms a 

hydrophobic pocket that allows close association of the AMP part of the cofactor. 

However, this glycine-rich segment has other functions in short-chain alcohol dehydrogenases. Tanaka 

et al. [37] and our 3D modeling [60] indicate that this glycine rich segment has an important role in 

cofactor specificity and binding of the nicotinamide moiety to 11β-HSD. 

B. 11β-HSD-1 Preference for NADPH 

Figure 4 shows our 3D model of human 11β-HSD types 1 and 2. These models identify residues 

important in preference of 11β-HSD-1 for NADPH and 11β-HSD-2 for NADH. In 11β-HSD type 1, 

lysine-44 and arginine-66 have favorable coulombic interactions with the 2'-phosphate on NADP + that 

stabilize binding (Figure 4a). Moreover, their positively charged side chains compensate for the negative 

interaction between glutamic acid-69 and the 2'-phosphate group. Tanaka et al. [37] found a similar 

function for lysine-14 and arginine-39 in the preference of mouse carbonyl reductase for NADPH. 

C. 11β-HSD-2 Preference for NAD+ 

The 3D structure of 11β-HSD-2 shows that NAD + has stabilizing interactions between the ribose 

hydroxyl and aspartic acid-91, serine-92, and threonine-112. Replacement of NAD + with NADP + 

reveals a coulombic repulsion between the 2'-phosphate group and aspartic acid-91. However, 11β-HSD 

type 2 lacks a nearby amino acid with a positively charged side chain that could compensate for the 

negative charge on aspartic acid-91. This explains the preference of 11β-HSD-2 for NAD +. 

D. Amino Acids Important in Binding the Nicotinamide Ring and Carboxamide Moiety 

Both 11β-HSD types 1 and 2 contain residues in the C-terminal half that interact with the nicotinamide 

ring and carboxamide moiety to limit rotations about the N-glycosidic bond. These intersections are 

important in positioning the cofactor for proS hydride transfer at C4. 

In 11β-HSD-1, cysteine-213 stabilizes the nicotinamide ring; threonine-220 and threonine-222 stabilize 

the carboxamide moiety. In 11β-HSD-2, there are more interactions: proline-262, phenylalanine-265, 

threonine-267, serine- 


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269, and valine-270 are close to either the nicotinamide ring or the carboxamide moiety. In addition, the 

face of the side chain of phenylalanine-94 is below the nicotinamide ring and its carboxamide group. 

This latter interaction is unusual because phenylalanine-94 is between the two canonical glycine 

residues in the βαβ fold, which is usually thought of as interacting mainly with the AMP part of the 

cofactor. In 11β-HSD-2, there is an interesting configuration of amino acids with aromatic side chains 

that are below the nicotinamide ring and which provide a hydrophobic cushion for NAD +. 

E. Catalytic Site 

Comparison of 11β-HSD-1 with homologs identifies tyrosine-183 and lysine-187 as being highly 

conserved residues. Mutagenesis of these residues [40] and the homologous tyrosine and lysine in 17β- 

HSD-1 and Drosophila alcohol dehydrogenase [44,45] shows that these residues are important for 

catalytic function. The 3D model of 11β-HSD-1 presented in Figure 4 shows that tyrosine-183 is 3.6 Å 

from the nicotinamide C4, where hydride transfer occurs. Similarly, in 11β-HSD-2, tyrosine-232 is 4 Å 

from C4 on NAD +. Their positions support the notion that tyrosine is the catalytically active residue. 

However, a problem with this model is that the pKa of tyrosine is about 10, which would make this 

residue a poor nucleophile at neutral pH. To resolve this problem for the homologous tyrosine in 

Drosophila alcohol dehydrogenase, Chen et al. [44] proposed that the pKa of tyrosine is lowered by a 

nearby positively charged lysine. The 3D structure of the two 11β-HSDs shows that lysine-187 and 

lysine-236 are close to the proposed catalytically active tyrosine residues, which supports the hypothesis 

of Chen et al. [44]. 

F. Dimer Interface and the Catalytic Site 

Most short-chain alcohol dehydrogenases are active as either dimers or tetramers. Analysis of rat 

dihydropteridine reductase by Varughese et al. [33] indicates that the dimer interface consists of α helix 

E and α helix F from each monomer arranged in a four α helix bundle, a structure in which the 

hydrophobic surfaces on the helices form a core that yields very stable structure in a wide variety of 

proteins [56–59]. A four-helix bundle also appears to stabilize S. hydrogenans 20β-hydroxysteroid 

dehydrogenase, a tetrameric enzyme [34]. The α helix F contains the conserved tyrosine and lysine 

residue, which adds a constraint to changes in the sequence of this helix. It has at least two functions: 

stabilizing the dimer and orienting tyrosine and lysine and other residues for optimal interaction with 

substrate and nucleotide cofactor. 

The role of a specific site on the outer hydrophobic surface of α helix F in dimerization was suggested 

recently when a Drosophila ADH mutant that does 


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not form stable dimers was sequenced [48]. This ADH mutant has alanine-159 replaced with threonine. 

A 3D model of Drosophila ADH shows alanine-159 on the opposite surface of α helix F from tyrosine- 

153 and lysine-157 [48]. Alanine-159 along with alanine-158 form a hydrophobic anchor that stabilizes 

the dimer interface. These two residues of ADH and the homologous residues in other short-chain 

alcohol dehydrogenases have been overlooked in sequence analyses because they are not absolutely 

conserved. In fact, at least five amino acids are found in these positions among the different sec-alcohol 

dehydrogenases. 

G. Dimer Interface in 11β-and 17β-Hydroxysteroid Dehydrogenases 

Because α helix F at the dimer interface also contains the catalytic tyrosine and the nearby lysine 

residue, any structural analysis of the catalytic site must also consider the structure of this part of the 

dimer interface. For this reason, we modeled α helix F on 11β-HSD-1 and -2 and 17β-HSD-1, -2, -3, 

and -4 to gain an insight into stabilizing interactions and how they may affect the catalytic site. 

H. Human 11β-HSD-1 

Figure 5 shows the modeled structure for the α helix F interface in human 11β-HSD-1, in which 

phenylalanine-188 and alanine-189 form an anchor. Alanine-189 is 3.5 Å and 4.7 Å from alanine-189 

and alanine-185, respectively, on the other subunit. The phenylalanine-188 side chain is 3.2 Å from 

glycine-192. There is a hydrogen bond between serine-185 and serine-196, which are 3.2 Å apart. 

Alanine-185 is 4.7 Å from phenylalanine-193. There also is a hydrophobic interaction between 

phenylalanine-193 and alanine-181, which are 3.9 Å apart. 

This web of interaction between side chains on the outer surface of α helix F on each subunit influences 

residues that have side chains oriented to the interior where catalysis occurs. Alanine-185, which is 

stabilized by interactions with phenylalanine-193, and serine-184, which interacts with serine-196, are 

between the conserved tyrosine-183 and lysine-187. Phenylalanine-193 is adjacent to phenylalanine- 

194, which is positioned into the catalytic site. 

I. Human 11β-HSD-2 

Figure 5 shows the α helix F interface of human 11β-HSD-2. Alanine-237 is about 3 Å from leucine- 

241; alanine-238 is about 3.7 Å from both alanine-238 and threonine-234. Threonine-234 has a 

stabilizing hydrophobic interaction 


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Figure 6 

Structure of α helix F interface of mammalian 17β-hydroxysteroid dehydrogenases. 

The α helix F part of the dimer interface on 17β-hydroxysteroid dehydrogenases is 

shown along with side chains of the highly conserved tyrosine and lysine residues 

and three other residues that are oriented into the cavity that binds substrate and nucleotide 

cofactor. (a) Modeled structure of human 17β-hydroxysteroid dehydrogenase type 1. 

(b) Modeled structure of human 17β-hydroxysteroid dehydrogenase type 2. (c) Modeled 

structure of human 17β-hydroxysteroid dehydrogenase type 3. (d) Modeled structure of 

porcine 17β-hydroxysteroid dehydrogenase type 4. 

with leucine-242, which is 4.2 Å distant. Threonine-245 is 4.2 Å from the C α carbon of glycine-230. 


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The type 1 and type 2 enzymes preferentially catalyze the reduction of the 11-keto group and the 

oxidation of the 11-hydroxyl group, respectively, on glucocorticoids. The chemistry of the side chains 

on methionine-243 in the type-2 


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enzyme and of phenylalanine-194 on 11β-hydroxysteriod dehydrogenase type 1 is quite different; it may 

be important in the different catalytic properties of these two enzymes. 

J. Human 17β-HSD-1 

Figure 6a shows the modeled α helix F interface in human 17β-hydroxysteroid dehydrogenase type 1 in 

which phenylalanine-160 and alanine-161 form an anchor. Both residues have important stabilizing 

interactions across the dimer interface. Alanine-161 is 4.1 Å from alanine-161 on the other subunit. 

Alanine-161 has a hydrophobic interaction with alanine-157, which is in the segment between the 

conserved tyrosine-155 and lysine-159. There is a hydrophobic 


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interaction between alanine-157 and leucine-165, which are about 3.8 Å apart. Phenylalanine-160 is 4 Å 

from glycine-164. There also is a hydrogen bond between cysteine-156 and serine-168, which are 3.2 Å 

apart. This is an interesting structural property of residues in the segment between the conserved 

tyrosine and lysine residues: this segment is important in stabilizing dimers. This pattern is repeated in 

the other 11β- and 17β-hydroxysteroid dehydrogenases suggesting conservation of this stabilizing 

structure, although the residues are not as well conserved as the tyrosine and lysine. 

K. Human 17β-HSD-2 

Figure 6b shows the modeled α helix F interface in human 17β-hydroxysteroid dehydrogenase type 2. 

Alanine-237 is 3 Å from the hydrophobic part of the side chain of methionine-241 on the other subunit. 

Methionine-241 is 3.2 Å from serine-234. Alanine-230 is 3.7 Å from phenylalanine-242 and 4.5 Å from 

valine-245. Alanine-238, the other anchoring residue, is 4.1 Å from alanine-238 on the other subunit. 

L. Human 17β-HSD-3 

Figure 6c shows the modeled α helix F interface in human 17β-hydroxysteroid dehydrogenase-type 3. 

Alanine-203 is 3.1 Å from alanine-207. Phenylalanine-204 is 3.2 Å from the other phenylalanine-204 

and alanine-200. These are the only stabilizing interactions that we find in our analysis. Human 17βhydroxys-teroid 

dehydrogenase type 3 has the weakest interactions across the α helix F interface among 

the four types of 17β-hydroxysteroid dehydrogenases. The conformation of this part of 17βhydroxysteroid 

dehydrogenase type 3 could change upon binding of substrate, leading to other 

stabilizing interactions. And, of course other parts of the protein may have intersubunit interactions that 

stabilize the dimer. Alternatively, the hydrophobic surface of α helix F may interact with another protein 

or a membrane surface, a potentially important regulatory mechanism that we discuss later in this paper. 

M. Pig 17β-HSD-4 

Figure 6d shows the modeled α helix F interface in pig 17β-hydroxysteroid dehydrogenase type 4. 

Leucine-169 is 2.9 Å from glycine-173. Leucine-174 is 4.3 Å from alanine-162 and 3.3 Å from alanine- 

166. There also is a hydrogen bond between serine-165 and serine-175, which are 3 Å apart. 

N. Prospects for the Application of Structure-Function Analysis of Steroid Dehydrogenases in 

Hormone Therapy 

In the last two years there has been impressive progress in understanding the structure of steroid 

dehydrogenases that are important in regulating blood pres 


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sure and the actions of reproductive hormones. This progress has come from several directions. First, the 

cloning and sequencing the dehydrogenases that regulate the actions of aldosterone, cortisol, estradiol, 

and testosterone. Second, determination of the 3D structure of 17β-HSD-1 and several homologs. 

Analyses of their 3D structures confirm a general principle that structural similarity is much higher than 

sequence similarity. This supports proposed molecular models of medically important steroid 

dehydrogenases using the alignment of their sequences onto the templates of 3D structural homologs. 

Models of 11β-HSD-1 and -2 are beginning to reveal important properties about these enzymes. We 

now have a good picture of the structural basis for specificity for NADPH and NADH in 11β-HSD-1 

and -2. With this information, we can now turn our attention to modeling cortisol in these two enzymes. 

This information will open up the possibility for developing analogs to regulate the actions of these two 

enzymes for use in regulating blood pressure and other physiological processes. The development of 

carbenoxolone, a water-soluble synthetic analog of glycyrrhetinic acid, shows that chemists can create 

compounds that have high affinity for 11β-HSD. The next task is to synthesize compounds that are 

specific for 11β-HSD-1 or 11β-HSD-2. Molecular modeling can contribute important information to 

solving this kind of problem. 

Similarly important information will come from the 3D models of 17β-HSD-1, -2, -3 and -4. These 

models will be useful in developing compounds to regulate estrogen and androgen action, which have 

important application in reproductive medicine and in treating estrogen-dependent breast tumors and 

androgen-dependent prostatic tumors. Many compounds in plants have estrogenic and androgenic 

activity; some of these compounds are likely to work via inhibition of one of the 17β-HSD enzymes 

[27]. Analogous to the development of carbenoxolone to regulate 11β-HSD, we can seek synthetic 

compounds that regulate specific types of 17β-HSD, which may be useful in reproductive medicine and 

in treating cancers. 

Considering the explosive pace of biomedical research and the new developments in computers for 

sophisticated structural analyses, the next few years promise to yield important advances in design of 

new hormone therapies based on the knowledge of the structure of steroid dehydrogenases. 


We thank Drs. Tanaka, Nonaka, Nakanishi, Deyashiki, Hara, and Mitsui for providing us with the x-ray 

crystallographic coordinates of carbonyl reductase and 7α-hydroxysteroid dehydrogenase. The support 

of the Supercomputer Center of the University of California, San Diego is gratefully acknowledged. 


Document 

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8 

Design of ATP Competitive Specific Inhibitors of Protein Kinases Using 

Template Modeling 

Janusz M. Sowadski, * Charles A. Ellis, * Rolf Karlsson * 


Madhusudan 

Scripps Research Institute, La Jolla, California 

I. Protein Kinases and Diseases 

Page 213 

The protein kinase family encompasses more than three hundred members of critically important 

enzymes, each one with a specific role or function within the cell. These enzymes, ATPphosphotransferases, 

recognize target proteins and through the phosphorylation of specific sites either 

activate or deactivate a particular pathway of signal transduction. Many of these signaling pathways are 

associated with cell surface receptors, which are located in the membranes that surround cells. The 

difference between the families of protein kinases is that they have different targets and generally fall 

into two major classes: 

The serine/threonine protein kinases transfer a phosphate from ATP to a serine or threonine residue in 

the target protein. This class of enzymes are generally associated with cytoplasmic signaling events. 

The tyrosine protein kinases transfer phosphate from ATP to tyrosine residues in the target protein and 

are generally associated with receptors that become activated after binding a growth factor or other 

ligand. 

Protein kinases are significant targets for therapeutic drug development and have been implicated as the 

disease causing components of numerous tumor 

* Current affiliation: Tufts University, Boston, Massachusetts. 


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viruses. Specifically, it is the deregulation of the activity of protein kinases that leads to disease by 

tumor viruses. The importance of this deregulation can be dramatically illustrated by the large number of 

viral oncogenes (or cancer causing genes) that encode structurally modified protein kinases. These 

deregulated enzymes are able to bypass the normal tightly regulated processes of growth control, leading 

to acute malignant transformation. These oncogenes are one of the first examples of the identification of 

disease-causing genes. Many of these viral genes have subsequently been implicated in human diseases. 

Malignant tissues also share the common characteristic of an acquired independence from controls. The 

receptor—for example, PDGF and EGFR—can be stimulated by a ligand coming either from the cell 

itself (autocrine) or from nearby tissues (paracrine). Regardless of the mechanism leading to receptor 

activity, the resulting kinase activity results in a cascade of signals that turn on cellular proliferation 

programs. Therefore, selective inhibition of receptor tyrosine kinase will block tyrosine kinase driven 

cell proliferation resulting in antitumor activity. In addition to cancer, a growing number of 

nonmalignant proliferative diseases, (e.g., psoriasis, atherosclerosis, restenosis, fibrosis, etc.) or 

inflammatory responses (e.g., septic shock, asthma, osteo and rheumatoid arthritis, etc.) involve 

dysfunctional signaling pathways. Successful development of drugs that target this class of enzymes will 

depend on the discovery of selective inhibitors designed for the appropriate protein kinase within the 

family. 

In the past several years there has been an explosion of structural studies within the protein kinase 

family [1–8]. These studies, initiated by the crystal structure of Protein Kinase A [9–12] (CAPK) have 

shown that all members of the protein kinase family fold into a uniform three-dimensional catalytic core. 

Yet this uniform three-dimensional fold exhibits both different surface charges and at least two major 

conformations. 

II. Protein Kinase Template 

The stereo view of the ribbon diagram of cAPK is presented in Figure 1a. The overall topology of the 

core extending from strand 1 through helix h, Figure 1b, is identical (except helix B) with the eight other 

structures of protein kinases determined to this point. Furthermore, Figure 2 presents an overall 

structural comparison of the catalytic cores of the five kinases, cAPK, CDK2, CDK2-CYCLIN, IR, and 

MAP. The N-terminal helix A, which is present only in the cAPK crystal structure, is anchored by 

myristic acid in the mammalian bovine heart of cAPK. Myristic acid inserts itself into the hydrophobic 

pocket of the lower lobe of the enzyme, which results in the structural ordering of helixA and Ser10[13], 

one of the four autophosphorylation sites. 


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Figure 1 

Diagram of cAPK fold. (a) Stereo MOLSCRIPT diagram. (b) The key loops 

are as follows: phosphate anchor located between strand 1 and strand 2, 

catalytic loop located between strands 6 and 7, DFG motif located between 

strands 8 and 9, activation loop including P+1 site between strand 9 and 

helix F. Phosphorylation site Thr197 is indicated by a large circle, 

inhibitor PKI (5–24) is colored in dark, and the P site in the peptide 

is shown in the dark circle. This figure has been generated using 

MOLSCRIPT [27]. 

Page 215 

Following the connectivity diagram, helix A is connected to β-strand 1, then to the phosphate anchor 

encompassing signature motif Gly50XGly 52XXGly55. The β-strand 2 is followed by β-strand 3 

carrying invariant Lys72. Three antiparallel beta strands create the unique fold of the nucleotide binding 

site of the protein kinase. The β-strands 3 is followed by helix B, which is present only in cAPK, helix 

C, and β-strands 4 and 5. Helix C shows the largest displacements among many different protein kinase 

structures and consists of invariant Glu91, which forms a salt bridge with Lys72. This salt bridge is 

absent in the inactive CDK-2 structure [1] but present in the crystal structure of the complex of CDK-2 

and its activator-cyclin [7]. Displacement of helix C is perhaps most pronounced in the case of the 

insulin receptor tyrosine kinase structure (IRK) [3]. In PKA, Phe185 resides in the hydrophobic pocket 

formed by the hydrophobic residues of helix C (upper lobe) and Tyr164 (lower lobe). In the IRK crystal 

structure, the hydrophobic residues of helix C, which provide the pocket for invariant Phe185 (of DFG 

motif), no longer interact with 


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Figure 1 

(Continued) 

this residue. The DFG motif in IRK occupies the ATP site, which blocks the access of ATP. 

Page 216 

The division between the upper and lower lobes of the enzyme is well defined by the two major 

conformations of the upper lobe observed in the crystal structures of cAPK. One conformation has been 

observed in the orthorhombic crystals of recombinant cAPK [9,10,14] and another in the cubic 


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Figure 2 

The diagrams of the Cα trace of the five kinases, cAPK, CDK2, CDK2-CYCLIN, 

IR, MAP. The thin line represents crystallographically determined homologous 

regions of the five kinases (R. Karlsson and J.M. Sowadski, personal 

communication). 

Page 217 

crystals of bovine heart mammalian cAPK [15,16]. A comparison between the two structures has shown 

that there is rotation of the upper lobe by 15 degrees and a translation of 1.9 Å in the mammalian 

structure, which results in the opening of the nucleotide binding cleft. 

The motion of this lobe, which includes His87, one of the ligands to Thr197 [15], indicates that the 

phosphorylation of this site will be important for conformational diversity of the upper lobe. This is 

confirmed by the varying degrees of displacement of the upper lobe of all structures of the inactive 

unphosphorylated protein kinases (see review [17]). The lower lobe of the enzyme starts with helix D, 

which is followed by helix E and β-strands 6 and 7. The catalytic loop connecting both strands consists 

of a critical set of residues with Tyr164 and Arg165 at the beginning of the loop. The Tyr164 residue 

forms a hydrogen bond with invariant Asp220. The Arg165 residue, which is present in a great majority 

of protein kinases, provides two hydrogen bonds to the oxygens of the phosphate of Thr197. Invariant 

Asp166 (the catalytic base) and Asn171 (the ligand to one of the metal sites) are also located within this 

loop. 


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Page 218 

The catalytic loop is the region of divergence between Ser/Thr and Tyr kinases. In cAPK and all Ser/Thr 

Kinases, Lys168 interacts with the γ phosphate of ATP during catalysis [12]. The role of Lys is replaced 

by Arg [9] and the insulin receptor tyrosine kinase structure [3] shows Arg1136 in a similar position as 

Lys168 in the active site of cAPK. 

The catalytic loop and β-strand 7 are followed by β-strand 8 and a short DFG conserved motif. This 

conserved motif consists of invariant Asp184, a ligand to the metal site, and invariant Phe185. The DFG 

motif is followed by β-strand 9, an activation loop that includes Thr197. The activation loop differs 

considerably among unphosphorylated kinases, CDK-2, ERK-2, and insulin receptor kinase. 

Furthermore, two crystal structures, twitchin protein kinase [2] and phosphorylase kinase [5], both lack a 

phosphorylation site in this region. In the first structure, twitchin protein kinase, Thr197 and Arg165, are 

replaced by hydrophobic residues, Val6098 and Leu6062 respectively [2]. This was predicted using 

modeling [18] and subsequently confirmed by the three-dimensional structure. In the second structure, 

phosphorylase kinase, the Thr197 is replaced by Glu182, which interacts with Arg148 [5]. Hence, in 

both structures the regulatory function of the phosphorylation site is replaced by a stable scaffold 

secured by either hydrophobic or electrostatic interactions. Since it has been shown that this loop 

provides a stable template for PKI(5–24) binding in cAPK, the status of phosphorylation of the 

activation loop critically affects the substrate binding. This is demonstrated in c-Src, a homolog of the 

Rous Sarcoma virus oncogene by mutation of Arg385, which is predicted to interact with the phosphate 

of Tyr416, and results in loss of activity toward the exogenous substrate [19]. 

The activation loop is followed by a P+1 loop which accommodates the P+1 site of the substrate. The 

P+1 loop is followed by invariant Glu208, which forms a salt bridge with invariant Arg280. This 

conserved pair plays a structural role and as the structure of CK-1 [16] shows, it can be replaced by 

other charged residues that maintain the same fold of the lower lobe. The P+1 loop and Glu 208 are 

followed by helix F consisting of invariant Asp220, followed by helices G, H, and I. The helix J and the 

C-terminal tail of cAPK, which are absent in other protein kinases, undergo a large motion during the 

cleft opening. 

The opening of the cleft results in loss of hydrogen bonds provided by the γ phosphate of ATP and the 

peptide that would bridge the lower and upper lobe of the enzyme. The motion of the upper domain 

increases the accessibility of the ATP binding site and one can envision that in the “open” conformation 

ATP binds. Yet, in the “closed” conformation ATP and its γ phosphate are positioned for a nucleophilic 

attack on the substrate. The motion of this lobe—which includes His87, one of the ligands to Thr197 

[15]—indicates that the phosphorylation of this site will be important for conformational diversity of the 

upper lobe. This is confirmed by the varying degrees of displacement of the upper lobe 


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of all structures of the inactive unphosphorylated protein kinases (see review [17]). 

Page 219 

The various displacements of the conserved upper domain of the catalytic cores of various kinases 

documented by crystallographic work suggest that this is the important underlying mechanism of 

catalysis. Analysis of crystal contacts of various kinases is however required to define the extent of 

displacement due to the lattice forces. In the case of cAPK, the displacement as observed for mammalian 

cAPK in the cubic crystal form is due to the intermolecular interaction in the lattice [20]. Analysis of the 

two crystal structures of the cell cycle-controlling kinases clearly shows two binding modes of ATP. In 

the inactive state without cyclin, ATP binding of its triphosphate moieties is different from that in the 

active form with cyclin bound. The major difference is the re-arrangement upon cyclin binding of the 

conserved Lys33-Glu51 pair, which is responsible for the binding of the α and β phosphates of ATP. 

III. Crystallographic Analysis of Substrate Specificities of Individual Kinases 

The most important contribution of subsequent crystallographic studies has been the confirmation of the 

structural homology extending through the members of this family of enzymes. The crystal structures of 

CDK-2 [1], ERK-2 [5], twitchin [2], insulin receptor kinase [3], phosphorylase kinase, CK-1 [6], along 

with structure of calcium/calmodulin-dependent protein kinase I [8] provide solid proof for the structural 

conservation of the catalytic core in the family. This is further confirmed by the recent structure of the 

active complex of CDK2/cyclin, which shows that Lys33-Glu51 pair is at a distance of 3.0 Å [7] as 

predicted in the model of CDK-2 based on the cAPK structure [21]. The structure of the complex has 

also confirmed the binding of cyclin to helix C and to the upper lobe, demonstrating the mechanism of 

activation by cyclin that results in bridging the invariant residues into the common network of distances 

required by structural homology of the protein kinase catalytic core. 

The crystallographic analysis of the structural homology of protein kinases can now be carried out using 

structures of various kinases to find a common search model to be used in molecular replacement 

methods (J.M. Sowadski and R. Karlsson private communication). The structures of various kinases 

have been used as search models to solve the structure of the cAPK using cAPK diffraction data. The 

best search model consists of fragments of the catalytic core excluding the activation loop, inserts, and 

upper lobe due to rotational motion observed in each structure. A structure solution has been found for 

several protein kinases using this selected model as shown in Figure 2. One of the most critical aspects 

of this analysis is the presence of the structurally 


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Page 220 

conserved substrate-binding cleft as observed in the crystal structure of cAPK:PKI complex (Figure 

la,b). This finding allows the charges within this cleft to be predicted using the amino acid sequence of 

any given kinase. 

In the analysis of the structural data of other protein kinases, it is noted that only cAPK has been 

crystallized with its specific peptide inhibitor. Nevertheless, three other structures of protein kinases 

compared with the structure of the cAPK-PKI complex provide substantial evidence for the conservation 

of the substrate binding cleft. The substrate binding cleft of the phosphorylase kinase structure has been 

analyzed in detail and it is clear that all amino acids of the known specific substrate can be built into the 

PKI model and all required corresponding charges can be found in the cleft of the phosphorylase kinase 

structure. In the CK-1 structure determined without a peptide, the requirement of the peptide specificity 

resides on the P-3 site, which has to be phosphorylated. An analysis of the surface charges of the cleft of 

the CK-1 structure reveals the exact correspondence of the residues required to interact with a 

phosphorylated substrate at this site. 

Finally, the tripeptide of the pseudosubstrate site of IRK consisting of the Asp-Tyr-Tyr motif has 

corresponding charges in the structure of the enzyme's substrate cleft and confirms the data obtained 

from the degenerated peptide library for the unique sequence motifs of nine tyrosine kinases [22]. It is 

becoming increasingly clear that the wealth of structural data of protein kinases with cAPK as a 

prototype provides evidence for two important features concerning substrate binding. First, the substrate 

binding cleft is structurally conserved and second, the surface charges of this cleft and hydrophobic 

cavities on the surface are very diverse and correspond to the specificity requirement of the substrate for 

individual protein kinases (see Figure la). It is now possible to use the structural conservation of the 

substrate binding cleft to predict the charges and hydrophobic residues of the cleft to define substrate 

specificities for individual kinases. 

IV. Crystallographic Analysis of the ATP Binding Site Reveals Distinct Differences Utilized for the 

Further Design of Specific Inhibitors 

The diagram elucidating detailed interactions of ATP with the enzyme is presented in Figure 3a,b. Six 

out of nine invariant residues of the catalytic core of protein kinase are involved in ATP binding and 

catalysis. The key residues that hold the β and γ phosphates in position are the phosphate anchor, the 

metal sites, and Lys168. The amides of the residues—Phe54, Gly55, and Ser53—are essential for the 

position of the γ and β phosphates. The metal site coordinated by invariant Asp184 is also sequestered 

by the β and γ phosphates and the metal 


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Figure 3 

(a) Ternary complex of MnATP with the inhibitor peptide PKI (5–24). 

Glu121 and Val123 of the conserved linker region of the protein kinase catalytic core 

form the bidentate hydrogen bond with the 6-amino group and N1 nitrogen of the purine 

base. Thr183, non-conserved, forms a hydrogen bond with the N7 position of purine. 

2'-OH of ribose interacts with the side chain of Glu127 of helix D and P-3 Arg of the 

specific inhibitor while the 3'-OH interacts with Glu170 of the catalytic loop. (b) The 

local environment of serine nucleophile at P site (left site) and local environment of 

phosphorylated P site serine (right side). The side chain of the catalytic base is at 

hydrogen bond distance from-OH of the Ser nucleophile which defines the conserved 

substrate binding P site. 


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Page 222 

site coordinated by invariant Asn171 is also sequestered by the α and γ phosphates. The residue Lys168 

is at hydrogen-bond distance from the γ phosphate. The postulated in-line mechanism of 

phosphotransfer in cAPK [23] can be examined through an analysis of the MnATP and PKI (5–24), Ser 

substrate peptide and ADP complexes [24]. A comparison between cAPK and IRK structures indicates 

that Ser versus Tyr specificity is obtained by displacement of the substrate binding site in such a way 

that the hydroxyl of nucleophiles of both Ser and Tyr fall in the same point in the active site facing 

corresponding catalytic bases Asp1132 and Asp166 for IRK and cAPK respectively. 

The purine base of ATP is anchored to the enzyme by three hydrogen bonds, two of them involve the 6amino 

group and N1 nitrogen, which interact with the backbone atoms of Glu121 and Val123. the 6amino 

group and N1 nitrogen of the purine base form the hydrogen bonds also in the structure of CK-1 

and in the structure of phosphorylase kinase. Yet, in the structure of inactive CDK-2, the purine base 

forms only one hydrogen bond via its N6 position. While N7 nitrogen interacts directly in cAPK with 

the side chain of Thr183 in CK-1, this nitrogen interacts directly with Glu55 and Tyr59 via two 

hydrogen-bonded water molecules and in phosposhorylase kinase via one water molecule. Hence, the 

N7 nitrogen and its interaction with the enzyme is a region for potential modification of ATP 

competitive inhibitors. Ribose is held by both enzyme (Glu127 and Glu170) and inhibitor (P-3 Arg). 

While the side chain of Glu127 interacts with 2'-OH of ribose in cAPK, in CK-1 the 2'-OH interacts via 

two water molecules with Ser91 and Asp94. In ERK-2, 2'-OH interacts with Asp109. This region has 

been utilized to design ATP-based specific inhibitors by modifications of the ATP-competitive 

nonspecific inhibitor staurosporine [25], see Figure 4. 

In the model cAPK with bound staurosporine inhibitor, the lactam amide group of the inhibitor functions 

as a bidentate hydrogen bond donor-acceptor 

Figure 4 

Chemical structures of staurosporine inhibitor, left, and 

CGP52411. 


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Figure 5 

(a) Staurosporine molecule docked in the ATP binding site of PKA. 

hydrogen bonds anchoring staurosporine molecule in the active site of PKA 

consists of the 6-amino group and N1 nitrogen and carbonyl of Glu121 and 

the amide hydrogen atom of Val123. This bidentate hydrogen bond formation 

has been observed in all complexes of protein kinases and ATP solved so far. 

(b) Inhibitor of CGP 52411 and ATP docked on the active site of PKA. 

Residues of Glu127 and Glu170 are also shown and these are not 

conserved in the EGFR kinase. 

Page 223 

(Figure 5a). This key observation is supported by chemical data of lactam amide derivatives, which 

provide a plausible model of staurosporine inhibition. This is in the protonated boat-type conformation 

found to fit in the ATP binding cleft with minimal steric hindrance. In this model, the 4-amino group 

forms hydrogen bonds with the backbone carbonyl of Glu170 and the carboxylate group of Glu127 of 

cAPK. A model of the EGFR kinase shows that Glu127 and 170 are replaced by Cys and Arg, 

respectively (Figure 5b). Replacement of Glu127 by Cys is critical according to the model and explains 

the several-fold decrease of potency of staurosporine inhibitor toward the EGFR kinase (IC 50=630 nM 


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Figure 5 

(Continued) 

Page 224 

versus IC 50=15 nM for cAPK). Yet enhancement of specificity utilizing the inhibitor CGP52411 (Figure 

4), whose selectivity originates in the occupancy by one of the anilino moieties of the inhibitor in the 

region of the enzyme cleft that normally binds the ribose ring of ATP, is considerable. The inhibitor 

CGP52411 inhibits the EGFR tyrosine kinase with an IC 50 value of 300 nM while it is less active by at 

least two orders of magnitude on a panel of protein kinases including cAPK, phosphorylase kinase, 

casein kinase, protein kinase C (most isoforms), and v-abl, c-lyn, c-fgr tyrosine kinases. 

Hence, the three-dimensional model of EGFR tyrosine kinase rationalizes the specificity of the 

CGP52411 inhibitor. This model suggests that analysis of the putative regions of the ribose binding of 

ATP in other kinases through template modeling would provide the required chemical modification of 

pharmacophores to enhance their selectivity. In modeling, the use of the bidendate hydrogen bonding 

provided by the linker region (Figure 3a) of the conserved 


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protein kinase core is essential. Yet to predict the region of the structure of the inhibitor that will form 

these strong Watson-Crick hydrogen bonds remains difficult. 

Page 225 

The use of the 6-amino group and N1 nitrogen of the purine base to model the pharmacophore, as seen 

with staurosporine, is not possible in two other adenine-type inhibitors. Both isopentenyl adenine, a 

nonspecific inhibitor of protein kinases, and olomoucin, a more specific inhibitor of Ser/Thr protein 

kinases, are modified only at the 6-amino group position. Thus, bidentate hydrogen-bond formation as 

seen in the ATP purine base is not possible. Furthermore, there are inhibitors that do not contain the 

chemical structure of adenine, for example des-chloro-flavopyridol, a potent inhibitor of cdc-2 cell cycle 

kinase. 

In crystallographic analysis of the binding of three inhibitors, olomoucin (OLO), isopentenyl adenine 

(ISO) and des-chloro-flavopyridol (DFP) to inactive CDK-2 cell cycle protein kinase, Kim and coworkers 

[26] have provided additional insight into the binding of adenine-and nonadenine-based 

inhibitors. Inhibitors with purine rings (OLO and ISO) bind in relatively the same area of the binding 

cleft as the adenine ring of ATP. Relative orientation of each purine ring with respect to the protein is 

different for all three ligands. This is most likely due to the fact that the 6-amino group of adenine in 

ATP is replaced by an isopentenylamino group in ISO and by the bulky benzylamino group in OLO. In 

the case of the third inhibitor, which is not an adenine derivative, the benzopyran ring occupies 

approximately the same region as the purine ring of ATP. The two ring systems overlap in the same 

plane but benzopyran is rotated about 60 degrees relative to the adenine of ATP. In this orientation, two 

strong bidendate hydrogen bond are formed with the oxygens in the 4th and 5th positions of the 

inhibitor. Furthermore, these bonds are the same ones formed by the 6-amino group and N1 nitrogen of 

the adenine ring. 

Crystallographic analysis has shown that both the substrate and ATP-binding clefts are structurally 

conserved yet differ in the surface charges between individual protein kinases. The structural template of 

the protein kinase family as discovered in the structure solution of cAPK predicts these differences. 

Template modeling provides a rational basis for the design of specific inhibitors for protein kinases 

based on the ATP binding site [25]. This is the first significant step in the design of specific inhibitors 

targeted at the ATP site. 

Recent work has now shown the conformational diversity of inhibitors binding in the interdomain ATPbinding 

cleft [26]. Although the residues of the protein kinase catalytic core that form the bidentate 

donor-acceptor bond with inhibitors are identical throughout different structures, the residues of the 

inhibitors vary greatly. All inhibitors use this common bidentate bond yet the specificity lies in several 

other bonds formed between the inhibitor and specific regions of the individual protein kinases. 

Furthermore, it is difficult to model 


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the bidentate bond-forming residues of the inhibitor and it is through protein crystallography that these 

are determined. Template modeling can then be used to determine the specific surface charges of the 

modeled kinase that can be exploited by the inhibitor for its specificity. Modeling and focused 

combinatorial chemistry are the tools to achieve the goal of inhibitor specificity. 


This work was supported by CTR grant 4237. We thank Drs. Furet, P. Traxler, and N. Lydon of Ciba for 

their drawings presented in Figure 5. We also thank Dr. Nikola P. Pavletich for the coordinates of the 

CDK2-CYCLIN complex used in Figure 2. 

References 

1. De Bondt HL, Rosenblatt J, Jancarik J, Jones HD, Morgan DO, Kim SH. Crystal structure of cyclindependent 

kinase2. Nature 1993; 363:595–602. 

2. Hu S-H, Parker MW, Lei JY, Wilce MCJ, Benian GM, Kemp BE. Insights into autoregulation from 

the crystal structure of twitchin kinase. Nature 1994; 369:581–584. 

3. Hubbard SR, Wei L, Ellis L, Hendrickson WA. Crystal structure of the tyrosine kinase domain of the 

human insulin receptor. Nature 1994; 372:746–754. 

4. Zhang F, Strand A, Robbins D, Cobb MH, Goldsmith EJ. Atomic structure of the MAP kinase ERK2 

at 2.3 Å resolution. Nature 1994; 367:704–710. 

5. Owen DJ, Noble MEM, Garman EF, Papageorgiou AC, Johnson LN. Two structures of the catalytic 

domain of phosphorylase kinase; an active protein kinase complexed with substrate analogue and 

product. Structure 1995; 3:467–482. 

6. Xu R-M, Carmel G, Sweet RM, Kuret J, Cheng X. Crystal structure of casein kinase-1, a phosphatedirected 

protein kinase. The EMBO Journal 1995; 14:1015–1023. 

7. Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, Massague J, Pavletich NP. Mechanism of CDK 

activation revealed by the structure of a cyclinA-CDK2 complex. Nature 1995; 376:313–320. 

8. Goldberg J, Nairn AC, Kuryian J. Structural basis for the autoinhibition of calcium/calmodulindependent 

protein kinase I. Cell 1996; 84:875–887. 

9. Knighton DR, Zheng J-H, Ten Eyck LF, Xuong N-H, Taylor SS, Sowadski JM. Structure of a peptide 

inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. 

Science 1991; 253:414–420. 


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10. Knighton DR, Zheng J-H, Ten Eyck LF, Ashford VA, Xuong N-H, Taylor SS, Sowadski JM. Crystal 

structure of the catalytic subunit of cyclic adenosine monophosphate dependent protein kinase. Science 

1991; 253:407–414. 

11. Zheng J-H, Trafny EA, Kninghton DR, Xuong N-H, Taylor SS, Ten Eyck LF, Sowadski JM. 2.2Å 

refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with 

MnATP and a peptide inhibitor. Acta Cryst 1993; D49:362–365. 


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12. Zheng J-H, Knighton DR, Ten Eyck LF, Karlsson R, Xuong N-H, Taylor SS, Sowadski JM. Crystal 

structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and 

peptide inhibitor. Biochemistry 1993; 32:2154–2161. 

13. Sowadski JM, Ellis C, Madhusudan. Detergent binding to unmyristylated protein kinase 

A—structural implications for the role of myristate. Journal of Bioenergetics and Biomembranes 1996; 

28:7–12. 

14. Knighton DR, Bell S, Xuong N-H, Ten Eyck LF, Taylor SS, Sowadski JM. 2.0Å refined crystal 

structure of catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and 

detergent. Acta Cryst 1993; D49:357–361. 

15. Karlsson RF, Zheng J-H, Xuong N-H, Taylor SS, Sowadski JM. Crystal structure of the mammalian 

catalytic subunit of cAMP-dependent protein kinase and an inhibitor peptide displays an open 

conformation. Acta Cryst 1993; D49:381–388. 

16. Zheng J-H, Knighton DR, Xuong N-H, Taylor SS, Sowadski JM, Ten Eyck LF. Crystal structures of 

the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed 

conformations. Proteins Science 1993; 2:1559–1573. 

17. Taylor SS, Radzio-Andzelm E. Three protein kinase structures define a common motif. Structure 

1994; 2:345–355. 

18. Knighton DR, Pearson RB, Sowadski JM, Means AR, Ten Eyck LF, Taylor SS, Kemp BE. 

Structural basis of the intrasteric regulation of myosin light chain kinase. Science 1992; 258:130–135. 

19. Senften M, Schenker G, Sowadski JM, Ballmer-Hofer K. Catalytic activity and transformation 

potential of v-Src require arginine 385 in the substrate binding pocket. Oncogene 1995; 10:199–203. 

20. Karlsson RF, Madhusudan Taylor SS, Sowadski JM. Intermolecular contacts in various crystal 

forms related to the open and closed conformational states of the catalytic subunit of cAMP-dependent 

protein kinase. Acta Cryst 1994; D50:657–662. 

21. Marcote MJ, Knighton DR, Basi G, Sowadski JM, Brambilla P, Draetta G, Taylor SS. A threedimensional 

model of the cdc2 protein kinase: identification of cyclin and suc1 binding regions. 

Molecular and Cellular Biology 1993; 13:5122–5133. 

22. Songyang, Z et al., Catalytic specificity of protein tyrosine kinase is critical for selective signalling. 

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group transfer catalyzed by cAMP-dependent protein kinase. J Am Chem Soc 1988; 110:2680–2681. 


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Crystallographic insights into substrate recognition and phosphotransfer. Protein Science 1994; 

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MOLSCRIPT—A 

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9 

Structural Studies of Aldose Reductase Inhibition 

David K. Wilson and Florante A. Quiocho 

Baylor College of Medicine, Houston, Texas 

J. Mark Petrash 

Washington University School of Medicine, St. Louis, Missouri 


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Aldose reductase (ALR2; EC 1.1.1.21) is an ~36 kDa enzyme that catalyzes the reduction of a wide 

range of carbonyl-containing compounds to their corresponding alcohols. It is a member of an extensive 

aldo-keto oxidoreductase enzyme family, a collection of structurally similar proteins expressed in both 

animals and plants. Most members of the enzyme family possess similarities in molecular mass, pH 

optimum, coenzyme dependence, and demonstrate overlapping specificity for many substrates and 

inhibitors. 

While no essential physiological function has been established for ALR2, extensive experimental 

evidence suggests that it plays an important role in the development of diabetic complications affecting 

the visual, nervous, and renal systems [1]. The linkage between ALR2 and pathogenesis of diabetic 

complications lies in the polyol pathway of glucose metabolism (Figure 1). In hyperglycemic tissues 

such as in diabetes mellitus, the capacity of hexokinase to shunt glucose to glycolysis and other major 

pathways of glucose metabolism is exceeded. Consequently, enhanced flux of glucose through the 

polyol pathway occurs. The enzyme ALR2 catalyzes the first step in this pathway, producing sorbitol, an 

active osmolyte. The polyol pathway is completed by the NAD +-dependent oxidation of sorbitol to 

fructose, mediated by sorbitol dehydrogenase. 

Extensive evidence exists to suggest a linkage between the pathogenesis of diabetic complications and 

enhanced glucose metabolism via the polyol pathway. The polyol pathway functions in all tissues 

susceptible to clinically 


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Figure 1 

Schematic of the polyol pathway showing the NADPH-dependent reduction of open 

chain D-glucose to sorbitol, which is catalyzed by ALR2. This step is followed by the 

NAD + -dependent oxidation of sorbitol by sorbitol dehydrogenase to yield D-fructose. 

Page 230 

significant diabetic complications. Transgenic animals overexpressing ALR2 in target tissues of diabetic 

complications are more prone to development of experimentally induced diabetic complications [2,3]. 

The most extensive body of evidence linking ALR2 to the pathogenesis of diabetic complications comes 

from numerous successes in the treatment of experimental animals with a variety of ALR2 inhibitors 

(ARI) [4]. Many of these studies demonstrated that ARIs substantially delay or in some cases prevent 

the onset of complications. 

Clinical trials of ARIs have yielded encouraging results in alleviating painful symptoms of diabetic 

complications. However, unacceptable side effects related to toxicity or inadequate pharmocokinetic 

profiles have rendered most of the drug candidates undesirable. Nevertheless, several ARIs are 

commercially available in some countries and more appear to be in the pipeline. The therapeutic 

rationale for treatment of human diabetics with ARIs to delay or prevent onset of diabetic complications 

is compelling. Animal models with experimentally induced hyperglycemia develop complications that 

are morphologically and functionally similar to that seen in the human diabetic patient. Many 

structurally 


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diverse ARIs have been shown to substantially delay or completely prevent the onset of such 

complications in experimental animal models. While some studies indicate that ALR2 may play a 

functional role in osmotic homeostasis in the kidney, evidence from animal studies suggests that it is 

metabolically dispensible. 

Page 231 

Long-term complications exact a terrible toll of morbidity and mortality on patients with diabetes 

mellitus. For example, patients with diabetes have about a 25-fold increased risk for becoming blind 

over that of the general population. Diabetic retinopathy is one of the most common causes of visual loss 

and accounts for about 12% of new cases of blindness each year in the United States alone [5]. 

II. Drug Design Prior to Structural Data 

Many inhibitors have been developed over the past two decades without the advantage of a structural 

understanding of the enzyme [4,6,7]. Significant improvement has been made since the discovery of the 

first such orally active compound to show in vivo activity, alrestatin (Figure 2), which had an IC 50 in the 

low micromolar range [8]. Many high-affinity inhibitors with IC 50s in the low nanomolar range are now 

under study. 

Recent drug-design efforts have yielded compounds usually with one of two chemical motifs: 

carboxylates or spirohydantoins. A number of these compounds such as tolrestat [9], ponalrestat [10], 

epalrestat [11], sorbinil [12], and zopolrestat [13] have progressed to the point of clinical trials. 

Unfortunately, clinical ineffectiveness and/or unacceptable side effects have limited the usefulness of 

most of those that had been shown to be effective in vitro. The latter problems may be associated with a 

lack of specificity since many aldose reductase inhibitors inhibit both ALR2 and aldehyde reductase 

[14]. For this reason, ALR2 as well as the other members of the aldo-keto reductase family have been 

the subject of crystallographic studies with the hope of determining a structural basis for inhibitor 

specificity and ultimately to provide a basis for enhancing binding affinity. 

III. Structural Studies of Aldose Reductase 

The first crystal structures available for ALR2 were those of the porcine form complexed with the 

NADPH analog 2'-monophosphoadenosine-5'-diphosphoribose [15] and the human enzyme complexed 

with the NADPH cofactor [16]. Further studies have been conducted on mutants of the human enzyme 

[17] and ternary complexes of the human enzyme with an inhibitor [18]. All of these 


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Figure 2 

A number of ALR2 inhibitors that have entered clinical trails. 

structures show the protein to fold into a (β/α) 8 barrel (Figure 3). This fold has emerged as the most 

common enzyme motif [19] although most of the proteins adopting this structure share no sequence 

homology. The ALR2 enzyme is, however, the first NAD(P)H binding protein to adopt this fold. It 

contains an extra β hairpin preceding the first β strand, which caps the N-terminal end of the barrel. It 

also has two helices that are not part of the regular barrel. One precedes α7 and the other follows α8. 

A. Cofactor Binding 

The NADPH cofactor is bound in an extended conformation across the C-terminal end of the β barrel. 

The catalytically active nicotinamide moiety is located 


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Figure 3 

C α trace of the ALR2 holoenzyme looking down the (β/α) 8 barrel. The NADPH 

cofactor is seen bound across the carboxy-terminal end of the β-barrel with the active 

nicotinamide moiety in the center. Figure produced using the MOLSCRIPT 

program [48]. 

Page 233 

at the center of the barrel while the adenosine extends away to bind between α7 and α8. A belt 

composed of residues 213 to 227 folds over the pyrophosphate of the NADPH to sequester a large part 

of the cofactor from the solvent. It is fastened to the other side of the NADPH binding site via Asp216 

on the loop that forms bifurcated salt links with Lys21 and Lys262. The dominant interactions holding 

the coenzyme in place are directional hydrogen bonds and salt links from positively charged side chains 

to the phosphates. The interaction between the 2' phosphate on the NADPH and the side chains from 

Lys262 and Arg268 account for the enzyme's preference for NADPH over NADH. Earlier biochemical 

studies had shown that it is the 4-pro-R hydride that is transferred from the nicotinamide to the substrate 

[20]. This is ensured by a hydrogen-bonding network using side chains from Ser159 and Asn160 and the 

main chain of Gln183 to orient the amide. It is also determined by the stacking interactions with Tyr209, 

which is adjacent to the 4-pro-S side of the nicotinamide. 

B. Mechanism 

The catalytic site was unambiguously identified using the location of the nicotinamide moiety of the 

NADPH cofactor in the holoenzyme structure. The region surrounding the catalytic site is a 12-Å deep 

groove that measures 


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approximately 7 by 13 Å and is lined primarily with hydrophobic side chains. This is entirely consistent 

with earlier experiments showing that the enzyme has a marked preference for lipophilic substrates 

versus polar substrates such as sugars [21]. 

The catalytic site also suggested a model for the enzyme's chemical mechanism, which is substantially 

similar to most other NAD(P)H-dependent oxido reductases. Upon binding of the reduced cofactor, the 

enzyme is able to form a ternary complex with the substrate. The pro-R hydride from the C-4 of the 

nicotinamide is transferred to the carbonyl carbon of the substrate, which in turn causes the carbonyl 

oxygen to abstract a proton from a general acid, which is presumably located on the protein, to form the 

alcoholic product. Three proton-donating side chains are located within 6 Å of the C-4 atom in the 

NADPH cofactor that could potentially fulfill this role: Tyr48, His110, and Cys298. Since it is not 

conserved in other members of the aldo-keto reductase family that exhibit enzymatic activity (Figure 8) 

Cys298 was unlikely as a candidate proton donor. The histidine is surrounded by several hydrophobic 

residues including Val47, Trp79, and Trp111, which would serve to lower the pK a of the side chain, 

making it less effective as a proton donor at physiological pHs. The tryrosine, which ordinarily has a pK a 

of approximately 11 engages in an interaction with the charged ammonium group of Lys77, which in 

turn charge-pairs with Asp43. This network serves to depress the pK a of the phenolic oxygen, increasing 

the exchangability of the proton. 

Subsequent activity studies involving site-directed mutants support this model [17,22]. The Tyr48 

rarrow.gif Phe mutation shows a complete lack of activity while the Asp43 rarrow.gif Asn, Lys77 

rarrow.gif Met, His110 rarrow.gif Asn, and Cys298 rarrow.gif Ser showed losses in catalytic 

efficiency of approximately 100-, 1000-, 10 6-, and 10-fold respectively when compared with the wildtype 

enzyme. These results correlate well with the functions predicted for each residue with the 

exception of the histidine. 

The structure of the ALR2 holoenzyme showed that the catalytic site was situated atop the nicotinamide 

moiety of the NADPH cofactor. The substrate binding site, which would determine the enzyme's 

specificity and also presumably bind inhibitors, appeared to be composed of a deep cleft (Figures 3 and 

4). It extended away from the catalytic site towards the loop composed of residues between β4 and α4 

and the last 20 residues of the carboxy-terminal meander. This hypothesis was supported by the 

appearance of poorly resolved density that occupied this region, which suggested the presence of an 

endogenously bound substrate or inhibitor in the structure of the holoenzyme [16]. Subsequent studies 

indicate that this electron density may be a citrate molecule, one of the components included in the 

crystallization mixture. Activity studies indicate that citrate is indeed one of the many inhibitors of the 

enzyme with a K i in the millimolar range [23]. 


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Figure 4 

Surface representation of the ALR2 holoenzyme in an orientation similar to Figure 3. 

The nicotinamide moiety that defines the active site of the enzyme is seen in the center. 

The groove that extends down from it is highly hydrophobic and was initially assumed 

to be the inhibitor binding site. Figure prepared using the GRASP program [49]. 

IV. Aldose Reductase Complexed with Inhibitor 

Page 235 

While a large number of high-potency inhibitors for ALR2 have been developed [4], a structural 

understanding of the exact molecular features that foster this affinity have been only vaguely 

understood. Several general chemical motifs such as hydrophobic ring systems, a spirohydrantoin group 

or carboxylate group are seen repeatedly when examining a list of known inhibitors (Figure 2) but little 

was known about the specific role for each in inhibitor binding. 

The structure of the ALR2/NADPH/zopolrestat ternary complex [18] has provided some answers about 

the mode of binding of zopolrestat (Figure 2), a high-affinity, carboxylate-containing compound 

developed by Pfizer, Inc. [13]. While the overall structure was preserved, the inhibitor binding induced a 

conformational change of the enzyme. This change, which involved the movement of several loops in 

the active site of the molecule, was large enough to cause a change in crystal packing relative to the 

holoenzyme. As a consequence of the shifting of the active-site loops, a cavity is created inside the 

protein in which the benzothiazole ring is seated and the groove that was implicated in substrate and 

inhibitor binding by the holoenzyme structure vanishes. This illustrates the unpredictability of 

conformational changes within a protein in response to substrate or inhibitor binding. It also implies that 

modeling compounds 


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Page 236 

in the active site of an enzyme or drug-design algorithms such as inhibitor docking may be inappropriate 

in some cases if they do not adequately model considerable plasticity in the binding site. 

A closer look at the binding of zopolrestat shows that it is dominated by extensive hydrophobic contacts 

between the protein and the inhibitor. These include side chains from Trp20, Tyr48, Trp79, Trp111, 

Phe115, Phe122, Trp219, Ala299, Leu300, Tyr309, and Pro310 (Figure 7). This is not surprising given 

the apolar nature of the enzyme's active site as determined by the holoenzyme structure. What is 

surprising is that the inhibitor created the part of its own binding site that the benzothiazole rings occupy 

by “burrowing” into the hydrophobic core of the protein to carve out a region with very good steric 

complementarity to this moiety. It does this rather than binding in the solvent-exposed hydrophobic 

binding groove that is seen in the holoenzyme structure. 

The remaining interactions involving hydrogen bonds and salt links also appear to be very important in 

inhibitor binding. With the exception of one of the fluorine atoms, all atoms that are able to engage in 

hydrogen bonding do so. The carboxylate, which is seen in so many aldose reductase inhibitors, is saltlinked 

to His110, which is located very near the catalytic site (Figure 7). Presumably, the carboxylate in 

the other inhibitors plays the same role and could be used as an anchor when modeling these into the 

active site. 

Inhibition studies involving ALR2 have indicated noncompetitive inhibition for virtually all compounds 

examined to date when the forward (reduction) reaction is monitored. This mode of inhibition is often 

interpreted as meaning that the inhibitor binds to a site on the enzyme that is independent of the catalytic 

site. Kinetic and competition studies have both led to this conclusion in the case of ALR2 [24,25]. The 

crystal structure of the enzyme complexed with both the NADPH cofactor and zopolrestat, however, 

clearly shows the inhibitor occupying the region directly above the nicotinamide of the NADPH and, 

therefore, the active site (Figures 5, 6, and 7). 

Most previous inhibition studies reported noncompetitive and/or uncompetitive inhibition patterns when 

aldose reductase inhibitors were examined in the forward direction, i.e. inhibition of NADPH-dependent 

aldehyde reduction. With the finding that the overall rate-limiting step in the direction of aldehyde 

reduction is at the level of structural isomerization following alcohol product release [26,27], it is not 

surprising that lack of competitive inhibition would be observed in such standard double reciprocal 

plots. To further complicate matters, many aldose reductase inhibitors were not recognized in previous 

studies as tight-binding inhibitors and were inappropriately evaluated using Michaelis-Menten kinetics. 

Thus, noncompetitive or uncompetitive inhibition patterns were previously reported for inhibitors that 

were subsequently shown to bind directly at the active site. Recent structure-function and kinetic studies 

have revealed important details concerning the structural basis for the catalytic 


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Figure 5 

Schematic representation of the ALR2/NADPH/zopolrestat ternary complex. The 

NADPH is bound across the enzyme from the center to the right while the zopolrestat 

binds atop the nicotinamide and extends to the lower left. Conformational changes are 

seen in the C-terminal loop below the zopolrestat in the picture and the loop to the right 

of the inhibitor between β4 and α4. 


Page 237

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Figure 6 

A surface representation of the ternary complex as seen in Figure 5. Note that the 

inhbitor creates part of its binding site by “burrowing” into the protein rather than 

binding entirely in the groove seen Figure 4. 


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Figure 7 

Stereo of zopolrestat binding to the active site of ALR2. The salt link made by the carboxylate of the inhibitor and 

hydrogen bonds are depicted with dashed lines. The remainder of the interactions are apolar with the residues shown. 


Page 238

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and inhibition mechanisms and provided a clarification for the mechanism by which inhibitors of both 

the carboxylate [28] and spirohydantoin [29] classes bind at the active site of ALR2. 

V. Aldo-Keto Reductase Family 

A. Effects of Sequence on Drug Design 

Page 239 

The problem of structure-based drug design for ALR2 and the drug-design effort in general is 

compounded by the fact that this enzymes is a member of a large family of aldo-keto reductases with 

overlapping substrate specificity. In humans at least three such enzymes have been found: ALR2 [30], 

aldehyde reductase (ALR1) [30], and chlordecone reductase [31]. Other members of the family that have 

been isolated in other species include rat 3α-hydroxysteroid dehydrogenase [32], murine fibroblast 

growth factor induced protein [33], bovine prostagladin F synthase [34], murine vas deferens protein 

[35], frog ρ-crystallin [36], the P100/11E gene product in Leishmania major [37], and Corynebactium 

diketogluconate reductase [38]. This large number suggests that there may be more such enzymes to be 

found in humans. The similarities between the proteins with respect to both the sequence and substrate 

specificity implies that the nature of the substrate binding sites are similar across the family. This has 

indeed been the case in all the structures determined from this family to date (see below). 

While detailed binding studies of various inhibitors with all the different enzymes have not been 

conducted, it is likely that drugs intended for ALR2 are likely to “cross react” with many of the other 

enzymes within the family. One such case that has recently been studied both crystallographically and 

biochemically is the murine FR-1 protein described below. 

The binding sites of all of these enzymes are characterized by their large size and hydrophobicity 

suggesting that ideal substrates may be steroids or molecules of a similar size and nature. Sequence 

comparisons of all the proteins, including those whose structure has not yet been determined, show that 

there is a large amount of similarity involving residues implicated in substrate binding (see Figure 8). 

One region that diverges somewhat is the 15-amino acid segment at the carboxy terminus of the protein. 

This segment is likely to be responsible for what little differences in substrate specificity exhibited by 

the enzymes. It is the same segment that is seen adopting a different conformation upon zopolrestat 

binding to ALR2. It may then be possible that it is not only the chemical nature of this loop that—in 

making positive and negative interactions with the substrate/inhibitor—modulates specificity, but also 

the flexibility conferred by the amino acid sequence. Such a difference is seen when contrasting the 

structures of ALR2 and FR-1 bound to zopolrestat. 


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B. Structures 

Figure 8 

Sequence alignment of several members of the aldo-keto reductase family. Abbreviations used 

are HALR2, human aldose reductase (30); HALR1, human aldehyde reductase [30]; 3α-HSD, 

3α-hydroxysteroid dehydrogenase [32]; FR-1, murine FR-1 [33]; BPGFS, bovine 

prostaglandin F synthase [34]; CCDR12, human chlordecone reductase [31]; CDGR, 

Corynebacterium diketogluconate reductase [38]; MVDP, murine vas deferens protein [35], 

JFRC, Japanese frog ρ crystallin [36]. 

Page 240 

Structures of several other members of the aldo-keto reductase family have also been determined. These 

include aldehyde reductase [39,40], FR-1 [41] and 3α-hydroxysteroid dehydrogenase [42]. Since each of 

these proteins retain a large amount of sequence identity and homology with human ALR2, it is not 

surprising to note that the overall tertiary structures are very similar. Root-meansquare C α deviations 

between the human ALR2 holoenzyme structure and the rest of the family are in the range of 1–2Å 


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Page 241 

Closer examination of the active site shows that the residues involved in catalysis (most notably the 

residues analogous to Asp43, and Tyr48, and Lys77 in ALR2) are structurally conserved among each of 

these proteins (Figure 8), suggesting that the mechanism is also conserved throughout the family. Many 

of the residues found in the binding site (defined as those making contact with the zopolrestat in the 

ternary complex) are also largely conserved with the exception of a number of residues in the carboxy 

terminus of the protein. This is indeed where the most structure variation appears to be concentrated 

among the proteins. These residues compose a loop that is the same loop that shifts upon binding of 

zopolrestat in ALR2. Inhibitors with improved specificity will very likely take advantage of the subtle 

structural differences that are introduced by the variation in sequences in this area. 

VI. Future Design of Aldose Reductase Inhibitors 

The availability of structural data for ALR2 in its holoenzyme and different ternary forms is likely to 

lead to improvements in the affinity of future generations of inhibitors. As the architecture and plasticity 

of the binding site are better understood, increasingly potent inhibitors may be designed to occupy it. 

Although these structures provide a positive target for drug design, there are a number of negative 

targets. Increased in vivo potency is likely to be derived from the specific inhibition of ALR2 that would 

entail the avoidance of other members of the aldo-keto reductase family. Determination of the structures 

of other members of the family may increase the specificity of compounds by providing structures of 

targets to avoid. While the incorporation of the negative targets in the drug-design process relies on the 

determination of other structures and is likely to be complicated, conventional computational techniques 

may be applied to the problem of the positive target. Two such methods that may hold promise are 

docking [43] and computational thermodynamic perturbation [44]. 

A. Inhibitor Docking to the Enzyme 

Our initial efforts to exploit the ALR2 holoenzyme structure for drug design utilized the program DOCK 

[43]. This program is capable of finding depressions on the surface of the enzyme that could serve as 

binding sites for substrates or inhibitors. Once the correct area is defined, the program rotates structures 

of candidate compounds within this space and scores each compound based upon its steric 

complementarity with the binding site. However, the program does not include the potential polar 

interactions between the inhibitor and protein when scoring. The search was further constrained by the 

inability to include conformational variations both in the test compounds as well as the protein, due to 

computational limitations. 


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This method was used to screen a significant portion of the contents of the Cambridge Structural 

Database [45] against the ALR2 holoenzyme binding site (D.K. Wilson, J. M. Petrash, and F. A. 

Quiocho, unpublished data). Among the approximately 30 highest scoring compounds were several 

aromatic aldoximes that had inhibition constants in the micromolar range. These were similar to 

aldoximes such as benzaldoxime, which has been previously observed to have similar inhibition 

constants [46]. 

Page 242 

A disappointing result was that this search did not “rediscover” any of the known high-affinity ALR2 

inhibitors that were contained in the search library. Before the determination of the ternary complex of 

the enzyme with zopolrestat, this was interpreted as meaning that these compounds bound to the enzyme 

in a conformation somewhat different than the one adopted in the crystal structure used for the search. 

The structure of the ternary complex showed this to be a wrong assumption; it was the protein that 

changed conformation upon inhibitor binding, creating a pocket that did not exist in the holoenzyme 

structure. When bound to the protein, zopolrestat is actually quite similar in conformation to its small 

molecule x-ray structure. It is therefore very possible that different ALR2 inhibitors and substrates may 

cause the enzyme to flex in different ways, creating binding sites that may be different in size and 

chemical nature. 

B. Computational Thermodynamic Perturbation 

Computational thermodynamic perturbation is a powerful, albeit computationally expensive, group of 

techniques that are designed to estimate relative binding affinities of two closely related drugs, given the 

structure of at least one of them complexed with the target protein [44]. This approach has the potential 

to assay candidate compounds in the computer for improvements in inhibitor binding, thereby removing 

the necessity to sythesize and assay these compounds in the lab. 

For a number of reasons, ALR2 promises to be a good system for the application of this technique and 

the experimental verification of the results. The structure is very well determined in complex with 

zopolrestat, a high-affinity inhibitor. A number of zopolrestat derivatives with various functional groups 

decorating the compound have been sythesized and partially characterized with respect to ALR2 

inhibition [13,47]. These compounds could serve as a sort of basis set of controls for the theoretical 

calculations. If parameters used in these calculations can be selected such that the computationally 

derived binding energies agree even qualitatively with the experimentally determined binding energies, 

serious consideration should be given to new compounds that are predicted to bind with enhanced 

affinity. Since ALR2 is crystallizable with zopolrestat bound, there is every reason to believe that 

crystals of the enzyme complexed with similar compounds will be obtainable. Such structures could 

provide the basis for further rounds of drug improvement. 


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VII. Conclusions 

Page 243 

The observation of a protein undergoing a conformational change when binding to an inhibitor, as seen 

with ALR2 and zopolrestat, illustrates a common problem associated with structure-based drug design. 

It is tempting to view proteins as static structures since their crystal structures are static. Attempts to 

design drugs to fit the apparent active site of an enzyme may fail when the plasticity of the protein is not 

taken into account. While the disorder associated with amino acid side chains can be modeled with a 

moderate computational effort, larger conformational changes—such as the loop movement seen in 

ALR2—are virtually impossible to predict. Until this becomes possible, x-ray crystal structures of 

complexes will continue to be indispensible. 

Finally, it can be easy to forget that a compound's affinity for the protein is not the only consideration 

when designing inhibitors of enzymes from a structural point of view. The structures of aldose reductase 

and the FR-1 protein complexed with the drug zopolrestat, a compound with a very high affinity for 

ALR2, can serve as a reminder of how specificity can also be a very important factor. This is 

particularly true when a protein is a member of a family of proteins that share sequence homology and 

are apt to have overlapping specificities. Structure may then play a key role in the determination of 

features that are unique to the target protein and therefore prime considerations when designing 

inhibitors. 


We thank T. Reynolds who assisted with the production of the figures. This work was supported by a 

grant from Research to Prevent Blindness, Inc. and grants EY05856, EY02687, and DK20579 to J. Mark 

Petrash. Florante A. Quiocho is an investigator of the Howard Hughes Medical Institute. 

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10 

Structure-Based Design of Thrombin Inhibitors 

Patricia C. Weber and Michael Czarniecki 

Schering-Plough Research Institute, Kenilworth, New Jersey 

I. Roles of Thrombin in Hemostasis and the Therapeutic Utility of Thrombin Inhibitors 

Page 247 

Thrombin is a serine protease that plays critical roles in both blood clot formation and anticoagulation. 

In the penultimate step of the coagulation cascade, thrombin cleaves soluble fibrinogen to form 

insoluble fibrin. Thrombin also activates other coagulation factors including Factor XIII, the enzyme 

responsible for crosslinking fibrin to further stabilize the thrombus. Additional clot-promoting functions 

include stimulation of platelet aggregation by cleavage of the thrombin receptor. In contrast to its roles 

in clot formation, thrombin participates in anticoagulant functions. For example, thrombin-mediated 

activation of protein C, a protease involved in anticoagulation, is enhanced when thrombin is complexed 

with thrombomodulin, and in this complex, thrombin can neither cleave fibrinogen nor activate platelets. 

The interrelationship among thrombin's many roles in hemostasis is complex and presents several 

mechanisms for inhibition of thrombus formation. For recent reviews see References 1 through 5. 

Most drug discovery efforts focus on thrombin inhibition as a means to prevent the serious 

consequences of thrombus formation in myocardial infarction and stroke. Thrombin inhibitors may also 

prevent clot formation in patients prone to deep vein thrombosis or repeat heart attack. In combination 

with thrombus dissolution therapies, thrombin inhibitors may decrease the incidence of reocclusion due, 

in part, to the release of active clot-bound thrombin. 

In this article, recent examples of small molecule inhibitors interacting at the fibrinogen primary 

specificity pocket and with residues of the catalytic triad 


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are given. Inhibitors designed to make more extended interactions with thrombin are also presented. 

II. Structure of Thrombin 

Page 248 

Thrombin consists of two polypeptides, an A chain of 36 residues and a 259-residue B chain, linked by a 

disulfide bond. The crystallographic structure of thrombin reveals a globular protein organized about 

two β barrels with the overall folding pattern of the chymotrypsin serine protease family [6,7]. The 

catalytic triad and nearby oxyanion hole are located roughly between the β barrels and adopt the 

geometric arrangement required for serine-protease-assisted, peptide bond cleavage (Figure 1). 

Thrombin's multifunctionality and regulation of activity are achieved by specialized subsites on the 

enzyme's surface (Figure 2). Fibrinogen cleavage, for example, involves interactions at the primary 

specificity pocket, the extended fibrinogen recognition exosite, and an additional specificity pocket. 

Subsite interactions differ for cleavage of other thrombin substrates including the thrombin receptor and 

protein C. Additional and overlapping subsites exist for thrombin effector molecules including heparin, 

antithrombin III, and heparin cofactor II [8,9]. 

Figure 1 

Stereoscopic view of the crystallographic structure of thrombin complexed with 

N-acetyl-(D-Phe)-Pro-boroArg-OH. Helical regions are represented in the standard 

way and arrows indicate regions of β sheet. Solid lines show the thrombin 

bound conformation of N-acetyl-(D-Phe)-Pro-boroArg-OH (taken from Reference 10). 

Active-site residues, His57 and Ser195, are shown with a ball-and-stick representation. 

The authors thank Dr. C. L. Strickland for the drawing. 


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Figure 2 

Schematic representation of Subsite Utilization in Thrombin Complexes (after 

Reference 8). Fibrinogen interacts with three thrombin subsites (here thrombin is 

represented by a large oval and the interconnected subsites by an irregular 

three-armed shape). Physiological effectors of thrombin and thrombin inhibitors form 

distinct interactions at these subsites. Additional subsites, such as the 

heparin-binding site, exist on the thrombin surface and are not indicated here. 

The catalytic triad is represented by three circles at the vertices of a triangle. 


Page 249

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III. Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket 

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The majority of synthetic thrombin inhibitors interact at the fibrinopeptide A binding pocket, which 

includes the catalytic residues Ser195 and His57, hydrogen-bonding capabilities within the oxyanion 

hole, peptide backbone functional groups that hydrogen bond with the peptide backbone of the substrate, 

and residues involved in amino acid recognition (Figure 3). Many of these binding determinants are 

utilized by N-acetyl-(D-Phe)-Pro-Arg-chloromethylketone (PPACK [7]) and its boronic acid analog 

(DUP714 [10]). The crystallographic structures of these molecules complexed with thrombin have both 

served as starting points for structure-based drug design and as reference structures for comparison of 

binding modes of other inhibitors. 

The use of arginine boronate esters as transition-state mimetics results in potent peptidyl thrombin 

inhibitors. These inhibitors, however, exhibit significant affinity for other serine proteases that have in 

common a specificity for substrates with basic residues at P1 (e.g. trypsin, Factor Xa, and plasmin). 

Earlier work demonstrated that neutral side chains of P1 boronate esters impart greater selectivity for 

thrombin. The boropeptide shown in Figure 4 was investigated as the prototype of neutral side chain, 

tripeptide thrombin inhibitors [11]. It had a K i against thrombin of 7 nM and shows selectivity relative to 

other trypsin-like plasma proteases. Since these inhibitors have a neutral residue at the P1 site, Deadman 

and coworkers [11] sought to demonstrate the mode of binding to thrombin in the absence of a salt 

bridge with Asp189. 

Figure 3 

Schematic diagram of binding determinants within 

the fibrinopeptide A binding pocket of thrombin and 

their utilization by N-acetyl-(D-Phe)-Pro-boroArg-OH. 


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Figure 4 

Schematic representation of the active-site orientation of a 

“neutral” P1 boronic acid thrombin inhibitor. 

Page 251 

Boron-11-NMR, a sensitive probe of the chemical environment around boronate esters, can distinguish 

between trigonal and tetrahedral forms of boron. The 11B-NMR spectrum of this inhibitor complexed 

with thrombin showed a single peak at -17 ppm that remained constant for 7 hours. The chemical shift 

suggests boron adopts a tetrahedral geometry on binding to thrombin and is consistent with the 

orientation of the inhibitor in the active site shown in Figure 4. While the 11B-NMR revealed an 

interaction within the catalytic site, it could not distinguish between bonding with Ser195 or His57. 

Kahn and coworkers [12] recently investigated the application of synthesized peptidomimetics as novel 

inhibitors of thrombin. Fibrinogen peptide A mimetic (FPAM, Figure 5) incorporates a bicyclic 

peptidomimetic within the turn region of fibrinogen peptide A. The bicyclic peptidomimetic confers 

conformational stability to the turn region as suggested by x-ray crystal structures of fibrinogen peptide 

complexes as well as complexes of BPTI with thrombin. 

X-ray crystallographic studies of FPAM complexed with thrombin (Figure 5) showed that the S1 subsite 

is occupied by the arginine guanidinium [12]. The Val group of FPAM makes extensive hydrophobic 

contacts within the S2 apolar binding site. The Gly at P3 interacts with thrombin via a β-sheet-type 

hydrogen bond with the carbonyl group of Gly216 and appears to be important in the positioning of the 

bicyclic ring corresponding to the β bend. This bicyclic ring, although not aromatic, forms an edge-toface 

contact with Trp215. One of the phenyl rings shows hydrophobic contact with lle174, while the 

other shows no significant interactions with thrombin. By comparison to other inhibitors complexed to 

thrombin, FPAM appears to have a new binding mode that differs from that of substrate, or hirudin, or 

argatroban. 


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Figure 5 

Schematic representation of the principal intermolecular 

interactions of a fibrinogen peptide A mimetic within the active site 

of thrombin. 

Page 252 

Obst et al. [13], departing from the peptide template, designed and synthesized novel nonpeptide 

inhibitors of thrombin. They began with a cyclic template having attachment sites for three side chains 

that would be complementary to the S1, S2, and S3 sites in thrombin. Important to the design was a rigid 

template that would avoid hydrophobic collapse of the side chains and loss of conformational degrees of 

freedom upon complex formation with thrombin. Using computational approaches (Insight 

II/Discover/CVFF force field), possible templates were modeled within the active site of thrombin. 

These studies resulted in the synthesis of thirteen analogs that shared a common template. 

The most active molecule (K i = 90 nM, 8-fold selective versus trypsin) was studied further by x-ray 

crystallography (Figure 6). The positively charged benzamidine binds into the S1 pocket of thrombin 

forming a bidentate hydrogen bond with Asp189. The proximal carbonyl of the rigid template acts as a 

hydrogen-bond acceptor for the amide NH of Gly216. The methylene dioxybenzyl group at P3 interacts 

with thrombin in two ways. An edge-to-face interaction was observed with Trp215, and an oxygen of 

the methylenedioxy group acts as an acceptor for a hydrogen bond with the OH hydrogen of Tyr60A. 

Recent communications from Bristol-Myers Squibb [14,15] describe peptidomimetic inhibitors (Figure 

7) that were designed to bind thrombin with an N- to C-polypeptide chain sense opposite that of the 

substrate and form interactions similar to those made by the first three residues of hirudin (lle1, Thr2, 

Tyr3). In the x-ray crystal structure of BMS-183507 (K i = 17.2 nM) with thrombin [15], the N terminus 

is facing the catalytic site while the methyl ester is 


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Figure 6 


interactions of a nonpeptide bicyclic inhibitor within the 

active site of thrombin. 

exposed to solvent. No specific interactions were observed with the catalytic triad. A bound water 

molecule hydrogen bonded to the Ser195 hydroxyl. 

Page 253 

The complex is stabilized by a network of hydrogen bonds as well as hydrophobic interactions. The 

Phe1-O and the Phe3-NH form hydrogen bonds with Gly216, and the Phe1-NH hydrogen bonds to the 

backbone carbonyl of Ser214. The Phe1 phenyl group occupies the S2 site, while Phe3 interacts within 

the S3 site. 

The retro-inhibitors contain a 4-guanidinobutanoyl group that extends into the S1 specificity site. Rather 

than forming two hydrogen bonds between the guanidine and Asp189 in a manner similar to PPACK, 

BMS-183507 forms only one, with the second hydrogen bond being directed to the carbonyl oxygen of 

Gly219. Binding affinity, as evidenced by loss of more than two orders of magnitude in affinity on 

addition of one or two methylene groups, was sensitive to chain length at this position. 

The allo-Thr hydroxyl oxygen accepts a hydrogen bond from the backbone NH of Gly219. This 

additional interaction accounts, at least in part, for the increase in affinity when compared to the 

inhibitor with Leu in this position. Comparison of the crystal structures of thrombin complexed with 

BMS-183507 and with hirudin reveals that the hirudin residue, Thr2, and the allo-Thr of BMS-183507 

interact differently with thrombin. The hirudin Thr2 binds at S2, 


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Figure 7 

Schematic representation comparing the principal 

inhibitor-to-thombin interactions of related inhibitors with either 

Leu (7a) or allo-Thr (7b) at P3. 

Page 254 

whereas the allo-Thr sidechain is oriented toward the protein exterior and is partially exposed to solvent. 


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Cyclotheonamide A (CtA), a macrocyclic marine natural product derived from the Japanese sponge, 

Theonella sp., inhibits thrombin with an IC 50 value of 


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Figure 8 


interactions of cyclotheonamide A within the active site of 

thrombin. 

Page 255 

100 nM and represents a novel structural class of serine protease inhibitors. An x-ray crystal structure of 

CtA complexed with thrombin was used to determine the molecular basis for this inhibition (Figure 8 

[16]). The Arg-Pro unit binds to the S1 and S2 sites in a manner similar to the Arg-Pro of PPACK. The 

Arg guanidinium group forms a bidentate hydrogen bond with Asp189 while the Pro establishes a βsheet 

interaction with the Ser214-Gly216 backbone. The α-ketoamide acts as a transition-state mimetic 

forming a tetrahedral hemiketal with the hydroxyl of Ser195. Within the complex, CtA adopts a 

relatively open conformation with the Pro orthogonal to the macrocycle and confined by a hydrophobic 

pocket defined by Tyr60A, Trp60D, and Leu99. Two aromatic residues are involved in stacking 

interactions with Tyr60A and Trp60D. Cyclotheonamide A, however, does not effectively match the S3 

interactions provided by the D-Phe group found in PPACK. In CtA, the formamide group is too polar to 

effectively complement the S3 site adjacent to Trp215. The authors note that the complex of CtA with 

thrombin does not appear optimal and suggest that synthetic analogs could significantly improve both 

potency and selectivity. 

Starting with the known thrombin inhibitors Argatroban and Nα(2-naphthyl-sulfonyl-glycyl)-DL-pamidinophenylalanyl-piperidine 

(NAPAP), a group at Roche initiated a medicinal chemistry program to 

develop thrombin inhibitors with reduced toxicity and an improved hemodynamic profile [17]. 


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Page 256 

The discovery program proceeded in four iterative phases which are shown in Table 1. Initial screening 

of low molecular weight organic bases led to the discovery of 1-amidinopiperidine (1–1) as a new 

surrogate for the guanidine and amidine functionality in Argatroban and NAPAP, respectively. A 

distinct advantage of 1-amidinopiperidine is its intrinsic selectivity for thrombin over 


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Page 257 

trypsin. Application of three-fold iterative strategy of design involving synthesis, x-ray crystallography, 

and molecular modeling, this group elaborated the 1-amidinopiperidine from structures that inhibited in 

the micromolar range to some inhibiting in the picomolar range. In doing so significant improvements in 

the selectivity of thrombin relative to trypsin were also achieved. In the case of the D-amino acid series 

(1–2), a “second inhibitor binding mode” that differed from that of Argatroban was identified. In this 

new and unexpected binding mode, the S2 pocket is unoccupied and the napthalenesulfonyl group fills 

the S3 site and overlaps the front of the S2 site. The benzyl group of the phenylalanine is oriented 

toward the protein surface and is partially exposed to solvent. The Argatroban or “inhibitor binding” 

mode was favored by the more potent L-amino acid series (1–3 and 1–4) where the piperidide (1–3) or Nbenzyl 

(1–4) binds to the S2 site and the aryl groups are found in the S3 site. 

IV. Bivalent Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket and the 

Fibrinogen Recognition Site 

A strategy to prepare highly selective thrombin inhibitors involves linkage of molecules capable of 

interacting at distinct subsites. This approach should result in inhibitors more specific for thrombin: 

while serine proteases possess common structural features related to catalysis and some serine 

proteases—including the coagulation enzyme Factor Xa—also exhibit primary substrate specificity for 

positively charged residues, only thrombin possesses recognition subsites for fibrinogen and effector 

molecules such as thrombomodulin. Nature has used this strategy in the evolution of hirudin, the 

anticoagulant protein produced by the medicinal leech. When this effective anticoagulant binds 

thrombin [18–20], the N-terminal domain blocks the primary specificity pocket while the C-terminal 

residues adopt an extended conformation and make multiple interactions within the fibrinogen 

recognition exosite. 

Guided by structural and biochemical information, small molecules capable of simultaneous interactions 

with both the primary specificity pocket and the fibrinogen recognition exosite were designed and 

synthesized. These bivalent inhibitors are composed of three regions: a group to block the primary 

specificity pocket, a sequence to bind the fibrinogen recognition site, and a chemical linker. The bivalent 

inhibitor approach was first executed with peptides [21–22]. In 1990, DiMaio et al. (3–3 [22]) used the 

peptide sequence from hirudin to link (d-Phe)-Pro-Arg-Pro, known to bind at the primary specificity 

pocket [23], with hirudin C-terminal residues, known to bind at the fibrinogen recognition site. 

Polyglycine linkers were also used to connect these sequences (Maraganore 


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et al. [21]). Among these hirudin analogs, the tetraglycine linker appeared optimal (3–8, K i = 2.3 nM, 

Table 2). 

Page 260 

Most of the peptide-based bivalent inhibitors were slowly cleaved by thrombin. Incorporation of a 

ketomethylene pseudo peptide bond (3–4) resulted in a noncleavable bivalent inhibitor that retained high 

thrombin affinity [24]. Decreased proteolysis in bivalent inhibitors increasingly nonpeptide in character 

continues to be observed. 

Chemically simpler linkers were made using multiple methylene-containing glycine variants [25]. The 

dependence of affinity on placement of amide linkage within linkers containing the same number of 

atoms indicated some specific thrombin-to-linker interactions (3–13,14,15,16). This was confirmed in 

the crystal structure of hirutonin-6:thrombin complex (3–26 [26]) where continuous electron density was 

observed for the entire bivalent inhibitor including the linker region. 

The extended nature of the fibrinogen recognition site complicates attempts to reduce inhibitor 

molecular weight while maintaining affinity. Although of similar molecular weight, substitution of the 

sequence -Asp-Tyr-Glu-Pro-lle-Pro-Glu-Glu-Ala-cyclohexylalanine-(D-Glu) for -Asp-Phe-Glu-Glu-lle- 

Pro-Glu-Glu-Tyr-Leu-Gin increases affinity an order of magnitude (compare 3–17 and 3–18). Within a 

series of bivalent inhibitors, inclusion of sulfated tyrosine, the naturally occurring residue of hirudin, 

increases affinity 5 to 6 fold (3–8 compared to 3–11, and 3–1 to 3–2). Only seven residues are present in 

one of the smallest bivalent inhibitors (3–26). 

Increasingly nonpeptide substituents have been incorporated into the primary specificity pocket binding 

portion of the bivalent inhibitors. Higher affinity for thrombin was achieved by replacement of the (D- 

Phe)-Pro-Arg with either dansyl-Arg-(D-pipecolic acid) (3–17, [27]) or 4-tert-butylbenzenesulfonyl-Arg- 

(D-pipecolic acid) (3–18, [27]). While the arginine side chain of these and the (D-Phe)-Pro-Argcontaining 

inhibitors make similar interactions with the aspartic acid within the S1 specificity pocket, 

the dansyl-Arg-(D-pipecolic acid) inhibitors bind in a nonsubstrate mode [27]. This initial result suggests 

that other nonpeptide thrombin inhibitors may be successfully incorporated into bivalent inhibitors. 

Recently, a pyridinium methyl ketone bivalent inhibitor capable of forming a reversible covalent 

complex with thrombin was synthesized (3–26, [28]). Crystallographic analysis of its complex with 

thrombin showed the ketone carbonyl becomes tetrahedrally coordinate by bonding to the side chain of 

thrombin's active site residue, Ser195. Substitutions of cyclohexylalanine for phenylalanine (3–4 

compared to 3–5) and the cyclohexylalanine-containing fibrinogen recognition peptide for the hirudin 

sequence (3–17 compared to 3–18) also contribute to the increased affinity of this bivalent inhibitor. 


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V. Inactivated Thrombin as an Inhibitor of Clot Formation 

Page 261 

A means to selectively inhibit thrombin's role in coagulation while preserving its anticoagulant functions 

involves site-directed mutagenesis of thrombin itself. By introduction of a single mutation, Gibbs et al. 

[29] altered thrombin's relative specificity for fibrinogen and protein C. The engineered thrombin's 

increased activation of protein C over fibrinogen cleavage offers the possibility of inhibiting clot 

formation with a modified human protein, a molecule likely to exhibit few side effects. 

VI. The Role of Structural Information 

The discovery of thrombin inhibitors has benefited from available protein structural information. Models 

of the thrombin overall structure and its active site geometry, constructed from available structures of 

related serine proteases [30], aided in the design of the mechanism-based inhibitors such as PPACK [31] 

and its boroarginine analog [10]. The unexpected, nonsubstrate binding mode of early thrombin 

inhibitors such as NAPAP was revealed by x-ray crystallographic analyses [32]. Iterative structurebased 

design methods have been critical in the optimization of bivalent inhibitors and inhibitors directed 

at the primary specificity pocket. Structures of inhibitor:thrombin complexes are essential for the 

optimization of substitutents forming interactions within the aryl-binding site of the primary specificity 

pocket. In some cases (e.g. Table 1), seemingly minor alterations of the inhibitor can result in dramatic 

changes in the inhibitor's overall interactions with thrombin [17]. 

Drug discovery efforts have also been strongly influenced by results of structural studies of thrombin 

complexed with effectors and substrate peptides. For example, recently the structures of thrombin 

complexed with fibrinopeptide A [33] and human prothrombin fragment F1 [34] have been determined. 

In addition to their role in design of high-affinity inhibitors, these structures provide valuable insights 

for design of drugs specific for the various subsites and conformational states of thrombin. 

VII. Conclusion 

Discovery of therapeutically effective thrombin inhibitors involves issues such as affinity and 

selectivity, bioavailability, and formulation. In addition to these relatively common concerns, the 

complex in vivo mechanisms designed to 


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balance its pro- and anticoagulant activities present additional challenges in the discovery of 

therapeutically effective thrombin inhibitors. 

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11 

Design of Antithrombotic Agents Directed at Factor Xa 

William C. Ripka 

Corvas International, Inc., San Diego, California 


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Serine proteases have long been recognized as important players in a number of biochemical processes 

and their specific and selective inhibition provides multiple therapeutic opportunities [1]. In particular, 

the blood coagulation process is the result of an amplified cascade of proteolytic events in which several 

specific zymogens of serine proteases in blood are activated sequentially by selective cleavages to 

produce active enzymes [2,3]. This process, in pathological circumstances, may lead to the formation of 

a thrombus—an insoluble matrix of fibrin and platelets. Thrombosis is a serious medical problem in the 

United States and Europe as exemplified by the fact that half the people who die each year die of 

cardiovascular related problems. While much recent work in antithrombotic therapeutic approaches has 

focused on inhibition of thrombin, the central role that Factor Xa plays in the coagulation response to 

vascular injury also makes it an ideal pharmacological target for antithrombotic drug development. The 

recent report of the x-ray crystal structure of native Factor Xa [4] allows, for the first time, a wellfounded 

structure-based drug design approach for inhibitors. A number of reviews describing the 

biology [5–10] and chemistry [11,12] of Factor Xa inhibitors have appeared. 

II. Coagulation Cascade 

In the coagulation cascade (Figure 1), a highly amplified process leads to the formation of thrombin, 

which is the primary mediator for the conversion of fibrinogen to fibrin, as well as activation of platelets 

through the thrombin 


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Figure 1 

The coagulation cascade. 

Page 266 

receptor. Thrombin generation is itself, however, the result of Factor Xa in complex with Factor Va on a 

phospholipid surface (the prothrombinase complex) acting on prothrombin. Vascular injury is the 

initiating event in the coagulation process, causing the activation of Factor Xa by the Factor VIIa/tissue 

factor complex. Factor Xa is, therefore, a central and crucial enzyme directly leading to the production 

of thrombin and its inhibition should be effective in blocking thrombogenesis. As a consequence of its 

key role early in the coagulation cascade process Factor Xa represents a potentially valuable therapeutic 

target for potent and specific inhibition. 

III. Proof of Principle for a Factor Xa Inhibitor 

In recent years the method by which certain hematophageous organisms maintain blood flow during 

feeding has been determined. Interestly, several of these organisms utilize Factor Xa inhibitors to 

prevent coagulation [13–15]; the tick anticoagulant peptide (TAP), a small protein isolated from the 

Ornithidoros moubata tick [13], and antistasin isolated from the Haementeria officinalis leech [14] are 

both potent and selective inhibitors of Factor Xa. As expected, these molecules are effective 

antithrombotics in several animal models of thrombosis (Table 1) and provide an important proof of 

principle with regard to the potential effectiveness of Factor Xa inhibitors as therapeutic anticoagulants. 


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Table 1 Factor Xa Inhibitors (TAP, Antistasin) in Experimental Models of Thrombosis 

Rat and rabbit models of venous thrombosis [25] 

Canine model of high shear, coronary arterial thrombosis [6,26] 

Canine model of femoral arterial thrombosis [27,28] 

Rhesus monkey model of acute disseminated intravascular coagulation [29,30] 

Baboon model of platelet dependent arterial thrombosis [9,31,32] 

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In an alternate approach, it has been shown that a covalently blocked, activesite-modified Factor Xa 

(DEGR-Xa) [16] as well as a catalytically impaired recombinant form [17] can be effective 

anticoagulants in models of deep-vein thrombosis [18] and in canine arterial thrombosis models [8]. In 

these examples, the active-site-inactivated Factor Xa competes with the active form for incorporation 

into the active prothrombinase complex since the binding of Factor Xa to Factor Va in this complex is 

independent of the active site [20]. These studies with Factor Xa inhibitors suggest that inhibiting earlier 

in the coagulation cascade, as well as inhibiting the production of thrombin by inactivating Factor Xa in 

the prothrombinase complex, may have certain therapeutic advantages. 

IV. Factor Xa—Structure and Function 

Factor Xa is a 59 kilodalton protein synthesized in the liver and secreted into the blood as an inactive 

zymogen (Figure 2) [21]. Prior to secretion the singlechain molecule undergoes co- and posttranslational 

modifications including removal of a signal sequence [22–24], gamma carboxylation of 

several glutamic acids (Gla) in the N-terminus [33], beta hydroxylation of Asp 63 [34], N-glycosylation at 

two sites [35], and cleavage at two sites, Arg 139 and Arg 142, to give a two-chain molecule [36]. The 

mature form of Factor X consists of a light chain (139 amino acids) and a heavy chain (303 amino acids) 

held together by a single disulfide (Figure 2). The Gla residues are responsible for calcium and 

phospholipid binding and the second EGF domain is thought to mediate binding to Factor VIIIa and 

Factor Va [37,38]. The heavy chain contains the catalytic domain with the prototypic serine protease 

active site triad, His 226, Asp 279, and Ser 376. During coagulation, Factor X is converted to the active 

protease, Factor Xa, by a complex of Factor VIIa/tissue factor or a complex of Factor IXa/Factor, 

VIIIa/phospholipid, and calcium, both of which cleave a specific Arg-Ile bond to release an activation 

peptide (Figure 2) [39]. Similar to the activation of chymotrypsin, trypsin, and thrombin, the newly 

formed N-terminal Ile folds into the interior of the protein to form an ion pair at the active site with 

Asp] 375 [39,40]. In the presence of calcium ions the newly formed Factor Xa associates with Factor Va 

on a phospholipid membrane surface to form the 


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Figure 2 

Factor Xa structure. Residues of the catalytic triad (His 226 , Asp 279 , Ser 376 ) are circled. 

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prothrombinase complex that rapidly converts prothrombin (PT) to thrombin by cleavage at two sites in 

PT, Arg 271–Thr 272 and Arg 320–Ile 321 [41]. The x-ray structure of human des(1–45) Factor Xa at 2.2 Å 

resolution has now been reported [4] (Figure 3). 

V. Natural Inhibitors of Factor Xa 

Several small, potent, and naturally occurring Factor Xa inhibitors—tick antico-agulant protein 

(TAP)[3], tissue factor pathway inhibitor (TFPI) [42], antistasin (ATS) [43], Ecotin [44,45]—have been 

isolated and characterized (Table 2). All but TAP apparently inhibit the enzyme in the extended 

substrate conformation referred to as the standard mechanism of inhibition (Figure 4) [47]. In this 

standard mechanism the inhibitor presents a conformationally constrained binding loop with a partial 

beta sheet motif to the target enzyme that mimics the required substrate conformation and, after binding 

to the enzyme, can undergo a reversible proteolytic hydrolysis at the reactive site peptide bond (P1–P1'). 


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Figure 3 

Stereo ribbon diagram of Factor Xa [4]. Residues of the catalytic triad are shown (His 57 , Asp 102 , Ser 195 ) as well as residues 

of specific interest for the binding of small molecules to the active site: Glu 192 ; S4 pocket residues Tyr 99 , Phe 174 , Trp 215 ; and 

S1 pocket residue, Asp 189 . Residues are designated with the chymotrypsin numbering. 


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Table 2 Naturally Occurring Inhibitors of Factor Xa 

Inhibitor Source K i Structural information 

TFPI Human 3 pM [49] 

90 nM [50] 

Ecotin Escherichia coli 50 pM [44] X-ray; complex with trypsin [46] 

TAP Ornithidoros moubata (tick) 135 pM [13] 2D-NMR [57,58] 

Antistasin Haementeria officinalis (leech) 61 pM [51] (X-ray in progress) [52] 

AcAP5 Ancylostoma caninum 43 pM [19] homology to Ascaris lumbricoides 

var.suum [79,80] 

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Studies of these natural inhibitors can be useful in defining the active site requirements for Factor Xa 

inhibition, and importantly, can indicate the level of inhibition that may be necessary for an effective 

Factor Xa inhibitor, recognizing that TAP and antistasin have evolved to yield functional, in vivo 

antithrombotics. Table 3 shows the reactive-site sequences of these substratelike inhibitors as well as the 

cleavage site sequences recognized by Factor Xa in the activation of prothrombin (PT), Factor VII, and 

Factor V. 

A. Tissue Factor Pathway Inhibitor (TFPI) 

The mature tissue factor pathway inhibitor (TFPI) is a 276-residue protein consisting of three tandom 

domains with homology to the Kunitz-like protease 


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Figure 4 

Extended binding modes for substrates and inhibitors. Sites in the enzyme (S) and in the 

inhibitor (P) are designated by the Schecter-Berger notation [48]. 


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Table 3 Active Site Sequences for Factor Xa Substrates and Inhibitors 

Substrates P4 P3 P2 P1 P'1 P'2 P'3 P'4 

PT 271,272 Ile Glu Gly Arg Thr Ala Thr Ser 

PT 320,321 Ile Asp Gly Arg Ile Val Glu Gly 

FVII Pro Gln Gly Arg Ile Val Gly Gly 

FV Lys Lys Tyr Arg Ser Leu His Leu 

Inhibitors 

Antistasin Val Arg Cys Arg Val His Cys Pro 

Ecotin Val Ser Thr Met Met Ala Cys Pro 

TFPI-II Gly Ile Cys Arg Gly Tyr Ile Thr 

AcAP5 Cys Arg Ser Arg Gly Cys Leu Leu 

AcAP6 Cys Arg Ser Phe Ser Cys Pro Gly 

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inhibitors [42]. A potent inhibitor of both Factor VIIa and Xa as well as trypsin, TFPI does not, 

however, have significant activity against leukocyte elastase, urokinase, activated Protein C, tissue 

factor plasminogen activator, thrombin, or kallikrein [53,54]. The second Kunitz domain from the Nterminus 

of TFPI has been identified as primarily responsible for the Factor Xa inhibition while both the 

first and second domains contribute to inhibition of Factor VIIa [42] (Figure 5). The proposed 

mechanism for this Factor-Xa-dependent inhibition of FVIIa/tissue factor involves the formation of a 

quarternary FXa-TFPI-FVIIa/TF complex [42]. The recombinant, isolated second domain, TFPI-II, has 

a K i for Factor Xa of 90 nM [50] compared to 3 pM [49] for the intact protein. The sequence of the 

P4–P5' region of the Factor Xa inhibitory second Kunitz domain (Table 3) has been incorporated into a 

prototypic Kunitz inhibitor, bovine pancreatic inhibitor (BPTI), to produce potent and selective Factor 

Xa inhibitors [75,76]. 

B. Antistasin (ATS) 

Antistasin is one of several anticoagulants isolated from the Mexican leech, Haementeria officinalis 

[14]. It is a 119-amino-acid cysteine-rich protein with a primary structure that shows a two-fold 

sequence symmetry suggesting the molecule possesses two separate and distinct domains [51]. 

Mutagenesis studies have shown that ATS binds to Factor Xa in a substratelike manner in the 

P3(Arg 32–P'3(Cys 37) regions and is cleaved only in the first domain [55]. 


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While the x-ray structure of antistasin has not been reported, it is known that a Factor-Xa-induced 

cleavage occurs between Arg 34 and Val 35 suggesting this peptide loop conforms to the conformationally 

rigid substratelike conformation suggested by other known protein inhibitors of serine proteases [55]. A 

hallmark of this mode of inhibition is the rigid structure around the cleaved 


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Figure 5 

Predicted secondary structure of tissue factor pathway inhibitor (TFPI) showing the Factor 

Xa and Factor VIIa inhibitory domains. The arrows point to the P1 sites. 

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bond, often imposed by cysteine crosslinks constraining the two ends of the cleavage site to be in close 

proximity even after cleavage [47]. Antistasin has cysteines at the P2(Cys 33) and P'3 (Cys 37) positions. 

C. Tick Anticoagulant Peptide (TAP) 

The tick anticoagulant peptide (TAP) is a 60-amino-acid polypeptide isolated from the soft tick 

Ornithodorus Moubata and is a potent (K i = 2–200 pM) and selective inhibitor of Factor Xa, both as the 

free enzyme and in the prothrombinase complex [13]. The TAP anticoagulant does not inhibit trypsin or 

other trypsinlike serine proteases and, importantly, is not cleaved by Factor Xa. The mechanism by 

which TAP inhibits Factor Xa appears to be unique and it apparently does not utilize the substratelike 

binding modes characteristic of antistasin and the Kunitz inhibitors. Mutagenesis studies have shown 

that the primary interaction of TAP with Factor Xa occurs at the N-terminous where Arg 3 appears to 

play a key role [56]. The solution structure of TAP has been deter- 


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Figure 6 

Stereo diagram of the NMR solution structure of the tick anticoagulant peptide. The crucial 

N-terminus Arg 3 is indicated along with the pattern of cysteine bonds. 

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mined by 2D-NMR studies [57,58] (Figure 6). With the exception of the region neighboring the 

Cys 15—Cys 39 bond in TAP, these studies support the originally proposed idea that there are significant 

structural similarities between TAP and Kunitz proteinase inhibitors [10]. Nevertheless, it is clear that 

TAP inhibitors FXa by a fundamentally different, and as yet, not fully understood mechanism. 

D. AcAP's 

In addition to ticks and leeches, other hematophagous organisms such as hook- worms have also evolved 

potent and selective Factor Xa inhibitors as anticoagulant strategies. Two such proteins, AcAP5 and 

AcAP6, have been isolated from the Ancylostoma caninum hookworm [19]. The AcAP5 protein is a 77amino-acid 

polypeptide with 10 cysteine residues. It inhibits the amidolytic activity of Factor Xa with a 

K i of 43 ± 5 pM. Incubation of rAcAP5 with its target enzyme Factor Xa results in partial cleavage of 

the Arg 40–Gly 41 peptide bond suggesting the sequence around this cleavage site can adopt the restricted 

conformational requirements of substrates [47]. 

The AcAP6 protein is a 75-amino-acid polypeptide, also with cysteines, with a K i for Factor Xa 

inhibition of 996 ± 65 pM. Alignment of the sequences of AcAP5 and AcAP6 suggest the P1 residue in 

AcAP6 is Phe 38 and not the basic residue usually associated with Factor Xa specificity [19]. Substitution 

of Phe 38 in AcAP6 with Arg resulted in a mutant that inhibited Factor Xa with a potency similar to 

rAcAP5. Both AcAP6 and ecotin suggest that an Arg or 


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Lys in the P1 position is not an absolute requirement for potent and selective activity. 

E. Ecotin 

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Ecotin, a protein isolated from Escherichia coli, is a promiscuous protease inhibitor that potently inhibits 

kallikrein, urokinase, Factor XIIa, granzyme B, trypsin, chymotrypsin, and elastase (reviewed in 

Reference 46). As with most protein inhibitors (hirudin [59] and TAP [10] being the exceptions) ecotin 

presents a ‘baitlike’ substrate sequence to the target protease resulting in a reversible cleavage of the 

P1–P1' peptide bond. However, unlike other Factor Xa inhibitors that require basic residues such as Arg 

or Lys in the P1 position, ecotin is cleaved between two hydrophobic amino acids, Met 84 and Met 85 [44]. 

Three preliminary crystal structures of ecotin with the serine proteases chymotrypsin, trypsin, and 

fiddler crab collagenase have been described [46]. The conformation of the sequence around the reactive 

site is similar to the bovine pancreatic trypsin inhibitor (BPTI) but differs in that the Cys at P' (not P2 as 

in BPTI) provides the rigidifying function for the reactive-loop sequence. Interestingly, antistasin has 

cysteines at both the P2 and P3' positions. The Pro at P4' is commonly found in FXa inhibitors including 

antistasin. 

In its complexes with the serine proteases for which structures are available [46] there is a sub van der 

Waals contact between Met 84-C and the enzyme Ser 195-O. The Met 84-O faces the oxyanion hole and 

forms hydrogen bonds with Ser 195 and Gly 193. In the trypsin structure the Met 84 side chain extends into 

the S1 site in a manner similar to Lys 15 in the BPTI-trypsin complex. Ecotin also forms both beta sheet 

hydrogen bonds to the enzyme Gly 216, Ser 82-N and O to Gly 216 O and N. 

VI. Small Molecule Inhibitors of Factor Xa 

While the x-ray structure of native Factor Xa has been reported [4] the nature of its crystal packing, 

specifically the fact that the active site of one Factor Xa molecule is blocked by the N-terminus of a 

second resulting in a “continuous polymeric structure,” apparently has precluded diffusing inhibitors 

into the preformed crystals to obtain complexes. Complexes with inhibitors cocrystallized with Factor 

Xa also have not been reported [81]. Thus, efforts to do structure-based design with this enzyme have 

relied on molecular modeling. 

Since, to date, it has not been possible to directly obtain x-ray structures of inhibitor complexes with 

Factor Xa, the substantial information available with respect to how serine proteases, particularly 

thrombin, bind inhibitors can 


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be utilized to model known Factor Xa inhibitors using the x-ray coordinates of native Factor Xa. 

Preliminary modeling of the several inhibitors described below into the Factor Xa active site was 

accomplished by the author [69] by first superimposing the backbone atoms (N, Ca, C) of the catalytic 

triads (His 57, Asp 102, Ser 195 in the benzamidine:thrombin and PPACK:thrombin x-ray structures on the 

corresponding residues in Factor Xa. This allowed an excellent fit of the P1 basic groups, 

arylbenzamidine in the case of benzamidine and arginine for PPACK, into the S1 pocket of Factor Xa. 

These groups were then used as templates for positioning the appropriate P1 basic groups of the various 

synthetic inhibitors [69]. Holding these docked P1 groups fixed, the remaining rotatable bonds were 

manipulated to allow a reasonable and complementary fit of the inhibitor atoms to the solvent accessible 

surface of the Factor Xa active site. In those cases where it was possible, hydrogen bonds, particularly to 

Gly 216, were formed. To fit extended peptide sequences such as that for antistasin and the antistasinderived 

peptides described below, the backbone atoms of the residues around the cleavage site (e.g., 

P4–P4') were positioned using the corresponding BPTI backbone atoms as a template. This was done 

after first aligning the trypsin catalytic triad backbone in the BPTI:trypsin x-ray complex (vida infra) to 

Factor Xa. Where the side chains of the bound peptide segments differed from BPTI, their orientation 

was either modeled for maximum complementarity to the Factor Xa molecular surface or set by an 

algorithmic approach [78]. In some cases the structures obtained were energy minimized initially by 

steepest descent followed by conjugate gradient minimization. 

Recently, compounds based on a bisamidine motif (e.g., DX-9065a) have been reported as potent and 

selective Factor Xa inhibitors (1, DX-9065a) [60–63]. 

The position of the amidino group makes little difference to the Factor Xa potency of these compounds 

but, interestingly, has a dramatic effect on the selectivity towards thrombin [62]. It was also observed 

that one carboxylic acid isomer (CX-9065a) was 7 times more potent on Factor Xa than the other. A 

second set of analogs shows a similar SAR [60]. 


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In both cases the acids are much more selective for Factor Xa over thrombin. An initial modeling study 

using a homology-built Factor Xa structure proposed a fit of DX9065a to Factor Xa in which the 

amidinoary1 group occupies the S1 pocket and the acetimidoyl group is directed out of the S4 pocket 

[60]. 

Lin et al. [64] have provided a more systematic study of possible fits of compound 2 and DX9065a 

using the recently available Factor Xa coordinates [4]. After aligning the His 57, Ser 195, and Asp 102 

backbone atoms for Factor Xa and thrombin (in the benzamidine:thrombin x-ray structure [65]) the 

arylamidino group of 2 was superimposed on the benzamidine template and a systematic conformational 

search was performed on the rotatable bonds of inhibitor 2. Energetics and complementarity to the 

Factor Xa surface determined a saved set, about 300 low-energy conformations, for further study. The 

final result was an optimized structure in which the acetimino group of 2 fits into 

Figure 7 

Molecular modeling fit of compound 2 with the arylamidino group positioned in the S1 

pocket and the acetimino group in the S4 cation-π site [69]. 


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the S4 pocket formed by the aromatic residues Trp 215, Tyr 99, and Phe 174 (Figure 7). It was proposed that 

this collection of aryl residues forms the basis for a cation-π interaction that has recently been well 

documented in other cases [66]. Interestingly, this cation-π site is absent in thrombin's equivalent “aryl 

binding site” where the corresponding residues are Trp 215, Leu 99, and Ile 174—residues that cannot 

provide the π-electrons necessary for stabilization of the cation in the inhibitor. The apparent preference 

for FXa inhibitors to have cations in the P3,P4 position may be directly related to the ability of the 

Factor Xa S4 site to stabilize these cations via a cation-π interaction. Factor Xa appears to be unique 

among the coagulation factors in providing this electron-rich S4 pocket (Table 4). 

The initial discovery of bisamidine structures as potential Factor Xa inhibitors was actually made much 

earlier with the finding that compounds such as 4 showed an almost 300-fold preference for Factor Xa 

over thrombin with a K i of 13 nM (FXa) [67], 

As with the DX-9065 analogs and compounds 2 and 3, the Factor Xa potency was relatively insensitive 

to the positioning of the amidino groups (4,4' versus 3,3') while replacing the 7-membered cycloalkyl 

ring with the 5 or 6 membered ring analogs reduced potency by about 10 fold. Model building 

compound 4 and docking into Factor Xa [69], again using the x-ray benzamidine:thrombin complex [65] 

as a template, shows that the second aryl amidino group can be positioned into the S4 aromatic pocket of 

Factor Xa in a conformation closely related to the mode of binding proposed for DX9065a (Figure 8) 

[64]. 

In an effort to compare the relative efficacy of thrombin versus Factor Xa inhibitors, Markwardt et al. 

[67] synthesized a set of amidinoaryl compounds with moderate potency as Factor Xa inhibitors (5). 

Table 4 S4 Residues in Selected Serine Proteases 

Residue Position a Factor Xa Thrombin Factor VIIa Trypsin 

99 Tyr Leu Thr Leu 

174 Phe Ile Pro Gly 

215 Trp Trp Trp Trp 

aChymotrypsin numbering system. Sequence alignments by comparison of x-ray structures (sequence 

for Factor VIIa). 


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The n=3 chain length was optimal while the nature of the tosyl group on the α- nitrogen was relatively 

nonspecific, with tosylGly and Nα-β-naphthylsulfonyl- Gly being of similar potency. While the phenyl 

group on the amide nitrogen was best, other groups were also tolerated. Although the authors did not 

speculate on how these compounds were bound to Factor Xa, it is reasonable to suggest that the amidino 

phenyl group fits in the S1 pocket similar to the orientation determined by x-ray crystallography for 

benzamidine in the benzamidine:thrombin x-ray crystal structure. If the aryl amidino group of 5 is 

matched to that of benzamidine after alignment of the catalytic triad backbone atoms (N,Cα,C) for 

thrombin and Factor Xa, a proposed fit of 5 to Factor Xa can be made (Figure 9) [69]. This mode of 

binding is consistent with the structure activity relationships observed but does not suggest the reasons 

for the observed small preference for Factor Xa over thrombin. 

Figure 8 

Molecular modeling fit of compound 4 in the Factor Xa active site [69]. The carbonyl of the 

cycloheptanone makes a hydrogen bond (3.12 Å) to N-Gly 216 . 


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Figure 9 

Molecular modeling fit of compound 5 in the Factor Xa active site. 


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Figure 10 

Proposed model of dansyl-Glu-Gly-Arg-chloromethyl ketone in Factor Xa [4,69]. 

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Tulinsky and coworkers [4] have proposed a model for the complex of dansyl-Glu-Gly-Argchloromethyl 

ketone using the thrombin-PPACK crystal structure as a template for the fit to Factor Xa 

(Figure 10). 

Using antistasin as a starting point, Ohta et al. [68] have synthesized a series of cyclic peptides based on 

the antistasin sequence. Three of these peptides are shown below and represent the most potent in the 

series. 

ATS29-47 NH2-Ser-Gly-Val-Arg-Cys * -Arg-Val-His-Cys * -Pro-His-Gly-Phe-Gln-Arg- 

Ser-Arg-Tyr-Gly-OH 

K i (FXa) 

0.035 µM 

ATS29-40 NH2-Ser-Gly-Val-Arg-Cys * -Arg-Val-His-Cys * -Pro-His-Gly-OH 11.8 µM 

dR-ATS32-38 NH2-dArg-Cys-Arg-Val-His-Cys-Pro-OH 0.96 µM 

(The Cys * -Cys * are joined in disulfide bonds to form cyclic structures) 


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These molecules are cleaved by Factor Xa suggesting they bind in a similar manner to antistasin itself. 

Assuming the sequence around the cleavage site occupies the FXa active site locally in a manner similar 

to BPTI in the BPTI:trypsin complex, a modeled structure of the complex can be constructed 


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Figure 11 

dArg-ATS 32-38 modeled into the active site of Factor Xa utilizing the BPTI: trypsin x-ray structure as a template [69]. 


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using BPTI residues 11–19 as a template [69]. The modeled dR-ATS 32-38:FXa complex is shown in 

Figure 11. Interestingly, these peptides do not inhibit trypsin even at 1000-fold higher concentrations 

than their FXa inhibitory concentrations. Antistasin, on the other hand, inhibits trypsin with a K i of 10 

nM. 

Preliminary reports have appeared describing a pentapeptide that is a potent (K i=3 nM) and selective 

inhibitor of Factor Xa (SEL2711) [70,71]. 

Two possible modes of binding can be envisioned for this compound with either the methylpyridinium 

group occupying the S1 site in a “substratelike mode” or the p-amidinophenyl group in the S1 pocket, 

which would require a reversed binding reminiscent of the hirudin-thrombin interaction [59]. Figure 12 

shows the case for the amidinophenyl group in the Factor Xa S1 pocket [69]. In this mode of binding the 

methylpyridinium group easily fits the S4-aryl binding site and is well positioned for a π-cation 

interaction. 

Of interest from a drug-design viewpoint is the finding that cyclotheonamide, a compound isolated from 

a marine sponge and originally reported as a thrombin inhibitor, has been found to also inhibit Factor Xa 

with a K i of 50 nM [72]. Cyclotheonamide possesses a novel α-ketoamide transition state functionality 

and x-ray structures of cyclotheonamide with trypsin [73] and thrombin [74] provide templates for 

modeling this inhibitor into Factor Xa [69]. In the resulting fit (Figure 13) cyclotheonamide does not 

project functionality into the S4-cation-π site and would not be expected to show Factor Xa selectivity. 

VII. Defining the Requirements for Factor Xa Inhibition by Mutagenesis of BPTI 

It has been known for some time that many examples of naturally occurring Kunitz inhibitors exist, both 

isolated and as domains in larger proteins, which inhibit a variety of serine proteases [47]. This strongly 

suggests that this molecular framework is compatible with inhibition of this general class. The contact 

region between these inhibitors and their protease targets is known from a 


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Figure 12 

Modeled fit of SEL2711 in Factor Xa with the arylamidino group positioned in the S1 pocket and the methylpyridinium 

group in the cation-π S4 site. 


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Figure 13 

Cyclotheonamide modeled into Factor Xa utilizing the x-ray structure of cyclotheonamide:trypsin [73] and 

cyclotheonamide:thrombin [74] as templates [69]. 


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number of x-ray structures of complexes. Appropriate site-specific and random mutagenesis, particularly 

of the P3-P4' residues of a prototypic Kunitz inhibitor, bovine pancreatic trypsin inhibitor (BPTI), has 

been shown to result in potent and selective Factor Xa inhibitors [75,76]. 

A potent inhibitor of trypsin, kallikrein, and plasmin, BPTI does not inhibit Factor Xa. It binds to serine 

proteases such as trypsin in an extended substrate mode from residue 13 (P3) through 17 (P'2) [47]. A 

second loop from BPTI also extends into the active site bringing residues 34, 39, and 46 into contact 

with the protease-active site. In terms of spatial proximity of residues three clusters can be defined: 

cluster 1 (13,39); cluster 2 (11,17,19,34); and cluster 3 (16,18,20,46). While residue 39 is approximately 

in the same region of space as residue 13 (9.4 Å CB-CB) the CA rarrow.gif CB vectors are directed in 

different directions and substitution at 39 would not be expected to have a cooperative effect with 

residue 13. Residue 34 on the other hand is in a key position. It is centrally located between residues 

11,17, and 19 with CB-CB distances of 5.6, 5.7, and 6.5 Å respectively, and its CA rarrow.gif CB 

vector converges with the corresponding vectors from these residues to a common point in space. This 

residue is therefore expected to have a substantial cooperative effect with the other residues of cluster 2. 

Finally residue 46 is close to residue 20 (CB—CB of 6.5 Å) although the CA rarrow.gif CB vectors are 

approximately parallel and cooperative effects are expected to be minimal. The BPTI residues 

11,12,13,15–20, 34,39, and 46 were therefore the focus of the site-directed and random mutagenesis 

studies. Residue 14 is Cys in BPTI and was not modified in the mutants since it is required for structural 

reasons. 

A. Site Specific Mutagenesis 

As a starting point for the design of BPTI-based Factor Xa inhibitors, the second domain of TFPI (TFPI- 

II) was used as a template [75,76]. Table 5 shows the results of site-directed mutagenesis of BPTI. 

Mutant 50cl is a direct analog of TFPI-II with the exception of the Lys at position 46. The finding that 

4c2 and 4c10 are essentially equivalent in potency (K i 2.8 versus 1.8 nM) and are identical 

Table 5 Site-Directed BPTI Mutants with Factor Xa Inhibition 

K i(nM) 12 13 14 15 16 17 18 19 20 34 39 46 

r-TFPI-II 90 Gly Ile Cys Arg Gly Tyr Ile Thr Arg Lys Leu Glu 

50cl 205 Gly Ile Cys Arg Ala Tyr Ile Thr Arg Lys Leu Lys 

4c2 2.8 Gly Ile Cys Arg Ala Tyr Ile Thr Arg Val Leu Glu 

4c10 1.8 Gly Ile Cys Arg Ala Tyr Ile Thr Arg Val Leu Lys 

57c1 1.6 Gly Ile Cys Arg Ala Tyr Ile Ile Arg Val Leu Lys 


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w.t. 

BPTI 

>1 mM Gly Pro Cys Lys Ala Arg Ile Ile Arg Val Arg Lys 


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cal in sequence with the exception of the Glu 46 Lys switch, suggests this residue is of minor importance. 

Therefore, the potency of 50cl (within a factor of 3) is consistent with r-TFPI-II. On the other hand, the 

incorporation of Val for Lys at position 34 leads to a dramatic increase in potency (˜100 fold; cf. 4c10 

and 4c2 with 50c1). From cluster 2 above this suggests the importance of P5, P'2,P'4 for potency. The 

changes in wild type BPTI that result in a potent Factor Xa inhibitor are Lys 15 rarrow.gif Arg 15, Arg 17, 

rarrow.gif Tyr 17, and Arg 39 rarrow.gif Leu 39. 

B. Random Mutagenesis 

11Libraries of mutant BPTI were created by inserting mutagenic cassettes in the BPTI gene of 

filamentous phage PIII coat proteins [76]. These libraries produced large numbers of mutants (~10 6) 

with randomized amino acids in positions 11, 13, 16, 17, 18, 19, 20, 34, and 39. The mutants were 

panned against Factor Xa, which was affixed to a solid support by a nonneutralizing antibody and the 

most potent inhibitors were separately expressed as soluble proteins. By this process it was possible to 

determine consensus sequences at the reactive sites and to define the pharmacophore requirements of 

inhibitors of Factor Xa in both a functional and conformational sense from the P4 to the P5 positions. 

Inhibitor amino acid preferences from both site directed and random mutagenesis studies are shown in 

Table 6. 

VIII. Positional Requirements of Factor Xa Inhibitors (Table 6) 

Examination of models of BPTI-mutants bound to Factor Xa show the L-amino acids in the P3 position 

project into solvent. In the Factor Xa cleavage sites in thrombin these residues are polar and acidic (Glu, 

Asp); they are polar and basic in antistasin (Arg), and polar and neutral in Ecotin (Ser). The exception is 

TFPI- II, with this position occupied by Ile. The BPTI random mutant results are consistent with the 

TFPI-II case and show a preference for aliphatics or aromatics in this position. There is a hydrophobic 

pocket in the enzyme, formed by Trp 215, Tyr 99, and Phe 174, that would be accessible to a D-residue in 

this position. 

The accessibility of the S2 pocket of the enzyme by P2 groups would be expected to be influenced by 

the orientation of Tyr 99. As the x-ray structure shows its position this residue puts severe limitations on 

the size of the P2 group. Consistent with this is the observation that Gly is the sole residue in the 

FXa:thrombin cleavage sites. Little information is available from the BPTI mutants, TFPI-II, or 

antistasin, which all have a structural requirement for Cys at this position. Synthetic compounds show, 

however, that in potent inhibitors large bulky aromatics are, in fact, allowed at this position, a situation 

that requires Tyr 99 to move out of the way [75]. 


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Additionally, the binding of BPTI mutants also requires Tyr 99 to move because of potential severe 

interactions with the Cys 14–Cys 38 bridge of the BPTI mutants. While larger groups can be 

accommodated, structural requirements are fairly rigid in agreement with the limited mobility expected 

of the Tyr 99. The apparent requirement for a P2-Gly in the larger substrates may suggest that in these 

cases extended binding occurs that does not permit movement of Tyr 99. 

In the P1 position, as expected, a basic group is preferred. Interestingly, of the two naturally occuring 

basic amino acids, arginine is preferred over lysine. This is seen in both the BPTI mutants as well as the 

Arg rarrow.gif Lys switch in antistasin. This may be due to Ala 190 in the S1 pocket of FXa, which 

cannot orient and stabilize the BPTI lysine analog as Ser 190 does in trypsin. An interesting exception to 

the need for a basic group is in Ecotin where a methionine occupies this site. The x-ray of Ecotin with 

trypsin clearly shows this neutral residue in the P1 pocket, aligned very closely to that seen for lysine in 

the BPTI:trypsin complex, and proximal to the charged Asp 189 [77]. Apparently, extended binding over 

the rest of the site compensates for this energetically unfavorable situation. 

In the P'1 position, the natural cleavage sites use Thr and Ile while Ecotin has Met. In contrast to 

thrombin, FXa lacks the 60-insertion loop and can accommodate large groups at this position. The BPTI 

mutants, however, are forced to use a small residue (Ala) because of steric hindrance from the Cys 58- 

Cys 42 group residue 61 in the enzyme. The inhibitor TFPI-II has a Gly at P'1. 

The BPTI mutants, TFPI-II, and antistasin all show a preference for aromatic groups at P'2. In the BPTI 

panning experiments Tyr was selected more than 80% of the time at this position. It can be seen from 

Table 5 that the Arg to Tyr change at position 17 is one of three significant changes that converts wild 

type BPTI from a non-Factor Xa inhibitor to a ˜1.6 nM inhibitor. There is a possible hydrogen-bond 

interaction between Tyr 17 of the inhibitor and Gln 192 of the enzyme, which may explain the strong 

preference. 

While models suggest the P'3 residue is directed at solvent and the FXa thrombin cleavage sites have 

polar residues at this position (Thr, Glu), the BPTI mutant results show a clear preference for a 

hydrophobic group. It is possible that aromatic groups can pack to Phe 41 of the enzyme. Mutant results 

show Ile is favored over Phe, His, which in turn is selected over Tyr. 


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IX. Conclusions 

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Factor Xa is clearly an important component of the coagulation process and inhibition of this enzyme 

can lead to potent anticoagulant effects. Recently, a number of naturally occurring anti-Factor-Xa 

polypeptides have been isolated from several hematophagous organisms including ticks, leeches, and 

hook- worms. With the exception of TAP, these molecules appear to bind to Factor Xa by the standard 

mechanism of inhibition proposed earlier [49]. The sequence information derived from these inhibitors 

as well as the natural cleavage sites of substrates of Factor Xa can be used along with the conformational 

constraints imposed by the proposed substrate-like binding to define the pharmacophore requirements of 

the active site of Factor Xa. The structurally rigid BPTI mutants, which have been found to be potent 

Factor Xa inhibitors, also provide important conformational information particularly with regard to the 

specific binding interactions on the P' side of the Factor Xa active site. A number of small molecule 

inhibitors have also recently been reported which appear to take advantage of a unique cation-π S4-site 

available in Factor Xa to achieve good selectivity with moderate potency. The availbility of the X-ray 

structure of native Factor Xa has allowed molecular modeling approaches to suggest possible fits of 

these inhibitors to the Factor Xa active site. 

Note Added in Proof 

After this review was written, the x-ray structure of Factor Xa with DX-9065a was reported [81]. 

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serine proteases of the coagulation cascade—factor Xa. Eur J Med Chem 1995; 30 (Suppl):88s–100s. 

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Xa. Pat Appl WO 94/01461; 1994. 

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12 

Polypeptide Modulators of Sodium Channel Function as a Basis for the 

Development of Novel Cardiac Stimulants 

Raymond S. Norton 

Biomolecular Research Institute, Parkville, Victoria, Australia 


Page 295 

Cardiovascular diseases remain one of the major causes of premature death in western societies. Chronic 

congestive heart failure (CHF) in particular is a common disease with a poor prognosis, median survival 

times after the onset of heart failure being 1.7 years in men and 3.2 years in women [1]. Current 

treatment relies on diuretics to reduce fluid volume, vasodilators to decrease the work load of the heart, 

and positive inotropic agents to increase cardiac contractility [2]. The most commonly prescribed of the 

positive inotropes is the cardiac glycoside digoxin (Figure 1) [3]. Although this drug has been in 

therapeutic use for over two hundred years, its efficacy in patients with a sinus rhythm has remained 

controversial, and evidence for its beneficial effects is quite recent [3–5]. It is also possible that these 

beneficial effects are not due solely to the positive inotropic activity of digoxin and that its 

neurohormonal effects may also be important [2, 5–7] Nevertheless, digoxin remains a widely used drug 

[3] and it follows that a suitable replacement or adjunct would find access to a significant market 

worldwide. 

The incentive to develop such a replacement follows from the low therapeutic index of digoxin [8,9] and 

the relatively common occurrence of side effects due to digitalis toxicity. In the 1960s and 1970s, 

20–30% of patients receiving digitalis experienced serious toxicity and about one quarter of this group 

died [6, 10]. Digitalis toxicity is manifest in CNS side-effects such as 


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Figure 1 

Structures of the positive inotropes digoxin [3,4], DPI 201-106 [15], and BDF 9148 

[15–17]. In digoxin the R group is (O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1 rarrow.gif 4)-O-2, 

6-dideoxy-β-D-ribo-hexopyranosyl-(1 rarrow.gif 4)-2, 6-dideoxy-β-D-ribo-hexopyranosyl)oxy. In 

DPI 201-106 the configuration at the hydroxyl-bearing carbon influences cardiac 

activity. 

Page 296 

fatigue, visual disturbances, and anorexia, and in cardiac side-effects that depend on the nature and 

extent of the underlying heart disease [3]. Careful monitoring of digoxin serum levels and bioavailability 

have reduced the incidence of digitalis toxicity [3] and the recent introduction of digoxin-binding 

antibodies or antibody fragments has provided an effective means of treating severe digitalis toxicity 

[3,7]. Nevertheless the quest continues for a substitute for the cardiac glycosides in the treatment of 

chronic CHF. 

Positive inotropic compounds can be classified into three groups: cAMP generators, intracellular 

calcium regulators, and modulators of ion channels or pumps [11]. The cAMP generators such as 

dopamine, dobutamine, and milrinone (a phosphodiesterase inhibitor) may worsen ischemia, cause 

arrhythmias, and increase mortality [2,6]. Intracellular calcium modulators have not reached clinical use, 

possibly because of additional effects such as vasoconstriction, 


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whereas, calcium sensitizers such as EMD 57033 may be useful positive inotropic compounds, even in 

the diseased myocardium [12]. 

Ion channel modulation represents another approach to positive inotropy [13]. Sodium channel 

modulators increase Na + influx and prolong the plateau phase of the action potential; sodium/calcium 

exchange then leads to an increase in the level of calcium available to the contractile elements, thus 

increasing the force of cardiac contraction [13,14]. Synthetic compounds such as DPI 201-106 and BDF 

9148 (Figure 1) increase the mean open time of the sodium channel by inhibiting channel inactivation 

[15]. Importantly, BDF 9148 remains an effective positive inotropic compound even in severely failing 

human myocardium [16] and in rat models of cardiovascular disease [17]. Modulators of calcium and 

potassium channel activities also function as positive inotropes [13], but in the remainder of this article 

we shall focus on sodium channel modulators. 

II. The Anthopleurins 

Two decades ago “drugs from the sea” were the subject of high expectations and a good deal of effort in 

various centers around the world. The number of therapeutically useful compounds to have emerged 

from that effort has been rather limited, but with the advent of high-throughput screening it is likely that 

useful new leads will be found, even from species investigated previously. Notwithstanding, some 

valuable leads did emerge from work carried out in the 1970s, amongst which were the polypeptide 

cardiac stimulants known as the anthopleurins. These were isolated from sea anemones, where they are 

components of the animal's venom and are believed to have a function in defense and the capture of 

prey. The work that led to the isolation and characterization of these and related polypeptides from sea 

anemones is covered in earlier reviews [18,19] and will not be reiterated here. 

The best characterized of the anthopleurins is anthopleurin-A (AP-A), which was isolated from the 

northern Pacific sea anemone Anthopleura xanthogrammica and consists of 49 residues cross-linked 

by three disulfide bonds [18,20]. It is active as a cardiac stimulant at nanomolar concentrations in vitro, 

making it some 200 fold more potent on a molar basis that digoxin. Its positive inotropic activity is not 

associated with any significant effects on heart rate or blood pressure [21], and in conscious dogs its 

therapeutic index is 7.5, which is about three-fold higher than that of digoxin [8]. Anthopleurin-A is 

active under conditions of stress and hypocalcaemia [18,22], as well as in ischemic myocardium where 

many other positive inotropes give equivocal results [23]. The profile of activity for AP-A suggests that 

it is a potentially valuable lead in the development of an alternative positive inotrope to digoxin 


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for use in the treatment of chronic CHF. This chapter describes how this development is being tackled 

using the approach of structure-based drug design. 

A. Related Sea Anemone Toxins 

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Anthopleurin-A is a member of a family of sea anemone polypeptides [24] (Figure 2) that is steadily 

increasing in number. These polypeptides have been classified into two groups, designated Types 1 and 

2 [27], which are similar with respect to the locations of their disulfide bridges and a number of residues 

thought to play a role in biological activity or maintenance of the tertiary structure [24,27], but are 

distinguishable on the basis of sequence similarity (>>60% within each type but

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Figure 2 

Amino acid sequences of Type 1 sea anemone polypeptides. The residue numbering system is based on the sequences of AP-A and 

AP-B. Literature references are from Norton [24], except in the case of recently published sequences for Bc III [25], Bg II and Bg III 

[26]. Identical residues are shaded in grey and conserved residues are boxed. The sequences of Bg II and Bg III around residue 28 

could also be aligned to bring the Gly-Cys sequence into register with the other toxins if the Arg were treated as an insertion. Toxins are 

named in this figure according to their genus and species; common names relevant to the text are Ax I = AP-A; Ax II = AP-B; As Ia = 

ATX Ia. 


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polypeptides BDS I and II [40,41], which were claimed to have antihypertensive and antiviral activity, 

and the α-scorpion toxins [24,27,42]. As discussed below, we may expect to obtain some useful 

information from comparisons of the molecular surfaces of these different classes of polypeptides, but 

the utility of this approach depends on the extent to which their binding sites are identical and not just 

partially overlapping, as well as the issue of channel sub-type specificity of the different toxins. 

The synthetic agent DPI 201-106 has been evaluated extensively as a potential replacement for digoxin 

[43]. It is a potent positive inotrope that also acts by delaying inactivation of the sodium channel, but its 

binding site appears to be distinct from that of ATX II [44]. Moreover, it exerts antihypertensive and 

local anesthetic effects and may also antagonise the calcium channel [43]. At present we are not aware 

of any low molecular mass compound that binds to the same site as the anthopleurins. This offers the 

prospect that a mimetic based on the anthopleurins might have a pharmacological profile distinct from 

other positive inotropes. 

The structure of the receptor for the anthopleurins, the α-subunit of the voltage-gated sodium channel, is 

known only in schematic form [35–37]. As illustrated in Figure 3, it contains four homologous domains, 

each consisting of six transmembrane regions (assumed to be helices) designated S1 to S6. The S4 

segments are thought to act as the voltage sensors of the channel. All four 

Figure 3 

Schematic of the α-subunit of the voltage-gated sodium channel, based on its amino 

acid sequence [35–37]. The transmembrane segments S1–S6 in each domain are 

thought to form helices—with the positively charged S4 segment acting as a 

voltage sensor—and the S5–S6 loop of each domain is thought to contribute to the 

transmem-brane pore. Site 3 includes regions of the S5–S6 loops of domains I 

and IV, and the inactivation gate (h) is located on the intracellular segment linking 

domains III and IV. 


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domains contribute to formation of the transmembrane pore, which is believed to be lined by short 

segments from the loops linking S5 and S6 in each domain. Binding site 3 is located on the extracellular 

surface of the channel and involves regions of the loops between S5 and S6 in domains I and IV [37,45]. 

The binding sites for several modulators of sodium-channel activity, including the blocker tetrodotoxin, 

have been mapped quite precisely onto the sequence (and thus the structural model of the channel) by 

combining information derived from comparisons of naturally occurring variants of the channel and 

from site-directed mutagenesis [37]. A similar approach could be applied to the definition of site 3, but 

in the absence of a crystal structure for the α-subunit the three- dimensional structure of this site will 

remain speculative. Therefore current attempts to design a low molecular mass analogue of the 

anthopleurins must be based on the structure of the ligand rather than the receptor. 

C. Development of a New Positive Inotrope 

Being polypeptides, the anthopleurins have limited therapeutic potential in their own right, as they are 

not active following oral administration and are antigenic in experimental animals [18]. Recent advances 

in the field of peptide mimetics, however, lend credence to the concept of harnessing the favorable 

cardiotonic properties of the anthopleurins in a low molecular mass, nonpeptide, synthetic compound. 

The goal of such a development would be to retain the activity of the parent polypeptides in a molecule 

that had good bioavailability and was not antigenic. Ideally, it might also be possible to increase the 

cardiac selectivity of such a compound. 

In order to achieve this goal, a knowledge of the three-dimensional structures of the anthopleurins and 

their structure-function relationships is essential. Significant progress has been made towards these goals 

over the past few years and we are now in a position to commence analogue design and synthesis. The 

following sections in this chapter summarize our knowledge of the cardioactive pharmacophore of the 

anthopleurins and the prospects for mimicking this in a nonpeptide moiety. Aspects of the 

pharmacological profile which such a compound would need to display in order to be useful in CHF 

therapy are also discussed. 

III. 3D Structure 

The structures in aqueous solution of both AP-A [46] and AP-B [47] have been solved using highresolution 

1H NMR data. Structures have also been determined for the Type 1 toxin ATX Ia [48] and the 

Type 2 toxin Sh I [49,50] from NMR data. The main secondary structure element in each of these 

structures is a 


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Figure 4 

Richardson-style diagram of the polypeptide backbone of the 

individual structure of AP-A [46] that is closest to the average over the 

whole molecule. The locations of the sulfurs in the three disulfide bonds 

(4–46, 6–36, and 29–47) are shown in CPK format. The locations of 

reverse turns found in more than half the NMR-derived structures 

(6–9, 25–28, and 30–33) are indicated by darker backbone shading. 

The diagram was generated using MOLSCRIPT [51]. 

Page 302 

four-stranded, antiparallel β-sheet linked by three loops, as illustrated for AP-A in Figure 4. The first of 

these loops, spanning residues 8–16 in AP-A and 8–17 in AP-B, is the largest and least well defined in 

solution (Figure 5), although it contains several residues that are essential for activity, as described in the 

next section. 

Differences between the structures of AP-A, ATX Ia, and Sh I have been noted [46] but the overall 

picture that emerges is one of similar backbone folds for all three molecules, making it likely that 

differences among the potencies and species-specificities of these toxins are due to the presence or 

absence of particular side chains rather than significant structural differences. Given that ATX Ia and Sh 

I have weak or negligible activities on mammalian nerve and heart tissue [24,27], we have focused on 

the anthopleurins in an effort to define the structure of the cardioactive pharmacophore of the sea 

anemone polypeptides. There are several challenges in this endeavor. One is that the structures are based 

on NMR data that, because of the paucity of NOE restraints for surface residues compared with those in 

the core of the structure, do not define the locations of the solvent-exposed side chains very precisely 

(although it should be borne in mind that this may be a more accurate picture of the actual structure in 

solution than one in which the side chains are fixed in a single 


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Figure 5 

Stereo views of 20 structures of AP-B [47] superimposed over the backbone heavy 

atoms (N, C α , C) of residues 2–7 and 17–49. The three disulfide bonds are shown in 

lighter shading. The lower view is related to the upper one by an approximately 180° 

rotation about the vertical axis. 


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location). Another is that both the backbone and the side chains of residues 8– 16 are less well defined 

than the bulk of the structure due to a lack of medium- and long-range NMR restraints between residues 

in this loop and the rest of the molecule, as illustrated in Figure 5. These problems are exacerbated in the 

case of AP-A and AP-B by the presence of multiple conformers in solution, one cause of which is cistrans 

isomerism about the Gly40-Pro41 peptide bond [52]. The additional peak overlap caused by these 

conformers limited the number of NOE restraints that could be obtained from the spectra. Finally, the 

structures available at present are for the free ligand and we have no information on how much these 

structures might change upon binding to the sodium channel. 

One way of addressing the issue of a lack of precision in the locations of functionally important side 

chains is to determine the range of conformational space available to them in different ligands. To this 

end, we undertook a detailed comparison of the structures of AP-A and AP-B in solution [47]. This 

proved to be a useful exercise both in terms of defining the positions of side chains known to be 

important for cardiotonic activity and identifying neighboring residues which might also be involved 

[47]. 

Models have been described in the literature for AP-B [53] and Bunodosoma granulifera toxins II (Bg 

II) [26]. The AP-B model was derived from the structure of Sh I using energy minimization and the Bg 

II model from that of BDS I using energy minimization and 10 ps of dynamics. In both cases the 

calculations appear to have been carried out for the molecule in vacuo without the use of a distancedependent 

dielectric, under which conditions the positions of the charged side chains on the surface are 

likely to be distorted. Visual comparison of the model of AP-B [53] with the experimentally determined 

solution structure [47] indicates significant differences in side-chain orientations. 

IV. Residues Essential For Cardiotonic Activity 

Information about which residues are essential for the cardiac stimulatory activity of the sea anemone 

toxins has been obtained from selective chemical modification and proteolysis studies, comparisons 

among naturally occurring sequences, and, most recently, site-directed mutagenesis. Although there are 

some discrepancies among the inferences drawn from different studies and different techniques, a 

consensus is emerging regarding the location of the cardioactive pharmacophore. Ideally, only effects on 

cardiac tissue should be considered, but doing so would exclude some useful data on activity against 

mammalian nerve preparations. However, data obtained on the Type 2 toxins or on the activity of Type 

1 toxins on nonmammalian tissues will not be discussed in detail. 


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A. Chemical Modification 

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A number of chemical modification studies have been carried out on the sea anemone toxins. As 

discussed previously [24], the results of these studies have to be interpreted with some caution because 

of less than rigorous characterization of the reaction products in many cases. Nevertheless, in the Type 1 

toxins it appears that one or both of the Asp7 and Asp9 carboxylates are required for activity, as well as 

one or both of the Lys37 and Lys48 ε-ammonium groups [24,27,54]. The C-terminal carboxylate 

appears not to be essential, whereas the N-terminal ammonium appears to have some role, although the 

various studies give a confusing view of its importance. There are also conflicting data on the 

importance of His34 and His39 but it seems that at least one of them might be important. Both are 

located in the vicinity of other residues found to be necessary for activity, but His39 is in close contact 

with Asp7 and Lys37 and on this basis may be expected to be the more important. 

Although the available evidence points to a role for one or both of the Asp7 and Asp9 carboxylates in 

cardiotonic activity, it has not been established that either residue makes contact with the sodium 

channel. As indicated above, the carboxylate of Asp9 participates in a hydrogen bond to the backbone 

amide of Cys6, so its role may be structural. The carboxylate of Asp7 is close to the side chains of 

Lys37 and His39 and is exposed to the solvent, making it a more likely candidate for direct interactions 

with the sodium channel. The only evidence for its importance, however, is indirect, coming from the 

observation that its replacement by Asn in synthetic Sh I abolished toxicity to crabs [55]. 

Considerable confusion has surrounded the role of Arg14, which is conserved throughout the Type 1 and 

Type 2 toxins. A recent study has shown, however, that modification of Arg14 in AP-A with 1,2cyclohexanedione 

under conditions where the positive charge is maintained did not affect positive 

inotropic activity [54]. This study also showed indirectly that any contact the Arg14 side chain makes 

with the sodium channel must be relatively loose: although the adduct is active, it is no longer 

susceptible to tryptic proteolysis, indicating that the modified side chain cannot be accommodated in the 

substrate binding site of the protease. The conclusion that the positive charge on Arg14, but not its exact 

spatial location, might be important for activity is consistent with the results of site-directed mutagenesis 

experiments discussed below. 

B. Selective Proteolysis 

When AP-A was treated with trypsin only the Arg14 to Gly15 peptide bond was cleaved [56]. The 

resulting derivative lacked cardiotonic activity but its binding affinity for the rat brain sodium channel 

was reduced by less than an order of magnitude (Llewellyn LE et al., unpublished results). Its overall 

structure, as 


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monitored by NMR, was unchanged, although it should be noted that the structure of the loop containing 

Arg14 was not well defined in either native or trypsinised AP-A. That the backbone structure was 

largely unaffected was confirmed by the observation that the NH and CαH chemical shifts were 

unaltered except in the immediate vicinity of the cleavage (which is the site of a reverse turn in AP-A 

[46]) and the N-terminal regions of the loop and the second strand of the sheet. It is possible that 

perturbations of functionally important groups near the start of the loop may be responsible for the lack 

of activity of the trypsinised derivative, and now that a high-resolution structure is available for AP-A a 

more detailed comparison with the cleavage product would be useful. Other possible explanations for 

the lack of activity are that the position of the Arg14 side chain relative to other key residues in the 

molecule is more important than suggested by chemical modification and site-directed mutagenesis data, 

or that the introduction of additional charges associated with the new termini affects activity. 

Endoproteinase LysC cleaved AP-A between Lys37 and Ala38 to yield a derivative with cardiotonic 

activity an order of magnitude lower than that of the parent molecule (Monks SA and Norton RS, 

unpublished results). This reduction in activity could be a consequence of local conformational 

perturbations. Treatment of AP-B with carboxypeptidase B removed Lys49, resulting in only a two-fold 

reduction in cardiotonic activity (Monks SA and Norton RS, unpublished results). 

C. Sequence Comparisons 

Among the Type 1 toxins shown in Figure 2, the Bc and Bg toxins (from the genus Bunodosuma) form a 

subgroup with characteristic differences from the Anemonia and Anthopleura toxins at residues 5, 12–13 

and 37–42. In addition, the Bg toxins also have Asp7 rarrow.gif Lys and Gly27 rarrow.gif Arg 

substitutions. A potent toxin in mice [26], the cardiac stimulatory activity of Bg II has not been reported. 

The potent activity of Bg II was ascribed to its abundance of positive charges [26]. Ignoring the 

histidines, which at least in AP-A and ATX II would be predominantly in their neutral forms at 

physiological pH [57], Bg II has six positively charged side chains and only one negatively charged side 

chain, whereas, AP-A has three and two respectively, and AP-B has five and two. As discussed below, 

however, it may be that an abundance of positive charge is associated with a lack of discrimination 

between the neuronal and cardiac sodium channels, as found for the scorpion α-toxins. 

Comparison of the activities of Af I and Af II is useful because of their close similarity. Differences 

between Af I and Af II are Ala3 rarrow.gif Pro, Asn12 rarrow.gif Ser, Thr21 rarrow.gif Ile, and an 

additional Gly at the N-terminus. Inspection of the sequences in Figures 2 shows that the identity of 

residue 3 correlates with that of residue 21, Ala or Ser at 3 co-occurring with Thr21, and Pro 3 with 

Ile21 


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(the exception is ATX Ib [19,24], which has Pro 3 and Thr21, but it is not known if this has the same 

local structure). Residues 3 and 21 are juxtaposed in the β sheet [46–48] and the effect of switching 

Ser3/Thr21 to Pro3/Ile21 can be assessed by comparing the structures of AP-A [46] and AP-B [47]. It 

appears that this leads to a distortion of the sheet between the second and third residues but causes no 

significant perturbations to the overall structure. In the calculated structures of AP-A a Va12 NH 

rarrow.gif Leu22 CO hydrogen bond was found but this was not present in the AP-B structures, 

presumably as a result of the local differences. Comparing Af I and Af II, the combination of the 

Ala3/Thr21 to Pro3/Ile21 switch plus the addition of an extra Gly at the N-terminus of Af II would be 

expected to alter the local conformation at the N-terminus. The fact that the cardiac stimulatory activities 

of the two are the same [58] therefore implies that the N-terminus is not important functionally. This 

inference is conditional on the effect on activity of the only other difference between Af I and Af II, 

Asn12 rarrow.gif Ser, being minimal. In the Type 1 toxins, Ser is found more often than Asn at position 

12, while in the Type 2 toxins, replacement of Asn12 by Tyr increases toxicity in mice by a factor of 

two [24,27]. Thus, it is possible that the presence of Ser in Af II slightly favors cardiac activity while the 

changes at the N-terminus might reduce it slightly; the main conclusion to be drawn, however, is that 

neither change has a significant effect. The lack of effect of changes at the N-terminus is consistent with 

the results from expression of AP-B [59], where a Gly-Arg extension at the N-terminus had no effect on 

activity. 

At seven locations AP-B differs from AP-A. Two of these, Ser3 rarrow.gif Pro and Thr21 rarrow.gif 

Ile, were discussed above. Two more, Leu24 rarrow.gif Phe and Thr42 rarrow.gif Asn, are 

conservative changes located in or at the start of loops that are not in the immediate vicinity of the 

sodium channel binding surface and would not be expected to have a direct effect on activity. This 

leaves Ser12 rarrow.gif Arg, Val13 rarrow.gif Pro, and Gln49 rarrow.gif Lys, which do have 

functional significance, as discussed in the following section. 

It is important to note that there are several residues that are common to all of the long toxins and serve 

to maintain the biologically active conformations of these molecules. The clearest examples of residues 

in this category are the six half-cystines, although we believe that the Gly10-Pro11 sequence may 

influence the structure and flexibility of the Arg14-containing loop [46] (at the other end of this loop 

Thr17 and/or Ser19-Gly20 may also be important). Also conserved, Trp33 is probably important 

structurally even though its surroundings are different in the Type 1 and Type 2 toxins. 

It is also interesting that in ATX Ia, which is a potent crustacean neurotoxin but a poor mammalian 

cardiac stimulant, Lys37 and both histidines are missing, suggesting that one or more of these side 

chains may be important in promoting specificity for the mammalian cardiac sodium channel. 


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D. Site-Directed Mutagenesis 

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The complete synthesis of AP-A has been reported [60] but so far this approach has not been pursued to 

generate analogues. More productive has been the analysis of analogues produced by site-directed 

mutagenesis, following the successful cloning and expression of AP-B [59]. A series of single-site 

mutations [61,62] showed that R14A, K48A, and K49A had affinities for the cardiac sodium channel 

that were, respectively, only 3.2-, 2.9-, and 2.4-fold lower than that of native AP-B, while R12A had an 

8.5-fold lower affinity. These results suggested that amongst the cationic side chains only Arg12 was 

significant for activity. Recent data on double mutations [53] indicates that the situation is not quite that 

simple. For example, the R12S-R14Q double mutant had a 72-fold lower affinity for the neuronal 

sodium channel whereas the R12S and R14Q mutants individually had 5.9- and 2.4-fold lower affinities, 

respectively, which should combine to produce only a 14-fold effect. It appears that the presence of one 

cationic side chain in the Arg14 loop may be sufficient for activity and that its exact location can vary 

somewhat. It would be interesting to know if the same applies to the C-terminus, where Lys48 and 

Lys49 may be able to compensate for one another. However, the proposal that the cationic side chains of 

residues 12, 14, and 49 form a cluster [53] is not consistent with the solution structure of AP-B [47]. 

A similar situation appears to exist in the ω-conotoxins, which possess 5–7 net positive charges. 

Substitution of individual cationic groups had a relatively minor effect on affinity for the voltage-gated 

calcium channel, whereas replacement of several had a significant effect, greater than that expected from 

the sum of the individual effects [63,64]. 

A further outcome of analyses of the double mutants was that the cationic side chains at positions 12 and 

49 in AP-B seem to favor binding to the neuronal over the cardiac sodium channel. Thus, the R12S- 

R49Q double mutant, in which residues 12 and 49 in AP-A, had a 37-fold lower affinity for the neuronal 

channel but only a 5-fold lower affinity for the cardiac channel relative to native AP-B [53]. In fact, the 

affinity of this double mutant for the cardiac channel was lower than that of AP-A, implying that one or 

more of the other five differences between AP-A and AP-B might decrease affinity for the cardiac 

channel. It seems, therefore, that while AP-A is slightly less potent than AP-B on cardiac channels, it 

has greater selectivity for the cardiac channel when measured in sodium flux experiments. In voltageclamp 

experiments AP-A and AP-B favor the cardiac channel to similar degrees [53]. 

V. Other Ligands for Site 3 on the Sodium Channel 

A. Sea Anemone Toxins 

Apart from the “long” sea anemone polypeptides that are the main focus of our interest, there are two 

other classes of anemone polypeptides that bind at or near 


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site 3 on the sodium channel. The first of these are the short anemone polypeptides [19,24] such as ATX 

III [38] and PaTX [39], which are neurotoxic to crustacea. The polypeptide ATX III, which consists of 

27 residues cross-linked by three disulfide bonds, and PaTX, which has 31 residues and four disulfides, 

can be aligned such that 16 residues are identical. The three disulfides in ATX III are linked in a 

1–5/2–4/3–6 pattern in the same way as in the long polypeptides [65], but the only other similarity at the 

level of primary structure is a GCPXG sequence corresponding to residues 28–32 of AP-A. Although 

neurotoxic to crustacea, ATX III is inactive as a positive inotrope [28], suggesting that it possesses an 

appropriate structural scaffold to interact with site 3 but lacks key side chains required for interaction 

with the cardiac channel. Nevertheless, the smaller size of ATX III makes it an attractive candidate for 

further study, with the aim of engineering into it the ability to bind to the cardiac channel. The welldefined 

solution structure for this toxin [65] provides and essential basis for such an effort. 

The Anemonia sulcata polypeptides BDS I and II [40], which were claimed to have antihypertensive and 

antiviral activity, also bind to site 3 on neuronal sodium channels and have weak negative inotropic 

activity [41]. The points of similarity and difference between the solution structures of BDS I [40] and 

the long anemone polypeptides have been discussed previously [40,41] and will not be reiterated here; 

suffice to say that the overall folds are similar but the Arg14 loop in the long polypeptides is truncated in 

BDS I and the molecule lacks several residues that have been shown to be important for activity. 

B. Scorpion Toxins 

The scorpion α-toxins have been shown to bind to site 3 on the voltage-gated sodium channel 

[24,27,42]. These polypeptides contain up to 70 residues crosslinked by four disulfide bonds, but show 

no sequence similarity to the anemone polypeptides. Possible structural similarities have been discussed 

[24], and in a theoretical model of the anemone toxin Bg II, some of the cationic residues were in similar 

locations to those in the crystal structure of the scorpion toxin Aah II [26]. 

It is clear that positively charged residues play an important role in the interactions of sea anemone 

toxins and scorpion toxins with site 3 on the sodium channel (as indeed they do with other polypeptide 

toxins binding to other ion channels) but this role may be relatively more important for the TTXsensitive 

sodium channel in nerve and muscle than for the TTX-insensitive channel of the heart. For 

example, Bg II, which is more positively charged than AP-A and AP-B (see above), has a higher affinity 

for neuronal sodium channels [26], and replacement of Arg12 and Lys49 in AP-B with uncharged 

residues favors its binding to the cardiac channel [53]. Similarly, the scorpion α-toxins bind more tightly 

to the neuronal channel than the anemone toxins but, as with the Type 2 


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anemone toxins, do not discriminate between TTX-sensitive and TTX-insensitive channels. In fact the 

scorpion toxins are less potent cardiac stimulants than the Type 1 anemone toxins [29]. Thus, while it 

will be interesting to compare the sodium channel binding surfaces of the two classes of toxin as these 

surfaces become better defined in each case, more useful input to the design of a positive inotrope acting 

at site 3 is likely to come from direct studies on the anthopleurins. 

VI. Defining the Cardioactive Pharmacophore 

Now that high-resolution structures are available for both AP-A [46] and AP-B [47], we can begin to 

interpret the results described above in terms of an emerging picture of the cardioactive pharmacophore 

of these molecules. It is encouraging that most of the residues that have been shown hitherto to be 

important for activity lie on one face of the structures, as shown in Figure 6. Of the residues highlighted 

in Figure 6a, evidence to support their role in activity on the neuronal or cardiac channels (or both) has 

come from chemical modification or site-directed mutagenesis studies except in the case of Asn35. The 

reason for including this residue is that in AP-B it is close to the Asp7/Lys37/His39 region and its side 

chain is hydrogen bonded to the backbone carbonyl of Lys37 [47]. Moreover, it is conserved throughout 

the Type 1 toxins (Figure 2). 

We anticipate that many of the residues highlighted in Figure 6 will participate in interactions with the 

sodium channel binding site. One residue which may not is Asp9, the side-chain carboxylate of which 

hydrogen bonds with the amide of Cys6 in AP-A and AP-B. Thus, it is possible that this residue has a 

“structural” role rather than a “functional” one. Other residues in the vicinity of the pharmacophore that 

may also have a structural role are Gly10 and Pro11, as discussed above. The side chain of Ser8 is 

exposed and on the same surface of the molecule, placing it in a position potentially to interact with the 

sodium channel; it is also conserved throughout the Type 1 toxins. 


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Figure 6 

(a) CPK representation of the individual structure of 

AP-B [47], which is closest to the average over the 

whole molecule, showing residues thought to contribute 

to the cardioactive pharmacophore. The surfaces of 

residues 7 and 9 are shaded black, those of 14, 37, 39, and 

48 dark grey, and that of 35 lighter grey. As discussed in 

the text, the primary functions of Asp9 (the side chain of 

which is hardly visible in this view) and Asn35 may be to 

maintain the local structure in an active conformation, 

but it cannot be excluded that they also interact directly 

with the sodium channel. In AP-B the cationic side 

chains of Arg12 and Lys49 are also important, but it 

appears that their roles can be compensated for by nearby 

cationic side chains (Arg14 and Lys48, respectively) and 

that they favor binding to the neuronal sodium channel 

rather than the cardiac channel [53]. (b) Connolly surface 

of AP-B in the same orientation as in part a, with the 

charged residues Asp7, Asp9, Arg14, Lys37, and Lys48 

highlighted. This figure was generated using Insight 

(Biosym Technologies). 


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In peptide—protein and protein—protein interactions the size of the buried surface area ranges from 400 

Å 2 to 1400 Å 2 [66]. In the potassium channel blocker charybdotoxin, 5–8 residues with surface areas of 

530–850 Å 2 were found to be essential for binding, depending on the type of potassium channel 

investigated [67,68]. In the calcium channel blocker ω-conotoxin GVIA, an alanine scan identified only 

two residues, Lys2 and Tyr13, that were important for activity [69]. It is likely, however, that the 

number of residues contributing to the binding surface is greater than this, particularly given the high 

affinity of this toxin for its receptor. If we consider a larger ligand such as human growth hormone, eight 

of the 31 residues in the growth hormone-receptor interface contribute 85% of the binding energy and 

more than half make no significant contribution to the affinity [70]. A similar result was found for a 

growth hormone-monoclonal antibody complex, where only five residues were critical for binding [71]. 

In fact, the example of growth hormone may be more relevant to the anthopleurins than those of 

charybdotoxin and conotoxin, which function simply as ion channel blockers. Thus, we expect the 

essential residues in the anthopleurins to number between five and ten, a total somewhat less than 

previously anticipated [24]. 

If we ignore Asp9 and assume that only one of Arg12 or Arg14 and one of Lys48 or Lys49 are 

necessary, then it is likely that the residues highlighted in Figure 6 make a significant contribution to the 

sodium channel binding surface of the anthopleurins. They span a larger area on the surface than the 

essential residues in charybdotoxin, but it is important to note that the conformation of the Arg14 loop in 

solution is not fixed and that conformations in which the Arg14 side chain is closer to the region around 

Asp7 could be more representative of the sodium-channel-bound structure. For example, the distance 

between the Arg14 guanidino group and the Asp7 carboxylate varies from 13 to 26 Å over the family of 

structures of AP-A and 10 to 22 Å in AP-B. 

By analogy with other protein—protein interactions, it is likely that the sodium channel binding surface 

of the anthopleurins will include side chains that mutagenesis studies will not identify as having a 

significant role in binding or activity. As indicated above, alanine scanning of residues in the human 

growth hormone—receptor interface indicated that less than a quarter of the contact residues provided 

most of the binding energy [70]. Thus, we believe that the residues identified above will contribute to 

the sodium channel binding surface of the anthopleurins by virtue of their location on the protein surface 

in the immediate vicinity of residues, which clearly are important for activity. Nevertheless, some of 

them may make only modest contributions to binding affinity and could be altered without destroying 

activity. Other residues, as yet unidentified, may also make significant contributions. By analogy with 

other protein—protein interactions characterized to date, it is reasonable to anticipate that some of these 

residues will be hydrophobic, in contrast to the charged 


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residues that have been the main focus of attention hitherto. A key requirement now is to identify those 

residues that provide the majority of the binding energy and to differentiate these from others whose 

main role is to stabilize the binding residues in the active conformation. 

VII. Mimicking The Pharmacophore 

Significant progress has been made over the past few years in the field of peptide mimetics, although the 

most successful examples are those where a small peptide ligand or a linear segment of a larger protein 

has been the target [72–74]. An alternative approach to de novo design is to optimize a lead compound 

obtained by screening chemical libraries on the basis of a knowledge of the conformation of the 

polypeptide ligand, as in the case of the endothelin receptor antagonist SB 209670 [75]. 

The task of mimicking a pharmacophore is simplified where the contributing residues are contiguous in 

the amino acid sequence. This is not the case in the anthopleurins, with residues from at least four 

different regions of the sequence contributing to affinity. In charybdotoxin the essential residues come 

from two or three regions of the sequence, depending on which potassium channel is considered, while 

in growth hormone, binding site I is comprised of residues from three different regions of the protein 

and site II from two regions. Mimicking the pharmacophore of the anthopleurins therefore represents a 

task at least as challenging as those presented by these two examples. Strategies for achieving this goal 

include de novo design, conformationally directed data base searching and screening chemical libraries 

(synthetic and naturally occurring) for leads, which could then be optimized on the basis of our 

knowledge of the structure. Our approach is based on the first two of these. 

Initial attempts to mimic the pharmacophore of AP-A were based on linear and disulfide-cyclized 

versions of the Arg14-containing loop [76]. At that time, our level of understanding of the 

pharmacophore was inadequate and it is clear in retrospect that not enough of the key elements were 

present. Nevertheless, conformational analysis of these peptides by NMR was useful in showing that 

they retained several elements of local structure observed in the corresponding region of the native 

protein, thereby emphasizing the independence of this loop from the rest of the structure in solution. 

VIII. Conclusions 

In this chapter I have attempted to summarize the current state of our understanding of the structure and 

structure-function relationships of the type 1 sea 


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anemone toxins and to indicate how the favorable cardiotonic properties of the anthopleurins might be 

mimicked in a low molecular weight analog. 

Progress in defining the cardiotonic pharmacophore has been hampered by difficulties in determining 

high-resolution structures of the anthopleurins in solution due to the presence of multiple conformers, 

and by uncertainties concerning the exact location of some of the key residues in the Arg14 loop. Both 

of these problems would have been alleviated by isotopic labeling of the molecules in a high-yield 

expression system, which would have allowed for better definition of the structures in solution. The 

question of how the structure in solution might change upon binding to the sodium channel remains 

open and is particularly relevant to residues in the Arg14-containing loop. Conformationally constrained 

analogs of the ligand offer one means of addressing this problem. The definitive solution would be 

provided by a high-resolution structure for the sodium channel, determined by x-ray or electron 

crystallography, but this will not be available in the near future. In the meantime, the approach of 

complementary mutagenesis (of both ligand and receptor), which has been very informative in defining 

the charybdotoxin-potassium channel interface [68,77], can be employed to produce a crude model of 

the binding site. 

Once a lead compound is obtained, further development will almost certainly be required to optimize 

properties such as bioavailability and stability in vivo. A key requirement will also be good selectivity 

for the cardiac over other sodium channels, but the results of mutagenesis studies carried out so far on 

the anthopleurins suggest that this should be achievable. Lead compounds will also have to be rigorously 

evaluated in terms of their effects on cardiac arrhythmias to ensure that they ameliorate rather than 

exacerbate this problem, especially in the failing heart. Finally, the possibility that the beneficial effects 

of positive inotropes in vivo may be the result of inotropic and noninotropic activities [6] would need to 

be evaluated for mimetics of the anthopleurins. These requirements notwithstanding, there is good 

reason to be optimistic that a mimetic of the anthopleurins can be developed and that it may have 

significant benefits in the treatment of congestive heart failure. 


I am grateful to all the colleagues who have contributed to our sea anemone toxin work over the years, 

and, in particular, to Steve Monks, Paul Pallaghy, and Jane Tudor for assistance with the figures and for 

helpful discussions. I also thank Ken Blumenthal for communicating results prior to publication. 


Since completion of this chapter, two additional papers on site-directed mutations of anthopleurin-B 

have been published. In the first (Khera PK, Blumenthal 


Document 

KM. Importance of highly conserved anionic residues and electrostatic interactions in the activity and 

structure of the cardiotonic polypeptide anthopleurin B. Biochemistry 1996; 35:3503–3507), it was 

concluded that Asp7 may be important for folding, Asp9 may be important for protein folding and 

interaction with the sodium channel, and Lys37 for channel interaction. In the second (Dias-Kadambi 

BL, Drum CL, Hanck DA, Blumenthal KM. Leucine 18, a hydrophobic residue essential for high 

affinity binding of anthopleurin-B to the voltage-sensitive sodium channel. J Biol Chem 1996; 

271:9422–9428), Leu18 was shown to be important for binding, with several hundred fold losses in 

affinity being associated with its mutation to Val or Ala. This residue is adjacent to the surface 

highlighted in Figure 6. 

References 

2. van Zwieten PA. Pharmacotherapy of congestive heart failure. Currently used and experimental 

drugs. Pharmacy World and Science 1994; 16:234–242. 

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35. Catterall WA. Structure and function of voltage-sensitive ion channels. Science 1988; 242:50–61. 

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39. Warashina A, Ogura T, Fujita S. Binding properties of sea anemone toxins to sodium channels in the 

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40. Driscoll PC, Gronenborn AM, Beress L, Clore GM. Determination of the three-dimensional solution 

structure of the antihypertensive and antiviral protein BDS-1 from the sea anemone Anemonia sulcata: a 

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Pharmacol 1989; 14 (Suppl 3):S24–S29. 

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solution. Protein Sci 1994; 3:1121–1124. 

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13 

Rational Design of Renin Inhibitors 

V. Dhanaraj * and J.B. Cooper† ** 

Birkbeck College, London, England 


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There has been much interest in the development of therapies for hypertension and associated heart 

failure, which is a major cause of death in the western world. One of the key mediators in primary 

hypertension is the plasma octapeptide angiotensin II (AII), which plays a major role by causing 

vasoconstriction and stimulating aldosterone release, thereby increasing blood volume by its action on 

the kidneys. Angiotensin II is produced by a proteolytic cascade—known as the renin-angiotensin 

system—in which the aspartic proteinase renin catalyses the rate-limiting cleavage of angiotensinogen 

produced by the liver to yield the decapeptide angiotensin I (AI). The subsequent removal of the 

carboxy-terminal dipeptide from AI by angiotensin-converting enzyme (ACE), yielding AII, is the target 

for a number of drugs that are effective for treating hypertension, hyperaldosteronism, and congestive 

heart failure [1]. 

The development of potent low-molecular-weight orally active ACE inhibitors from natural and 

synthetic metalloproteinase inhibitors has been rapid, due in part to the relative lack of specificity of this 

enzyme. In contrast, renin cleaves only its natural substrate or very close analogs and although inhibition 

of an enzyme more specific than ACE may be desirable for reducing side effects in vivo, the selectivity 

of renin meant that during the early stages of drug development, potent inhibition required the use of 

large peptide-based compounds. These were often poorly absorbed and susceptible to gastric proteolysis 

and biliary excretion. Nevertheless, the commercial and clinical success of ACE inhibitors fueled 

interest in the search for therapeutic renin drugs. Most inhibitors have been developed by elaboration of 

the minimal substrate sequence (residues 6–13 of angiotensinogen), which exhibits weak competitive 

inhibition, and replacement of the scissile bond with various nonhydrolysable surrogates, some of which 

may be transition state analogues [2]. 

Current affiliation: University of Cambridge, Cambridge, England. 

Current affiliation: University of Southhampton, Southhampton, England. 


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Figure 1 

The three-dimensional structures of human (left) and mouse renins (right) showing 

oligopeptide inhibitors bound in the active site cleft. The cleft lies between the N- and 

C-terminal domains of the enzyme and is approximately perpendicular to the plane 

of the page. It can accommodate 9–10 residues with the substrate/inhibitor bound 

in an extended conformation. The catalytic aspartic acid residues (not shown) 

are centrally placed at the base of the cleft. 

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Renin is a member of the homologous group of enzymes known as aspartic proteinases that includes 

pepsin and a group of fungal enzymes such as endothiapepsin, penicillopepsin, and rhizopuspepsin. 

Their sequences all contain two aspartates (at positions 32 and 215 in porcine pepsin) that are essential 

for catalytic activity. The crystal structures of several aspartic proteinases have been solved by x-ray 

diffraction at high resolution, revealing a common bilobal structure with a large cleft between the N- and 

C-terminal domains that can accommodate up to nine residues of a substrate (Figure 1) [3]. The two 

essential carboxyls of Asp 32 and Asp 215 are within hydrogen-bonding distance and are approximately 

co-planar due to the constraints of a hydrogen-bonding network involving residues of the two highly 

conserved loops that contain the essential aspartates. The three-dimensional structures of the two 

domains are related by a topological two-fold axis passing between the catalytic residues where the 

pseudosymmetry happens to be strongest. Modeling studies based on the homology with other aspartic 

proteinases showed that human renin assumes a tertiary structure that is similar to the other enzymes and 

that the homology is greatest for the binding cleft region [4]. Subsequent x-ray analysis of the structure 

of renin (described later) revealed the specific interactions made with inhibitors and implicated certain 

loop regions covering the active site as being important for tight binding of peptides. 

One enigmatic feature of renin is its extreme substrate specificity, its only known natural substrate being 

a single Leu-Val peptide bond of angiotensinogen. The minimal synthetic analog is the 6–13 octapeptide 

that encompasses the scissile peptide bond of angiotensinogen between residues 10 and 11 [5]. It has 


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been suggested that specificity of proteinases in general is due to rigidity in the binding pockets [6]; 

broad specificity results from the ability of the pockets to change shape in response to different ligand 

side chains. However, renin is known to be inhibited by a wide variety of peptide analogs of different 

length and sequence indicating either that the active site may be somewhat flexible or that the strength 

of binding of a substrate does not determine the rate of subsequent turnover. This is emphasised by the 

existence of substrates that act as competitive inhibitors, e.g., RIP of Haber and Burton [7], which has a 

K i that is lower than the K m. Therefore hydrolysis of bound substrate appears to be more specific than 

the binding step. This may be because only certain substrate sequences allow correct positioning of the 

scissile bond for hydrolysis. Evidence for this effect was provided by comparison of 21 inhibitor 

structures of endothiapepsin [8], where it was shown that for inhibitors with different sequences but with 

the same transition state analog, the scissile bond analog can be disposed somewhat differently with 

respect to the catalytic carboxyls in each case. 

The availability of crystal structures of a number of renin inhibitors complexed with fungal aspartic 

proteinases [8, 9] allowed new compounds to be designed and modeled by such techniques as computer 

graphics, energy minimization, and molecular dynamics [10]. X-ray crystallographic analysis of aspartic 

proteinase inhibitor complexes has made a significant contribution to rationalizing the activity data for 

many of these compounds as well as understanding the catalytic mechanism of this class of proteinase. 

II. Strategies for Design of Renin Inhibitors 

Some of the parameters that have been varied in the search for therapeutically active renin inhibitors are 

outlined below. 

A. Elaboration of the Transition State Analog 

There is no evidence that aspartic proteinase catalysis involves a covalently bound intermediate [11] and 

major advances in the design of nonhydrolysable analogs have stemmed from attempts to mimic an 

intermediate of the following form. 


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This intermediate is derived by nucleophilic attack of a water molecule on the scissile-bond carbonyl. 

Szelke pioneered the use of reduced-bond analog (-CH 2-NH-), which were incorporated into the 6–13 

peptide of angiotensinogen [12]. Cocrystallisation with endothiapepsin revealed that the reduced-bond 

analog associates tightly with aspartate carboxyls (32 and 215) probably via a salt link. The naturally 

occurring transition-state analogue statine (-CHOH-CH 2-CO-NH-) [9] is a closer analogue of the 

putative intermediate and has been incorporated into many inhibitors [13]. The scissile bond has also 

been replaced by the ketone analogue (-CO-CH 2-) [12] or by a C-terminal aldehyde group in a series of 

tetrapeptides [14,15]. Although these appear to mimic the substrate more closely than the intermediate, 

the carbonyl probably binds to the enzyme in the hydrated gem-diol form (-C(OH) 2-CH 2) [16]. Use of 

the hydroxyethylene analog (-CHOH-CH 2-) has led to exceptionally potent inhibitors [17] as has 

substitution of fluorines into ketone analogs [16,18] giving, for example, -CO-CF 2- which undergoes 

hydration of the carbonyl to form -C(OH) 2-CF 2- and exhibits tight binding to renin. The hydrated gemdiol 

is thought to closely mimic the putative transition state -C(OH) 2-NH-. 

All inhibitors solved in complex with aspartic proteinases by x-ray diffraction are observed to adopt 

similar main-chain conformations and form a conserved set of hydrogen bonds involving the inhibitor's 

main-chain groups interacting with enzyme moieties. The inhibitors bind in extended conformations and 

residues in the P 3-P 1 region form antiparallel β-sheet-like interactions with residues 217–219 on the 

enzyme. A β-hairpin turn formed by residues 74–78 lies between the inhibitor and bulk solvent and 

forms a number of generally conserved hydrogen bonds with the bound peptide. These interactions are 

indicated in Figure 2. The binding pockets for the inhibitor's side chains are shallow and contiguous with 

a greater hydrophobic character towards the central region of the active site cleft. 

The elaboration of several classes of transition state analog are now considered in greater detail. 

Statine Analogs 

The natural transition analog statine possesses one less main chain atom than a dipeptide and has been 

shown by x-ray analysis of inhibitor cocrystals to occupy the S 1 and S 1' sites of the enzyme [19]. The 

hydroxyl group of statine binds symmetrically between the catalytic carboxyl groups displacing a 

solvent molecule bound to the native enzyme. The carboxyl diad, therefore, provides a stereospecific 

binding site for statine and hydroxyethylene analogues with preference for the S-enantiomer. 

Replacement of the hydroxyl by an ammonium group might be expected to improve the potency of an 

inhibitor by introduction of a salt link with the enzyme. The corresponding deoxy-aminostatine (ASTA) 

analogs have been synthesised [20, 21] and were found to be nearly as potent as 


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Figure 2 

A schematic diagram of the putative hydrogen bonds formed between oligopeptide inhibitors and the fungal aspartic 

proteinase endothiapepsin. The latter enzyme provided a useful model system for structural studies of interactions formed by renin 

inhibitors with the active site cleft of aspartic proteinases prior to the determination of the human renin structure. The inhibitor is shown 

horizontally with enzyme groups above and below. Intervening hydrogen bonds are indicated by dashed lines. Note the extensive 

hydrogen-bond interactions made between the transition state analogs and the catalytic apparatus of the enzyme. 


Page 325

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the equivalent statine analogs with the benefit of improved solubility. Preference for the S- versus Renantiomer 

at the C3 amino position is observed in accordance with the statine inhibition data [22]. 

Page 326 

Oxahomostatine (-CHOH-CH 2-O-CO-) and azahomostatine (-CHOH-CH 2-NR-CO-) analogs eliminate 

the main-chain frameshift that occurs with statine and, in addition, the azahomostatine conveniently 

reduces the stereochemical complexity of the dipeptide surrogate by introducing a 7-membered urea-like 

planar group into the S 1' binding region. The crystal structure of such a compound complexed with 

endothiapepsin has been solved at 1.8 Å resolution [23] and reveals that the large planar group is 

accommodated by the active site and that hydrogen bonds to the P 1' CO and P 2' NH groups, observed in 

other complexes, are retained. 

One shortened analog of statine -CHOH-CO-O-R referred to as norstatine (where R is a C-terminal alkyl 

group) has been found to be more potent than some equivalent statine analog [24]. The x-ray structure of 

such an inhibitor complexed with endothiapepsin reveals that the carbonyl oxygen of this analog is held 

by a hydrogen bond to the active site flap region of the enzyme involving the Gly76 >NH group (pepsin 

numbering) in much the same way as the P 1' >C=O group of other isosteres (Figure 2). 

Aminoalcohols 

In principle, a good analog of the putative intermediate would be the aminal –CHOH–NH– group but 

this would be in equilibrium with the aldehyde and amino fragments. Interposing a methylene group 

between the hydroxymethyl and amino groups stabilizes the analog and may still allow tight binding to 

the enzyme. Such aminoalcohols (-CHOH-CH 2-NH-) have been synthesized [25] and were shown to be 

potent inhibitors. 

Cocrystallisation of two such compounds extending from P 1 to P 3' with endothiapepsin allowed their 

bound structures to be solved at high resolution [26]. The bound structures revealed that despite the 

insertion of a methylene group in the analog a frameshift in the binding mode does not occur since the 

residue following the aminoalcohol occupies the S 1' pocket. In contrast, the single amino acid, statine, 

replaces two residues of the substrate. The hydroxyl of the aminoalcohol (S-enantiomer) is bound 

symmetrically to both essential carboxyls as is the case for the hydroxyl of the statine and 

hydroxyethylene analogs. 

Glycols 

Incorporation of glycol or vicinal diol analogs of the peptide bond (-CH(OH)-CH(OH)-) has led to 

potent inhibitors and the x-ray structure for one such compound complexed with endothiapepsin is 

available [27]. The first hydroxyl in 


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this analog interacts with the catalytic aspartate carboxyls in the same manner as statine or 

hydroxyethylene moieties; whereas, the second hydroxyl forms a hydrogen bond with the NH group of 

Gly 76, thereby mimicing the carbonyl oxygen at P 1' of other analogs (see Figure 2). 

Phosphorus-Containing Analogs 

Aspartic proteinase inhibitors in which the scissile bond is replaced by a phosphinic acid group (shown 

below) have been reported [28]. 

These may mimic the tetrahedral intermediate more closely than statine or hydroxyethylene analogs. 

One of the oxygens binds to the carboxyl diad and the other resides adjacent to Tyr75 (pepsin 

numbering) forming a hydrogen bond with the outer oxygen of Asp32 [27]. This isostere is very 

effective against pepsin. However, it ionizes at physiological pH and the resulting anion is ineffective as 

an inhibitor of renin [29]. 

Fluoroketone Analogs and Implications for Catalysis 

Fluoroketone analogs (-CO-CF 2-) have been reported [16, 30] and found to be substantially more potent 

than the unhalogenated statone molecules, presumably due to the ease of hydration and greater 

complementarity of the resulting hydrated gem-diol with the catalytic site. The structure of a 

difluorostatone inhibitor complexed with endothiapepsin [31] revealed interactions that indicate how the 

catalytic intermediate is stabilized by the enzyme (Figure 3). One hydroxyl of the hydrated fluoroketone 

associates tightly with the aspartate diad in the same position as the statine hydroxyl or the native 

solvent molecule and the other hydroxyl is positioned such that it hydrogen bonds to the outer carboxyl 

oxygen of Asp32. It has been suggested that the tetrahedral intermediate is uncharged, because if the 

carboxyl of Asp32 carries a negative charge instead, the latter can be stabilized by a full complement of 

hydrogen bonds donated by the gem-diol intermediate and surrounding protein atoms [31]. The current 

mechanistic proposals are based on the key suggestion by Suguna et al. [32] that, although transition 

state analogs appear to displace the active-site water molecule located between the two catalytic 

aspartate carboxyls, the more weakly bound substrate may not. Instead as the substrate binds, the water 

may be partly displaced to a position appropriate for nucleophilic attack on the scissile bond carbonyl. 

Details of the proposed mechanism are given in Figure 3. 


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Figure 3 

The catalytic mechanism proposed by Veerapandian et al. [31] based on the x-ray 

structure of a difluoroketone (geminal-diol) inhibitor bound to endothiapepsin. A water 

molecule tightly bound to the aspartates in the native enzyme is proposed to 

nucleophilically attack the scissile-bond carbonyl. The resulting geminal-diol 

intermediate is stabilised by hydrogen bonds with the negatively charged carboxyl of aspartate 32. 

Fission of the scissile C-N bond is accompanied by transfer of a proton from Asp215 to 

the leaving amino group. 

B. Complementarity of the Inhibitor 

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Optimizing the fit of a ligand to its binding site improves the potency by burying lipophilic residues and 

by maximizing the number of van der Waals contacts, hydrogen bonds, and charge—charge interactions. 

The principles that apply to ligand binding are similar to those involved in protein folding. Inhibitor 


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binding involves displacement of hydrogen-bonded water molecules from both the ligand and the 

binding cleft. This process is entropy favored since waters in the solvent lattice are more disordered than 

those bound to protein. The change in enthalpy on forming NH…CO bonds in the complex from 

>CO…HOH and >NH…OH 2 is favorable but relatively small [33]. The hydrophobic effect is therefore 

thought to play a dominant role in the energetics of binding with hydrogen bonds providing precise 

alignment of the ligand with respect to the catalytic apparatus. The main chain >CO and >NH groups 

from P 3 to P 3' of aspartic proteinase inhibitors are nearly always satisfied by hydrogen-bond interactions 

on formation of the complex. Therefore, given that polypeptides can form the same hydrogen bonds to 

the binding cleft regardless of amino acid sequence, differences in affinity for ligands of equal length 

must be due to other interactions at the specificity pockets, presumably those between the ligand's side 

chains and the enzyme. 

One example of optimizing these interactions for renin is the use of the cyclohexylmethyl side chain at 

P 1, which has been shown to improve the potency by two orders of magnitude relative to the equivalent 

leucine-containing inhibitor [13]. Structure/Activity Relationship (SAR) studies have shown that in 

many inhibitor types, the cyclohexylmethyl group is optimal for the S 1 pocket of human renin; whereas, 

other analogs such as cyclohexyl, cyclohexylethyl, and the very bulky dicyclohexyl and adamantyl rings 

generally have significantly reduced potency [10]. The use of a cyclohexylmethyl appears to introduce 

selectivity for renin versus other human aspartic proteinases. This has been partly rationalized for 

endothiapepsin where it was shown by x-ray analysis that the cyclohexylmethyl group at P 1 can force 

the Phe at P 3 to adopt a less energetically favorable X 2 angle. Hence, differences at the S 3 pocket in renin 

may allow the P 3 Phe to adopt a more favorable X 2 angle in the presence of a cyclohexyl at P 1. In 

contrast the S 2 site is able to accommodate a wide variety of side chains depending on inhibitor type, 

e.g., Phe and His are equipotent in some analogs [34]. The x-ray structures of a number of bound renin 

inhibitors complexed with endothiapepsin have shown that His at P 2 can adopt different X 1 angles 

separated by about 120 degrees [8]. In one conformation the imidazole is lying partly in the S 1' pocket, 

which has a definite hydrophobic character. In the other conformation, the His side chain is in a more 

polar environment. The ability of aspartic proteinases to accept a variety of both polar and hydrophobic 

groups at the P 2 position may be due to this bifurcation. Many inhibitors possess naphthylalanine side 

chains at P 3 and P 4 [14,24,35]. Compounds of this type are potent renin inhibitors with binding constants 

in the nanomolar range. Cocrystallisation of such an inhibitor with endothiapepsin revealed that one 

naphthalic ring is accommodated in the S 3 pocket by significant conformational changes of local enzyme 

side chains (Asp77 and Asp114). The other naphthalene lies in the S 4 binding region [36]. 


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C. Rigidification 

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The number of conformations that a peptide can adopt in solution is reduced by cyclization. This can be 

optimized, at least in theory, to lock the peptide in the conformation that has the highest affinity for the 

receptor, resulting in a gain of affinity, primarily for entropic reasons. The structures of the fungal 

aspartic proteinases reveal that the binding cleft is a wide channel with no obvious division between the 

pockets, e.g., S 1 and S 3 are contiguous. The bound structures of numerous inhibitors have shown that 

alternate side chains are in van der Waals contact due to the extended conformation that these ligands 

adopt. In addition, at certain positions, e.g., P 2, the side chains are allowed very different conformations 

due to the permissiveness of the pocket. Hence, the cross-linking of certain side chains may, at least, not 

be detrimental to inhibitory potency and may also reduce the susceptibility to degradation in the gut or 

plasma. It might therefore be expected that oligopeptide renin inhibitors would be suitable' candidates 

for cross-linking experiments. A similar philosophy of rigidification was pursued in the development of 

the ACE inhibitor cilazapril [37]. 

A number of statine-containing inhibitors possessing disulphide links between P 2 and P 5, and P 2 and P 4' 

have been synthesized [13] although the best potencies were slightly less than for the linear peptides. An 

alkyl cycle of varying length was introduced between the hydroxyl of a serine residue at P 1 and the main 

chain nitrogen of P 2 in a series of reduced-bond inhibitors [53]. Potencies similar to the uncrosslinked 

molecule were achieved but none were greater. This was attributed to the cis isomerisation of the P 3—P 2 

peptide bond giving a conformation that cannot fit the active site of the enzyme. Difficulties in 

achieving more potent cyclic inhibitors may be due to the tight binding environment provided by some 

pockets (especially S 1 and S 3), and the possibility that other unproductive conformations of the inhibitor 

become favorable. More recently similar findings have been reported for cyclic analogs of pepstatincontaining 

alkyl crosslinks of variable length between the P 1 and P 3 side chains [38]. 

D. In Vivo Stability 

Peptides, when administered orally, are susceptible to degradation in the stomach by gastric enzymes 

and the proteinases of the pancreas and brush border of the small intestine. Their lifetimes in the plasma 

are often short due to rapid proteolysis and other metabolic processes. Early efforts were made to 

improve the resistance of renin inhibitors to hydrolysis in vivo by the use of blocking groups at the N- 

and C-terminii [39] and replacement of susceptible peptide bonds other than the renin cleavage site. 

Studies of SAR have shown that various N- and C-terminal groups, some based on the morpholine 

nucleus and derivatives of it, have a favorable effect on the duration of inhibition in the 


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plasma. This may arise from reduced nonspecific plasma binding due to the relatively polar nature of 

these blocking groups [24]. 

Page 331 

Resistance of inhibitors to gut proteolysis has been improved by various methods such as replacement of 

phenylalanine at P 3 with O-methyl tyrosine (or naphthylalanine), which was shown to abolish 

chymotrypsin cleavage and yet retain high inhibitory potency for renin [40]. 

III. Structural Studies of Rennin Complexed With Inhibitors 

The three-dimensional structures of renin-inhibitor complexes had long been sought as an aid to the 

discovery of clinically effective antihypertensives [41]. X-ray analyses of recombinant human renin [42] 

and mouse submandibulary renin [43] have given an accurate picture of active-site interactions and 

largely confirm the predictions of models based on homologous aspartic proteinases [4]. A large number 

of questions concerning the specificities of renins have been answered by these x-ray analyses. The 

renin-inhibitor structures also make an important contribution towards the rational design of effective 

antihypertensive agents. 

A. X-Ray Analysis of Mouse and Human Renin Complexes 

For both of these renins multiple copies of the molecules have been independently defined in the x-ray 

analysis and shown to have very similar structures. These x-ray structures were refined to final 

agreement factors and correlation coefficients of 0.19 and 0.91 for human renin at 2.8 Å resolution and 

0.18 and 0.95 for mouse renin at 1.9 Å resolution. As expected from the high degree of sequence 

identity of human and mouse renins (approximately 70%), they have very similar three-dimensional 

structures as shown in Figure 1. 

The active-site cleft has a less open arrangement in renins than in the other aspartic proteinases. Many 

loops as well as the helix h c (residues 224–236) belonging to the C-domain (residues 190–302) are 

significantly closer to the active site in the renin structures compared to those of endothiapepsininhibitor 

complexes. This is partly due to a difference in relative position of the rigid body comprising 

the C-domain. For instance, there is a domain rotation of ~4 ° and translation of ~0.1 Å in the human 

renin complex with respect to the endothiapepsin-difluorostatone complex. 

The entrance to the active-site cleft is made even narrower in renins as a consequence of differences in 

the positions and composition of several well-defined loops and secondary structure elements. Unique to 

the renins is a cis proline, Pro111, which caps a helix (h N2) and contributes to the subsites S 3 and 


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Page 332 

S 5. This helix is nearer to the active site in renins than in other aspartic proteinases. On an equivalent 

loop in the C-lobe (related by the intramolecular pseudo 2-fold axis), there is a sequence of three 

prolines—the Pro292–Pro293– Pro 294 segment. This structure is also unique to the renins among the 

aspartic proteinases with Pro294 and Pro297 in a cis configuration. Such a proline-rich structure 

provides an effective means of constructing well-defined pockets from loops that would otherwise be 

more flexible. 

This rather rigid poly-proline loop, together with the loop comprised of residues 241–250, lies on either 

side of the active site “flap” formed by residues 72–81. Hence, in the renins, the cleft is covered by the 

flaps from both lobes rather than from the N-lobe alone as in other pepsin-like aspartic proteinases. This 

gives renin a superficial similarity to the dimeric, retroviral proteinases where each subunit provides an 

equivalent flap that closes down on top of the inhibitor [44,45]. 

B. The Role of Hydrogen Bonds in Inhibitor Recognition 

Whereas the mouse renin inhibitor extends from P 6 to P 4', the human renin inhibitor extends only from 

P 4 to P 1'. The cyclohexyl norstatine residue at P 1 in the human renin inhibitor mimics a dipeptide analog 

with its isopropyloxy group occupying the subsite for the side chain of P 1'. The mouse renin inhibitor 

(CH-66) possesses a Leu-Leu hydroxyethylene transition state analog [12]. Both inhibitors are bound in 

the extended conformation that is found in other aspartic proteinase-inhibitor complexes. Both inhibitors 

make extensive hydrogen bonds with the enzymes as shown in Figure 4. In general the two renin- 

inhibitor complexes described here demonstrate that a similar pattern of hydrogen bonding is probably 

used in the substrate recognition of all aspartic proteinases although their specificities differ 

substantially. 

There is also great similarity between aspartic proteinases in terms of interactions with the transitionstate 

analog inhibitors at the catalytic center. The catalytic aspartyl side chains and the inhibitor 

hydroxyl group are essentially superimposable in both renin complexes. The isostere C-OH bonds lie at 

identical positions when the structures of inhibitor complexes of several aspartic proteinases are 

superposed, in spite of the differences in the sequence and secondary structure. Most of the complex 

array of hydrogen bonds found in endothiapepsin complexes are formed in renin with the exception of 

that to the threonine or serine at 218, which is replaced by alanine in human renin. The similarity can be 

extended to all other pepsin-like aspartic proteinases and even to the retroviral proteinases [44,45]. This 

implies that the recognition of the transition state is conserved in evolution, and the mechanisms of this 

divergent group of proteinases must be very similar. 


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C. Specificity 

Figure 4 

The inhibitors complexed with human (top) and mouse (bottom) renin 

showing the putative hydrogen-bond interactions made with the enzyme moieties. 

Page 333 

If the main-chain hydrogen bonding of substrates is conserved among aspartic proteinases, how are the 

differences in specificities achieved? Table 1 defines the enzyme residues that line the specificity 

pockets for both mouse and human renin. In modeling exercises (e.g., Reference 4) it was assumed that 

specificities derive from differences in the sizes of the residues in the specificity pockets (S n) and their 

ability to complement the corresponding side chains at positions P n in the substrate/inhibitor. A detailed 

analysis now shows that this simple assumption only partly accounts for the steric basis of specificity. 


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For example, in the specificity subsite S 3 the phenyl rings of Phe P 3 occupy almost identical positions in 

both renin inhibitor complexes. Modeling studies have predicted the specificity subsite S 3 to be larger in 

renins than in other aspartic proteinases [4] due to substitution of smaller residues, Pro 111, Leu114, and 

Ala115, in place of larger ones in mammalian and fungal proteinases. However, a compensatory 

movement of a helix (h N2) makes the pocket quite compact and complementary to the aromatic ring as 

shown in Figure 5. Thus, the positions of an element of secondary structure differ between renin and 

other aspartic proteinases with a consequent important difference in the specificity pocket. 

The differing positions of secondary structural elements may also account for the specificities at P 2'. 

Mouse submaxillary and other nonprimate renins do not appreciably cleave human angiotensinogen or 

its analogs [46], which have an isoleucine at P 2', although they do cleave substrates with a valine at this 

position. In contrast, human renin not only cleaves the human and nonprimate substrates but also the rat 

angiotensinogen with tyrosine at P 2', albeit rather slowly [47]. This can be explained in terms of the threedimensional 

structures. In the mouse renin complex, the P 2' tyrosyl ring is packed parallel to an 

adjoining helix (h 3) in a narrow pocket and there is only limited space available beyond the Cβ 

methylene group. This appears to be able to accommodate a valine, but not the larger isoleucine at P 2', 

which will suffer greater steric interference from several residues that are conserved in identity and 

position in the two renins. On the other hand, in human renin differences in the orientation and position 

of 


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Figure 5 

The S 3 specificity pocket of human renin occupied by phenylalanine in the 

cyclohexylnorstatine inhibitor. 

Page 335 

helix h 3 bring it closer by (by ˜0.5Å) to the substrate-binding site than in mouse renin. It is orientated in 

such a fashion in human renin that, although it can accommodate the isobutyl side chain of isoleucine at 

P 2', aromatic rings on substituents such as phenylalanine and tyrosine will have severe short contacts 

with the side chain of Ile130 (valine in mouse renin). Thus the reorientation of a helix, coupled with 

subtle differences in the shapes of the side chains, makes significant changes in the substrate specificity 

at this subsite. It is interesting to note that in pepsin this helix is in a similar position with respect to the 

active site as in human renin. This provides a structural rationale for the negative influence of peptides 

containing phenylalanine [48], tyrosine, or histidine [49] at this subsite (S 2') on the rate of proteolytic 

pepsin cleavage, while isoleucine and valine enhance catalysis. 

Differences in the specificity subsites at S 1' in the human and mouse renins have a more complicated 

explanation. At first sight the situation appears to be explained by complementarity of the subsites to the 

valine and leucine at 


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P 1' in human and mouse angiotensinogens. Accordingly residue 213 is leucine in human renin and valine 

in mouse renin. The S 1' pockets of chymosin, pepsin, and endothiapepsin have an aromatic side chain at 

residue 189 while the renins have amino acids with smaller side chains (valine in human and serine in 

mouse renins). This would be expected to make the pocket larger in renins. However the structure of the 

mouse renin complex shows that the substrate moves closer to the enzyme in renins as a result of the 

smaller residue at 189 and the pocket is made even more compact due to a compensatory change in the 

position and composition of the polyproline loop (residues 290–297). Thus, the specificity difference at 

this site arises not only from a compensatory movement of a secondary structure, in this case a loop 

region, but also from the substitution of an enzyme residue that allows the substrate to come closer to 

the body of the enzyme. 

Elaboration of loops on the periphery of the binding cleft in renins also influences the specificity. This is 

most marked at P 3' and P 4', for which it has been particularly difficult to obtain complexes with welldefined 

conformations for other aspartic proteinases. In endothiapepsin, which has been the subject of 

the greatest number of studies, different conformations are adopted at P 3' and the residue at P 4' is 

generally disordered. In contrast these residues are clearly defined in mouse renin. This is mainly a 

consequence of the polyproline loop, illustrated in Figure 6, which occurs uniquely in renins. The x-ray 

analysis of the mouse renin complex shows that the S 3' and S 4' subsites are formed by the polyproline 

loop together with residues of the flap, and a similar situation is likely to occur in human renin. The welldefined 

interactions of P 3' described in the mouse renin complex explains the significant affinity when 

inhibitors have phenylalanine or tyrosine at P 3' as well as the importance of a P 3' residue for catalytic 

cleavage of a substrate by renin [50]. 

Hydrogen bonds between the side chains of the inhibitor and the enzyme do not play a major role in 

most specificity pockets. However, S 2 is an exception. This subsite is large and contiguous with S 1', so 

that in human renin the S-methyl cysteine (SMC) side chain of P 2 is oriented towards the S 1' pocket, 

which is only partly filled by the isopropyloxy group of the putative P 1' residue. The carbonyl oxygen of 

P 2 accepts a hydrogen from the O γ of Ser76, which is unique to human renin; residue 76 is a highly 

conserved glycine in all the other aspartic proteinases, including mouse renin. In mouse renin the P 2 

histidyl group has a different orientation and forms a hydrogen bond with the O γ of Ser222. If such a 

conformation were adopted by the human angiotensinogen in complex with human renin, the two 

imidazole nitrogens would be hydrogen bonded to the O γ of both Ser76 and Ser222. The observed 

reduction in the rate of cleavage of a human angiotensinogen analog containing a 3-methyl histidine 

substituent at P 2 [51] could be explained on the basis of the hydrogen bonding scheme proposed above. 


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IV. Rational Drug Design 

Figure 6 

The P 3' tyrosine residue of the mouse renin inhibitor complex showing 

the unique polyproline loop on the right. Specificity of this and 

neighboring subsites in renins must derive partly from this rigid loop 

region. 

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The pioneering work of Burton, Szelke, and others in developing peptide-based renin inhibitors has been 

followed by a worldwide commercial effort to elaborate such compounds into therapeutically active 

antihypertensives. The twin problems of insufficient oral bioavailability and rapid clearance has 

seemingly presented major obstacles to success. In addition, the possible advantages of renin inhibitors 

compared with ACE inhibitors remain questionable. Never-theless information from human-renin 

crystallographic studies—such as the more recent high resolution analyses [52] and algorithms for 

analysing voids in the complexes as potential sites for elaborating the drug molecule (e.g. Figure 

7)—may yet provide leads for compounds with suitable therapeutic characteristics. 

The detailed analyses of renin-inhibitor complexes reported here confirm the general structural features 

thought to contribute to renin's specificity but 


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Figure 7 

Schematic illustration of the voids between enzyme and 

inhibitor in the crystal structure of human renin complexed with a norstatine 

inhibitor. The figure was produced using the GapE software 

(Dr. Roman Laskowski). The inhibitor (dark bonds) is 

enclosed by a net surface and the gaps (where the 

enzyme and inhibitor are not in 

contact) are represented by solid surfaces [42]. 

Page 338 

demonstrate the need for careful, high-resolution x-ray analyses for more confidence in drug design. In 

particular, they show that even minor alternations in the positions of secondary structural elements can 

lead to major changes in the disposition of the subsites and thus the recognition of substrates. Since such 

molecular recognition defines the species specificity and determines the catalytic efficiency of the 

enzymes, a through understanding is indispensable for the synthesis of suitable inhibitors. The 

specificity pockets—the molecular recognition sites—are modified by elaboration, particularly of 

surface loops, which can be disordered in the uncomplexed enzymes and difficult to model with 

precision from homologous structures. These data establish a new foundation for the rational design of 

renin inhibitors and have provided a rational base for development of clinically successful HIV 

proteinase inhibitors. 

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renin acting on pure natural or synthetic substrates. Biochim Biophys Acta 1987; 913:10–19. 


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48. Powers JC, Harley AD, Myers DV. Subsite specificity of porcine pepsin. In: Tang J, ed. Acid 

Proteases-Structure, Function and Biology. New York: Plenum Press, 1977:141–157. 

49. Antonov VK. In: Tang J, ed. Acid Proteases-Structure, Function and Biology. New York: Plenum 

Press, 1977:179. 

50. Skeggs LT, Lentz KE, Kahn JR, Hochstrasser H. Kinetics of the reaction of renin with nine synthetic 

peptide substrates. J. Exp Med 1968; 120:130–34. 

51. Holzman TF, Chung CC, Edalji R, Egan DA, Martin M, Gubbins EJ. Krafft GA, Wang GT, Thomas 

AM, Rosenberg SH, Hutchins C. Characterisation of recombi- 


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nant human renin kinetics, pH-stability, and peptidomimetic inhibitor binding. J Protein Chem 

1991; 10:553–563. 

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52. Tong L, Pav S, Lamarre D, Pilote L, Laplante S, Anderson PC, Jung G. High resolution crystalstructures 

of recombinant human renin in complex with polyhydroxymonoamide inhibitors. J Mol Biol 

1995; 250:211–222. 

53. Sham HL, Bolis G, Stein HH, Fesik SW, Marcott PA, Plattner JJ, Rempel CA, Greer J. Renin 

inhibitors. Design and synthesis of a new class of conformationally restricted analogues of 

angiotensinogen. J Med Chem 1988; 31:284–295. 


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14 

Structural Aspects in the Inhibitor Design of Catechol O-Methyltransferase 

Jukka Vidgren and Martti Ovaska 

Orion Corporation Orion Pharma, Espoo, Finland 


Catechol O-methyltransferase (COMT) plays an important role in the catabolic inactivation of 

catecholamines. It is present both in extracerebral tissues and in the central nervous system. During the 

last few years there has been a remarkable interest in COMT. Basic biochemical and molecular biology 

research has given detailed insights into the function and nature of the enzyme. The knowledge of the 

crystallographic structure has allowed researchers to analyze the molecular mechanism of the catalytic 

reaction and to accomplish the structure-based design of inhibitors. The development of potent and 

selective inhibitors has provided effective pharmacological tools to investigate the physiological role of 

the enzyme. The main clinical interest has been the possible application of COMT inhibitors as adjuncts 

in the L-dopa therapy of Parkinson's disease. Parkinson's disease is a dopamine deficiency disorder. The 

dopamine-producing neurons in striatum are destroyed. The medication strategy is to replenish the 

missing dopamine. L-Dopa, given together with a peripheral inhibitor of dopa decarboxylase (DDC), for 

example, carbidopa, is a standard therapy in Parkinson's disease. While dopamine does not penetrate 

into the brain, L-dopa penetrates the blood-brain barrier and is decarboxylated into dopamine in the 

brain. The half-life of L-dopa is short and in the presence of DDC inhibitor a large amount of the drug is 

eliminated by COMT. The COMT enzyme produces the metabolite 3-methoxytyrosine (3-OMD), which 

has no benefit in the treatment of Parkinson's disease, but has a long elimination half-life and may be 

harmful during chronical treatment. Also a gradual loss of the efficacy of L-dopa occurs during longterm 

medication. Since the early 1980s active 


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Figure 1 

The rationale of COMT inhibition as adjunct in the L-dopa therapy of 

Parkinson's disease (reproduced by permission from COMT News, Issue 1, Orion 

Corporation, Orion Pharma, 1994). 

Page 344 

research in pharmaceutical companies has been carried out to develop new potent, selective, and orally 

active COMT inhibitors. Some of them (e.g., entacapone), are now in final clinical trials and the results 

have been promising. The rationale of COMT inhibition can be seen in Figure 1. It can be concluded 

that COMT inhibition in peripheral tissues improves the brain entry of L-dopa and decreases the 

formation of 3-OMD. The dose of L-dopa can be lowered and the dose interval prolonged. Also a 

decrease of the fluctuations of dopamine formation has been observed. The inhibition of COMT seems 

to be the next step in improving the L-dopa therapy of Parkinson's disease. This paper discusses the 

structure-based approach for the understanding of the enzyme function and inhibitor design. 

II. The Enzyme 

A. Physiological Role of COMT 

Catechol O-methyltransferase (COMT, EC 2.1.1.6) was originally detected in rat liver extracts [1]. Since 

then, COMT has been found in many species: 


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Figure 2 

The reaction catalyzed by catechol O-methyltransferase. Dopamine: 

R=CH 2-CH 2-NH 2; L-dopa: R=CH 2-CH(NH 2)-COOH. 

animals, plants, and procaryotes [2]. In mammals the highest COMT activities have been found in the 

liver and kidney, but COMT is common in almost all mammalian tissues [2–4]. 

Page 345 

The COMT enzyme catalyzes the transfer of the methyl group from the coenzyme S-adenosyl-Lmethionine 

(AdoMet) to one of the phenolic hydroxyl groups of a catechol or substituted catechol [1] 

(Figure 2). The presence of magnesium ions is required for the catalysis. The reaction products are Omethylated 

catechol and S-adenosyl-L-homocysteine (AdoHcy). Physiological substrates of COMT are 

catecholamine neurotransmitters, dopamine, noradrenaline, and adrenaline, and some of their 

metabolites. The COMT enzyme inactivates catecholic steroids such as 2-hydroxyestradiol, drugs with a 

catechol structure such as L-dopa, and a large number of other catechol compounds [1,2,5–7]. The 

general physiological function of COMT is the inactivation of biologically active or toxic catechols. A 

schematic view of the major catecholamine pathways in the brain is shown in Figure 3. L-Dopa is the 

dopamine precursor used in the treatment of Parkinson's disease [8]. 

B. Primary Structures 

There are no isoenzymes of COMT known in different mammalian tissues. Two distinct forms of 

COMT have been found: one is soluble (S-COMT) and the other membrane bound (MB-COMT) [9,10]. 

Both soluble and membrane-bound COMT have been cloned and characterized [11–16]. The soluble and 

membrane-bound COMT are coded by one gene using two separate promoters [17]. The soluble COMT 

contains 221 amino acids, whereas the membrane-bound form has a 50-(human) or 43-(rat) residueslong 

amino-terminal extension containing the hydrophobic membrane anchor region. The sequences of 

COMT enzymes from different species are highly similar (see Figure 4). The soluble human protein is 

81% identical with the rat enzyme. The 165-amino-acids-long fragment of porcine COMT has 82% 

homology with the human 


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Figure 3 

The main metabolic routes of dopamine and noradrenaline in 

the brain. COMT, Catechol O-methyltransferase; MAO, monoamino 

oxidase; DDC, dopa decarboxylase; DBH, dopamine 

β-hydroxylase; 3-OMD, 3-methoxytyrosine; Dopac, 

dihydroxyphenyl acetic acid. 

Page 346 

enzyme [13]. The existence of a thermolabile low-activity and a thermostable high-activity COMT in 

human population has been reported [18]. Interestingly, the two published sequences of human soluble 

COMT differ in only one amino acid. Recent kinetic studies have shown that this difference affects 

unambiguously the thermostability of the enzyme [19]. 

C. Kinetics of Human COMT 

The kinetic mechanism of the methylation reaction of human COMT has been studied exhaustively 

using recombinant enzymes [19]. The mechanism is sequential ordered: AdoMet binding first, then 

Mg 2+ and the catechol substrate as the last ligand. Human S-COMT and MB-COMT have similar kinetic 

properties. The main difference is the one-order lower K m value of MB-COMT for dopamine as 

substrate (S-COMT 207 μM and MB-COMT 15 μM). The COMT enzyme is a rather slow enzyme with 

a low catalytic number. At saturating substrate levels S-COMT has a double efficiency compared with 

MB-COMT (k cat=37 and k cat =17, respectively). At low substrate concentrations (

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Figure 4 

Amino acid sequence comparison of known COMT sequences (rat [11], human 

[14], pig [13]). Secondary structure elements in the sequence of rat soluble COMT 

are indicated as well as important active-site residues involved in binding of ligands 

(a, AdoMet; m, magnesium; s, substrate/inhibitor). The numbering of the residues 

corresponds to the soluble enzyme. The extension of the MB-COMT consists of the first 

50 amino terminal residues. In the human sequence of COMT determined by Bertocci 

[13], Val108 is replaced by Met108. 

Under physiological concentrations of catecholamines in the brain, MB-COMT may play a more 

important role than S-COMT [19,20]. 

D. Three-Dimensional Structure of COMT 

Backbone 

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The crystal structure of rat soluble COMT has been solved at 2.0 Å resolution [21]. The COMT enzyme 

has a single domain α/β-folded structure, in which eight α-helices are arranged around the the central 

mixed β sheet. The sheet contains five parallel β strands and one antiparallel β hairpin. An overview of 

COMT is illustrated in Figure 5. 


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The AdoMet binding motif is similar to the Rossmann fold, which is well known from the nucleotide 

binding proteins [22]. It has been shown that the known crystal structures of methyltransferases are 

strikingly similar in the AdoMet-binding regions [23], which indicates that all AdoMet-utilizing 

enzymes may share a common divergent evolution. 


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Figure 5 

Schematic stereo view of the three-dimensional structure of COMT. The ligands 

bound to COMT are the methyl-donating coenzyme AdoMet and the magnesium ion. 

Figures 5–7 and 13 were produced using the program MOLSCRIPT [50]. 

Figure 6 

Stereo view of the AdoMet binding to COMT. The most important amino acid 

residues are shown as well as the magnesium ion and the inhibitor 

3, 5-dinitrocate-chol (DNC). 


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Active Site 

The active site of COMT consists of the AdoMet binding domain and the catalytic site. The structural 

elements and individual interactions between COMT residues and the enzymatic-action-participating 

ligands are demonstrated in detail in Figures 5 to 8. 

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AdoMet Binding by COMT. The active-site residues, which have significant interactions with the 

coenzyme, are shown in Figure 6. The loop region between strand β1 and helix α4 forms the AdoMetbinding 

consensus sequence (in COMT GAxxG) that is conserved in methyltransferases [23]. In this 

region the terminal amino and carboxyl groups of AdoMet are bound. The last residue of the strand β2, 

Glu90, forms a hydrogen bond to the ribose hydroxyls. The residue Met91 has face-to-face van der 

Waals contacts on one side, and His142 has edge-to-face contacts on the opposite side of the adenine 

ring. The residue Trp143 closes the adenine of AdoMet into the protein with face-to-edge contact. 

Furthermore, the N-6 atom of the adenine hydrogen binds to Ser119. The Met40 residue holds the 

sulphur of AdoMet with the methyl group in right position towards the hydroxyl group of the catechol 

substrate. As a result of the various hydrogen bonds and van der Waals contacts, AdoMet has a high 

affinity to COMT with a dissociation constant of 23 μM [19]. 

Catalytic Site. The catalytic site of COMT is a rather simple environment formed by the metal ion and 

by the amino acids important for substrate binding and catalysis of the methylation reaction. 

The magnesium ion plays a crucial role for the catalytic activity of COMT. Figure 7 shows the binding 

of magnesium to COMT as derived from the 

Figure 7 

Magnesium binding in COMT. The magnesium ligands are Asp141, Asp169, Asn170, 

both hydroxyls of 3, 5-dinitrocatechol (DNC) and a water molecule (W). 


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Figure 8 

The catalytic machinery of COMT. Shown are the COMT residues 

important for catalysis, Mg 2+ binding, the catechol as substrate, and the 

methyl-donating coenzyme AdoMet. The hydrophobic walls are defined 

by two tryptophane residues and a proline residue. 

Page 350 

crystallographic studies. Magnesium has an octahedral coordination to two aspartic acid residues 

(Asp141 and Asp169), to an asparagine residue (Asn170), to both catechol hydroxyls of the substrate, 

and to a water molecule. In addition to the Mg 2+ ion, Lys144 and Glu199 participate directly in the 

methylation reaction as shown in Figure 8. The “gate keeper” residues Trp38 and Pro174 form the 

hydrophobic “walls” and define the selectivity of the enzyme to different side chains of the substrate. 

They play a significant role in the binding of the substrates and inhibitors of COMT [19, 21]. 

III. Mechanism of the Catalytic Action of COMT 

The catalytic site is a shallow groove with the catalytic machinery at the bottom as illustrated in Figure 

8. The two hydroxyl oxygens of a catechol substrate bind directly to the Mg 2+ ion. The active methyl 

group of AdoMet is near one of the hydroxyl groups, on one side of the catechol ring. The amino group 

of Lys144 is also located near this hydroxyl group, on the other side of the catechol ring from AdoMet. 

The Glu199 residue is near the other hydroxyl group. 


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Figure 9 

The energy profile (as calculated at the 3-21G/PM3 level [24]) for the proton 

transfer step A rarrow.gif B and the methyl transfer step B rarrow.gif C for catechol as a 

substrate. 

Page 351 

The pK a of the catecholic hydroxyl is about 9.8. The role of the Mg 2+ ion bound to the enzyme is to 

make the hydroxyl groups more easily ionizable. It has been shown by quantum mechanical calculations 

that the hydroxyl protons can be transferred to Lys144 and Glu199 [24]. The proton transfer OH 

rarrow.gif Lys144 activates the hydroxyl group for the methyl transfer AdoMet rarrow.gif O -. Thus 

the reaction coordinate for methylation of catechols by COMT consists of a proton transfer from a 

hydroxyl group to Lys144 and a subsequent methyl transfer from AdoMet to the hydroxyl group (Figure 

9). The Lys144 residue acts as a typical catalytic base in a general base-catalysed SN2-like nucleophilic 

substitution reaction [24]. 

IV. Inhibitors of COMT 

A. First-Generation Inhibitors 

First generation COMT inhibitors such as pyrogallol, U-0521 (3, 4-dihydroxy-2-methylpropiophenone), 

tropolone, and 8-hydroxyquinoline (Figure 10) were 


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Figure 10 

Structures of some first generation COMT inhibitors: pyrogallol, 

U-0521 (3, 4-dihydroxy-2-methylpropiophenone), tropolone, 

and 8-hydroxyquinoline. 

Figure 11 

Structures of second-generation COMT inhibitors discussed in this 

paper: nitecapone (OR-462), entacapone (OR-611), tolcapone 

(RO-40-7592), and 2-((3, 4-dihy-droxy-2-nitrophenyl) vinyl) phenylketone. 


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Table 1 Inhibitor Constants of Selected COMT Inhibitors 

K i (nM) Human b K i (nM)Pig c 

Nitecapone 1.0 700 

Entacapone 0.3 N.D. 

Tolcapone 0.3 N.D. 

Vinylphenylketone 4.0 a 200 

a T. Lotta, unpublished results 

b Reference 19. 

c Reference 41. 

N.D.: not determined 

Page 353 

used as in vitro tools to investigate COMT inhibition, but because of the lack of potency and selectivity 

and because they were toxic, they were not clinically useful [2]. These inhibitors have inhibition 

constants (K i values) in the micromolar range. Many of them contain the catechol structure and are also 

substrates of COMT. 

B. Second-Generation Inhibitors 

The invention of a new structural family of COMT inhibitors in the late 1980s lead the COMT research 

into a new active epoch [25,26]. The most potent second-generation inhibitors are nitrocatechol 

derivatives; some examples are shown in Figure 11. Entacapone (OR-611) and tolcapone (RO-40-7592) 

are now in clinical trials for the treatment of Parkinson's disease, and have been extensively studied 

[27–40]. Both compounds are very potent and selective tightbinding inhibitors of human COMT with K i 

values of 0.3 nM [19]. They differ mainly in their pharmacokinetic properties. Entacapone acts 

peripherally while tolcapone inhibits COMT both peripherally and centrally. 

Recently it was reported that 2-((3,4-dihydroxy-2-nitrophenyl)vinyl) phenylketone is a tight-binding 

inhibitor of pig COMT [41], and it is also a potent inhibitor of human COMT (Table 1). This inhibitor 

has a vinylphenylketone group at position 4 of the catechol ring, i.e., in the position ortho to the nitro 

group. 

V. Enzyme Inhibitor Interactions 

A. Background 


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From quantitative structure activity relationship studies (QSAR) of COMT inhibitors it became evident 

that the acidity of the catechol hydroxyl group is the most important factor that influences the inhibitory 

activity of catechol 


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derivatives [26,42]. Structures containing a catechol ring optimally substituted with a nitro group in 

position 3 and other electron-withdrawing substituents in position 5 showed high-potency inhibition 

[25,26,42,43]. It was also detected that the side-chain hydrophobicity at position 5 correlates 

significantly with the inhibitor activity [42]. 

B. X-Ray Structures of COMT-Drug Complexes 

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The structure of 3,5-dinitrocatechol complexed with COMT has been solved [21]. This inhibitor is a 

typical nitrocatechol derivative with a high affinity for COMT. The excellent electron density of the 

inhibitor in the active site of COMT is represented in Figure 12. The planar structure of this compound 

fits well into the active-site cavity of the enzyme and forms nearly ideal contacts 

Figure 12 

A portion of the structure model and the 2F 0-F c electron 

density map contoured at 1.0 standard deviation. The region 

containing the inhibitor, magnesium, parts of AdoMet, and 

the residue Glu199 is shown. The sphere marked with “W” 

represents a water molecule. 


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Page 355 

with the tryptophane residues 38 and 143. The hydroxyl groups of the inhibitor are coordinated to the 

Mg 2+ ion. The hydroxyl group in position 1 has an important hydrogen bond to the carboxyl group of 

Glu199. The other hydroxyl is near the methyl group donated by AdoMet. The pK a of this hydroxyl 

group is low (about 3.4) [26]. The 3-nitro group of the inhibitor has favourable van der Waals 

interactions with Trp143. The Trp38 residue is located edge-to-face with the catechol plane, which 

allows an ideal aromatic hydrophobic contact. Such aromatic hydrophobic interactions have been 

described to be important in proteins and for the binding of ligands [44,45]. The binding mode of the 

catechol ring of other crystallographically determined potential nitrocatecholtype inhibitors complexed 

with COMT is essentially the same as with 3,5- dinitrocatechol (J.Vidgren, unpublished results). 

C. Differences in the Active Site of Human, Rat, and Pig COMT 

Models for human and pig COMT are easy to build using the experimental structure of the rat COMT, 

due to the high degree of homology between the rat, human, and pig COMT enzymes (Figure 4). The 

active sites are especially well conserved—the few differences in the active-site residues are collected in 

Table 2. The kinetic data show that the K m values of common substrates for rat and human COMT are 

very similar. Pig COMT shows, however, a considerably higher K m value for catechol [46]. The same 

difference is apparent for inhibitors represented by the K i values in Table 1. 

The model for the binding of vinylphenylketone to pig and rat COMT is shown in Figure 13. Assuming 

that the catechol part of the inhibitor adopts the same position as found in the crystal structure with 

dinitrocatechol, the vinylphenylketone substituent has enough room to bind to both enzymes. The most 

significant difference between these enzymes lies in residue 38, the hydrophobic tryptophan in rat (and 

human) COMT and the polar arginine in pig COMT. If Arg38 is directed towards the hydrophobic core 

of the enzyme in a similar conformation as Trp38 (shown in Figure 13), it causes repulsion with the 

catechol ring of the inhibitor. However, it is probable that the polar Arg38 is directed towards the 

solvent. In this case the substrates and inhibitors will lack the favorable contacts that exist with Trp38 in 

human and rat enzymes. Obvi- 

Table 2 Differences in the Active Sites Between Rat, Human, and Pig COMT 

Position Rat Human Pig 

38 Trp Trp Arg 

173 Val Cys Cys 

201 Met Arg Ser 


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Figure 13 

The inhibitor 2-((3,4-dihydroxy-2-nitrophenyl)vinyl)phenylketone modeled 

into the active site of rat (a) and pig (b) COMT. 

Page 356 

ously this one amino acid differences in the active site of the isoenzymes can explain the significant 

differences in the inhibitory potency of vinylphenylketone against these enzymes. From the structural 

point of view it seems that the position of the side-chain substitution (for example at C5 in entacapone 

and C4 in vinylphenylketone) is not critical for the inhibitor binding. In both cases the substituent has 

sufficient space to adapt to the protein structure, and in fact, large substituents reach from the active site 

cavity to the solvent (Figure 13). 

The tenfold higher inhibitory activity of entacapone compared with vinylphenylketone against human 

COMT can be accounted for by the electron- withdrawing effect of the side-chain substitution. In the 

case of entacapone, the side chain at position C5 has a more beneficial electronic influence on the 2- 

hydroxyl of the inhibitor producing a better inhibition (see Section VI). 

VI. Mechanism of the COMT Inhibition By Nitrocatechols 

As described above, catechols with strong electronegative groups are potent inhibitors of COMT. These 

compounds seem to bind well to the active site, but in spite of that they are very poor substrates. It has 

been shown, with nitecapone 


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Figure 14 

The energy profile (as calculated at the 3-21G//PM3 level [24]) for the proton 

transfer step A rarrow.gif B and the methyl transfer step B rarrow.gif C for 3,4-dinitrocatechol as a 

substrate. 

in a rat COMT assay, that the rate of nitecapone methylation is equivalent to about 1% of the rate of 

dopamine methylation [47]. 

Page 357 

The energy profile for the hypothetical methylation of 3,5-dinitrocatechol is shown in Figure 14 [24]. 

The electronegative nitro groups strongly stabilize the ionized catechol–COMT complex, and the energy 

barrier for the methylation step is high (see Figure 9 for comparison). This can be understood as 

decreased nucleophilicity of the hydroxyl oxygen, due to the electron-withdrawing properties of the 

nitro groups. 

The electronic effect of the substituents of the catechol ring to the nucleophilicity of the hydroxyl group 

at the active site can be readily seen from the molecular electrostatic potential (MEP) surfaces of the 

system. The MEP surfaces were calculated at the PM3 level and plotted at –20 kcal/mol for catechol and 

3,5-dinitrocatechol at the active site of COMT [24]. The results are summarized in Figure 15. In the case 

of catechol the effect of the proton transfer form OH to Lys144 is seen as a remarkable increase in the 

negative potential between AdoMet and the substrate. 3,5-Dinitro substitution of the catechol ring 


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Figure 15 

The energy profiles and MEP surfaces (–20 kcal/mol) for catechol (black) and 

3,5-dinitrocatechol (grey). 

decreases the activating negative potential substantially. As a consequence, the OH group is weakly 

nucleophilic and 3,5-dinitrocatechol is not a substrate of COMT but a potent inhibitor. 

VII. Inhibitor Design 

Page 358 

The drug-design process of COMT inhibitors started long before the structure of the target molecule was 

available. The most important results were extracted from the QSAR studies of substituted catechols 

[26,42]. Those investigations clearly indicated the importance of the acidity of one of the two hydroxyl 

groups in the catechol ring. The ionization of the hydroxyl was greatly influenced by electronwithdrawing 

substituents in the positions ortho and para to the hydroxyl. The lipophilicity of the side 

chain was predicted, but after the determination of the enzyme structure it became clear that the active 

site of COMT is a relatively shallow groove where ligands with longer side chains 


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Page 359 

easily reach the surface of the enzyme. The pharmacophore model constructed with biochemical and 

QSAR knowledge [42] was surprisingly good and realistic in comparison to the experimental structure. 

The crucial role of the magnesium ion for the binding of substrates and catalysis was not given enough 

attention. Even without the experimental knowledge of the three-dimensional structure of COMT, the 

correct decision for the direction of the drug-design process was possible. Many open questions were 

still waiting for the determination of the molecular structure. The most important drug-design aspects 

under consideration included the optimization of the pharmacokinetic properties of the molecules, 

especially the penetration across the blood brain barrier. The structure of COMT clearly showed that 

with catechol-type inhibitors, one of the positions ortho to the hydroxyls is optimally substituted by a 

nitro group, while the other ortho position has to be unsubstituted. Sterically, the two remaining sites can 

be substituted quite freely, so that these substitution positions can be used to modify the 

physicochemical properties of the inhibitors. The question of the possibility of designing an inhibitor 

without the catechol structure is important, because the potent inhibitors which rely on the ionization of 

a catechol hydroxyl, penetrate very poorly into the brain. In clinical trials of the therapeutic use of 

COMT inhibitors it has become evident that the beneficial effect to L- dopa metabolism is fully reached 

with peripheral inhibitors such as entacapone [27–29]. As demonstrated above, based on the threedimensional 

structure of COMT, the design of potent noncatechol type inhibitors may be very tedious. 

The active site of COMT is a rather simple environment with a few catalytic residues and a magnesium 

ion defining the structural limits of the catechol ligands. 

VIII. Clinical Possibilities of Inhibitors of COMT 

A. Parkinson's Disease 

Parkinson's disease is a neurological disorder that affects voluntary movement. The symptoms are 

slowness of movement, rigidity, and tremor. The reason for the disease is unknown. Parkinson's disease 

is a progressive disorder and involves the deterioration of dopaminergic nerve fibers in substantia nigra, 

which leads to a striatal deficiency of dopamine. The symptoms of Parkinson's disease are detected only 

after about 80% of the dopamine-producing neurons are degenerated. There is no known cure but the 

symptoms are treated with a combination of different drugs. 

Current and Future Therapy of Parkinson's Disease 

Dopamine does not penetrate the blood-brain barrier. L-Dopa is actively transported into the brain and 

then converted to dopamine. L-Dopa is rapidly metabolized and only about 1% of an oral dose reaches 

the brain. In the current 


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Page 360 

therapy of Parkinson's disease L-dopa (precursor of dopamine) is administered together with a 

peripheral dopadecarboxylase (DDC) inhibitor (such as carbidopa or benserazide). The major peripheral 

L-dopa metabolizing enzymes are DDC and COMT. After inhibition of DDC, COMT is responsible for 

the main catabolism of L-dopa. In the central nervous system COMT together with monoamino 

oxidase (MAO) participitates in the metabolism of L-dopa and dopamine. Large amounts of orally 

administered L-dopa are converted by COMT to 3-O-methyldopa (3-OMD). Having a long plasma halflife 

(approximately 15 h compared with the dopamine 1-h half-life), 3-OMD accumulates in the plasma 

during the L-dopa treatment. L-Dopa and 3-OMD also compete for the same active transport system into 

the brain. It has been proposed that 3-OMD could cause some side effects of the L-dopa treatment 

(dyskenisia, on-off phenomenon). Inhibition of COMT enzyme decreases the 3-OMD formation and 

improves the brain entry and bioavailability of L-dopa. The use of COMT inhibition should prolong the 

L-dopa effects and permit a decreased does [27– 29]. 

Preclinical and clinical results indicate that both entacapone and tolcapone are orally active, nontoxic 

and well-tolerated drugs. The adjuvant L- dopa therapy with DDC inhibitor + COMT-inhibitor (+ 

possible MAO inhibitor) may substitute for the present double therapy in the treatment of Parkinson's 

disease [27–40]. Together with the development of dopamine agonists and MAO inhibitors, the 

inhibition of COMT will constitute major progress in the treatment of Parkinson's disease in the near 

future. 

B. Other Possible Indications of the COMT Inhibition 

It has been proposed that COMT inhibitors co-administered with L-dopa could have beneficial effects in 

the treatment of depressive illness [40]. This can be caused by either the better availability of dopamine 

or by the elevated noradrenaline levels in the brain. Another hypothesis suggests that the increasing level 

of AdoMet caused by COMT inhibition may cause an antidepressive effect [33]. 

Dopamine has also natriuretic and diuretic effects in kidney. There has been evidence that abnormalities 

of the renal dopamine system can lead to salt- sensitive hypertension [48]. In rat kidney, deamination 

represents the major pathway in the metabolism of dopamine, but when MAO is inhibited, methylation 

appears to offer an alternative metabolic pathway [49]. Thus COMT inhibition may be important in the 

regulation of renal sodium excretion. 

References 

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cells. Biochimie 1971; 53:871–874. 

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15 

Antitrypanosomiasis Drug Development Based on Structures of Glycolytic 

Enzymes 

Christophe L. M. J. Verlinde, Hidong Kim, Bradley E. Bernstein, Shekhar C. Mande, * and Wim G.J. 

Hol 

University of Washington, Seattle Washington 

I. Trypanosomiasis 

A. Disease and Treatment 

Human African trypanosomiasis, also called sleeping sickness, is caused by the parasites Trypanosoma 

brucei gambiense and Trypanosoma brucei rhodesiense. These unicellular organisms live freely in the 

bloodstream of the human host and invade the brain during the later stage of the disease. Without 

treatment the disease is always fatal [1]. The course of the gambiense form may last from months to 

years, while T. brucei rhodesiense usually kills within weeks. Sleeping sickness occurs in thirty-six sub- 

Saharan African countries, putting fifty million people at risk. Each year 25,000 new cases are reported, 

but the actual number of cases is more likely to be about 250,000 [2]. The current state of 

antitrypanosomal chemotherapy is dismal; many parasitologists do not want to take the risk of being 

infected with T. brucei rhodesiense and prefer to study the parasite T. brucei brucei, which is harmless 

to humans [3]. 

Not more than four drugs are available to treat the disease: pentamidine, suramin, melarsoprol—all from 

the first half of this century—and eflornithine, introduced in 1990 (Figure 1). Except for eflornithine, 

which is an irreversible ornithine decarboxylase inhibitor [4], the mechanisms of the drugs are poorly 

understood [5]. All four drugs require administration by injection in a hospital setting, which is a major 

drawback in rural Africa [6]. Pentamidine is useful for treating early stage T. brucei gambiense 

infection, suramin for both early stage gambiense and rhodesiense infection. The permanent charges on 

pentamidine 

* Current affiliation: Institute of Microbial Technology, Chandigarh, India 


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Figure 1 

Available drugs for the treatment of human African trypanosomiasis. 

Page 366 

and suramin explain why they show poor oral absorption and do not cross the blood–brain barrier, 

making them unsuitable for the treatment of late-stage trypanosomiasis. Melarsoprol, an organoarsenical 

compound, was until 1990 the only drug effective in the late stage of both forms of trypanosomiasis. 

Unfortunately, it is also highly toxic, causing reactive encephalopathy in up to 10% of the patients, of 

which about one half die. This deadly complication is well known to villagers of areas where the disease 

is endemic and, ironically, discourages people from participating in diagnostic surveys. Eflornithine, 

heralded as the “resurrection drug” upon its introduction [7], cures patients infected with late-stage T. 

brucei gambiense but is ineffective against the more virulent rhodesiense form. Additionally, it causes 

bone marrow suppression in half of the patients and occasionally convulsions [6]. A serious concern is 

that resistance has been reported against each of the four antitrypanosomal drugs [1]. 


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Page 367 

Sleeping sickness has been largely ignored by the pharmaceutical industry because the poor 

socioeconomic situation in the part of the African continent afflicted by this debilitating disease offers 

little prospect of reasonable financial returns [8]. It is revealing that eflornithine was originally 

developed as an anticancer drug. It was screened for antitrypanosomal properties only when the 

biochemistry of the trypanosomal polyamine metabolism was understood [9]. Fortunately, the cell 

biology of trypanosomes is so extraordinary that they have been the subject of more fundamental 

research than most other protozoan parasites [5]. Each of these unique features of T. brucei may become 

a target for new drugs, provided they prove to be essential for the survival of the parasite in the human 

host. With such a wealth of biochemical information, structure-based drug design provides a tremendous 

opportunity to arrive at new drugs to cure sleeping sickness. 

B. Targets for Future Drugs 

Preventing trypanosomiasis would be a nobler goal than curing it. Unfortunately, trypanosomes are 

experts in evading our immune system. They achieve this by varying their dense surface coat. It is 

composed of ten million copies of a single protein, the variant surface glycoprotein (VSG), for which 

they have no less than a thousand different genes. In this way trypanosomes can change surface antigens 

more rapidly than the host can produce new antibodies [10]. Clearly, such a mechanism leaves little 

hope for preventing sleeping sickness by vaccination. 

In contrast to the small number of drugs available to treat trypanosomiasis, the opportunities for 

developing new drugs are ample, as can be seen from Table 1. They range from unique RNA processing 

to reduced metabolism, salvage systems, and different rates of protein turnover. All of these features 

were the subject of an outstanding review by C.C. Wang [5]. 

An inventory of the structural information waiting to be exploited by structure-based drug design reveals 

eight potential target enzymes (Table 2). Since for most trypanosomatid proteins there is a human 

counterpart it is mandatory that designed inhibitors be selective, i.e., have very little affinity for the 

equivalent enzymes of the human host. As a consequence, selective design requires pairs of equivalent 

structures from the parasite and from the host. A complication for selective design is that for three of the 

mammalian enzymes only the structure of one isoenzyme is known. For example, the structure of human 

aldolase A has been determined [23] but not the structures of isoenzymes B and C, which share only 69 

and 82% sequence identity to isoenzyme A [40–42]. Of course, homology modeling might be a way to 

overcome this problem. 

From Table 2 it is evident that only for three proteins, TIM, GAPDH, and trypanothione reductase the 

structures of the parasite and host enzymes are 


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Table 1 Trypanosomal Targets, Their Human Equivalents, and Current Leads for Drug Design 

T. brucei Human host Lead 

RNA editing by trans-splicing [11] cis-splicing none 

All energy from fast glycolysis [12] glycolysis and oxidative 

phosphorylation 

Glucose transporter [14] human erythrocyte glucose 

transporter 

Purine P2 transporter [15] none 

Purine salvage enzymes, e.g., HGPRT b [16] HGPRT none 

Slow rate of enzyme turnover [17], e.g., ornithine 

decarboxylase [18] 

Polyamine metabolism, e.g., S-adenosyl 

methionine decarboxylase [19] 

MMBA a [13] 

none 

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fast rate of enzyme turnover eflornithine (= drug) [9] 

S-adenosyl methionine 

decarboxylase 

MDL 73811 c 

Trypanothione reductase [20] glutathione reductase mepacrine 

VSG anchor: a myristate-containing GPI [21] 10-(propoxy)-decanoate d 

a MMBA = 2' -deoxy-2' -(m-methoxybenzamido)-adenosine. 

b HGPRT = hypoxanthine guanine phosphoribosyltransferase. 

c MDL 73811 = 5' -{[(Z)-4-amino-2-butenyl]methylamino}-5' -deoxyadenosine. 

dIt has not been established whether the trypanocidal effect of this compound is due to its incorporation in the GPI anchor 

[5]. 

known. For five more targets the crystal structure of only the mammalian enzyme is available. Efforts to 

solve the structures of trypanosmatid aldolase, PGK, and PK counterparts are underway in our lab. This 

review explains how we attempted to arrive at selective inhibitors of three trypanosomal glycolytic 

enzymes. 

C. Trypanosomal Glycolysis: Enzyme Inhibition as a Target 

In the bloodstream of the human host, trypanosomes are metabolically 


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“lazy”. Since there is plenty of glucose and oxygen available, they rely solely on glycolysis to the stage of 

pyruvate for their energy supply [12]. Their glycolysis proceeds at an amazing rate, which is about fifty 

times faster than in the cells of the human host [43]. This fast rate is a necessity because only two 

molecules of ATP are generated per molecule of glucose instead of the thirty-six produced by complete 

oxidation. These findings led to the proposal that inhibitors of trypanosomal glycolysis might be turned 

into drugs. Support for this idea comes from in vitro experiments where salicylhydroxamic acid (SHAM) 

was used to bring the parasite under anaerobic conditions, after which glycolysis was 


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Table 2 Three-Dimensional Structures Available for Trypanosomal Drug Design 

(a) Glycolytic: 

(b) Other: 

Trypanosomatid PDB code Mammalian PDB code 

PGIa — — Porcine 1PGI[22] 

Aldolase — — Human b 1ALD[23] 

TIM T. brucei 5TIM[24] Human 1HTI[25] 

GAPDH T. brucei 

L.mexicana 

1GGA[26,27] 

[30] 

PGK — — Horse 

Pig 

Human c 3GPD[28,29] 

2PGK[31]—[32] 

PK — — Cat d 1PYK[33] 

TR T. cruzi 1NDA[34] HumanGR 3GRS[35] 

C. fasciculata 1TYT[36] 

1PPR[37] 

HGPRT — — Human 1HMP[38] 

VSG T. brucei 1VSG[39] No equivalent 

a Abbreviations: PGI = phosphoglucose isomerase, PGK = phosphoglycerate kinase, PK = pyruvate kinase, TR = 

trypanothione reductase; GR = glutathione reductase, HGPRT = hypoxanthine-guanine phosphoribosyl 

transferase, VSG = variable surface glycoprotein. 

b A isoenzyme, from muscle; other mammalian isoenzymes are B in liver and C in brain. 

c Muscle isoenzyme; a liver isoenzyme exists. 

dM1 isoenzyme, from muscle; mammals also have M2 in kidney, adipose tissue and lung, L in liver, and R in 

rood blood cells. 


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blocked by mass action through the addition of glycerol (Figure 2). As a result trypanosomes were lysed 

within five minutes [44,45]. Treatment of infected rodents with the SHAM/glycerol mixture proved to 

be effective to clear the blood of the animals from T. brucei [46], although only with sublethal doses was 

permanent aparasitemia obtained [47]. If glycolysis could be blocked selectively, i.e., without affecting 

the equivalent enzymes of the host, one might have a promising therapy against trypanosomiasis. 

D. Beyond Enzyme Inhibition: Protein Routing as a Target 

In trypanosomes, seven enzymes involved in glycolysis, from hexokinase to phosphoglycerate kinase, 

are sequestered in specialized organelles, called glycosomes [48]. These microbodies are probably 

evolutionary relics of an endosymbiont [49] but are devoid of genetic material encoding for the 

glycosomal enzymes. Instead, these enzymes are encoded in the nucleus and are post-translationally 

imported into the glycosome. Since the import process likely involves unfolding one might envision 

blocking import by stabilizing the folded 


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Figure 2 

Glycolysis in bloodstream-form trypanosomes. All net energy production takes place 

in the cytosol as the result of ATP formation by pyruvate kinase. However, the 

majority of glycolytic enzymes are sequestered into a specialized organelle, the 

glycosome. There the net ATP synthesis is zero irrespective of the presence of oxygen. 

Under aerobic conditions the NADH produced by glyceraldehyde-3-phosphate dehydrogenase 

is reoxidized via the G-3-P/DHAP shuttle, which couples glycolysis to a 

mitochondrial glycerophosphate oxidase. Under anaerobic conditions or when the 

oxidase is blocked by SHAM, equimolar amounts of glycerol and 3-phosphoglycerate 

are formed. However, the addition of an excess of glycerol to the cytosol prevents the 

reoxidation of NADH. As a result trypanosomes treated with a mixture of SHAM and 

glycerol die in a matter of minutes. 


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state through the tight binding of ligands. In this way inhibitors might act at two levels, directly by 

blocking catalysis and indirectly by preventing proper enzyme routing in the parasite. Thus far, we have 

engaged in a collaborative effort to inhibit three of the glycosomal enzymes. (From Ref. 95. Copyright 

1988 by Elsevier.) 


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II. Three Glycolytic Enzymes Of T. Brucei: Molecular Biology, Biochemistry, and X-Ray 


A. Triosephosphate Isomerase (TIM) 

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TIM is a homodimeric enzyme that interconverts dihydroxyacetone phosphate and glyceraldehyde-3phosphate. 

It ensures that both trioses derived from glucose can be used for ATP production in the 

glycolytic pathway. Triosephosphate isomerase does not require any cofactor. Both the T. brucei and 

human enzymes have been overexpressed in Escherichia coli [50,25] and their crystal structures were 

solved in our group [24,25]. In addition, the structures of T. brucei TIM in complex with seven 

nonselective competitive inhibitors, with inhibition constants of 300 μM or higher were determined: 

monohydrogen phosphate [51], 2-phosphoglycerate [52], 3-phosphoglycerate [53], 3phosphonopropionate 

[53], glycerol-3-phosphate [53], 2-(N-formyl-N-hydroxyamino)-ethyl phosphonic 

acid [54], and N-hydroxy-4-phosphonobutanamide [55]. These studies gave an excellent picture of 

different ligand binding modes and of the conformational flexibility of the enzyme. 

All ligands interact with the main features of the catalytic machinery of the enzyme (Figure 3): (1) the 

phosphate is sequestered by the positive end of a 3 10-helix and Lys13; (2) polar groups on the carbon 

framework interact with His95 and Glu167, the catalytic electrophile and the catalytic base of the 

enzyme, respectively; (3) the entire inhibitor is shielded from the bulk solvent by a flexible loop, which 

normally closes over the substrate during catalysis to prevent phosphate elimination [56] (Figure 4). The 

only exception to flexible loop closure is N-hydroxy-4-phosphono-butanamide. It binds to the enzyme 

with the flexible loop in the open conformation because its size precludes loop closure. Thus, the 

crystallographic binding studies point out that it should be possible to design two very different classes 

of selective inhibitors: a class that binds to the enzyme in the closed loop conformation and one that 

binds to the open loop conformation. 

Selective inhibitor design in the case of TIM appears to be a formidable task. All residues within 10 Å 

of the active site are conserved [25]. This is also reflected in the similarity of the kinetic characteristics 

between trypanosomal and human TIM: for T. brucei TIM, K m (glyceraldehyde-3-phosphate) = 0.25 

mM, kcat = 3.7 × 10 5min -1 [57]; for human TIM, K m = 0.49 mM, kcat = 2.7 × 10 5 min -1 [25]. There are 

significant differences in the surface protein of the two enzymes about 15 Å away from the substrate 

phosphorus atom [58]. In a shallow cleft, T. bruceiTIM has Ala100-Tyr101, while the human 

counterpart of these residues is His-Val (Figure 5). The cleft is formed by the flexible loop of one 

subunit of the enzyme and a different loop originating from another subunit. When the flexible loop 

changes its conformation from the closed to the open form the cleft widens substantially. Moreover, the 

Ala-Tyr dipeptide becomes then directly accessible from the active site, the distance being about 


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Figure 3 

Schematic representation of the structure of a TIM monomer. Helices and strands 

are labeled as H and B, respectively. The view is along the axis of the β barrel, into 

the active site. Key catalytic residues Lys13, His95, and Glu167 are shown along with 

the helix that binds the substrate phosphate and the flexible loop that covers the 

substrate during catalysis. Black dots indicate residues in contact with the second 

monomer of the enzyme. (From Ref.24. Copyright 1991 by Harcourt Brace.) 

Page 372 

10 Å instead of the 15 Å in the closed loop conformation of the enzyme. In any case, selective inhibitor 

design for TIM appears to require de novo design as there are no leads known that interact with the Ala- 

Tyr region of the enzyme. 

B. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) 

Glyceraldehyde-3-phosphate dehydrogenase is a homotetramer that carries out the oxidative 

phosphorylation of glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate. During this reaction 

NADH is formed. Each subunit of the enzyme consists of two domains and has an NAD + binding site. 

The N-terminal domain anchors the adenosine portion of the cofactor while the nicotinamide portion is 

involved in the catalytic reaction at the C-terminal domain. T. brucei 


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Figure 4 

Stereoview of superimposed TIM monomers, one with an open flexible loop (full black) 

and another one with a closed flexible loop (open gray). The loop location is marked 

by an asterisk. Note the proximity of the active site, indicated by the catalytic residues 

Lys13, His95, and Glu167 (all sterofigures in this paper were drawn with 

MOLSCRIPT [93]). 


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Figure 5 

Exploitable structural differences between T.brucei(full) and human (dashed) 

TIM. The inhibitor 2-phosphoglycolate as observed in the structure of the human 

enzyme indicates the location of the active site. Drug design targets are the T.brucei 

Ala100-Tyr101, which are considerably different from their human counterpart 

His-Val. (From Ref.25. Copyright 1994 by Cambridge University Press.) 


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Figure 6 

Stereoview of NAD binding by trypanosomal GAPDH (full black). For clarity 

only 2 of the 4 subunits are shown. A substantial deviation of the protein backbone 

occurs in human GAPDH (open gray, only one subunit shown) near the adenosine 

part of the cofactor. The nearby cleft (marked by an asterisk) is important for 

introducing selectivity in inhibitor binding and has therefore been termed “selectivity cleft.” 

Page 374 

glycosomal GAPDH has been overexpressed in E. coli [59] while human erythrocyte GAPDH is 

available from commercial suppliers. The sequences of the two enzymes are only 55% identical [60]. 

The crystal structure of the parasite enzyme was solved from Laue data at 3.2 Å resolution in our group 

(Figure 6) and in a second crystal form at 2.8 Å [26]. The structure of the human muscle enzyme was 

solved at 3.5 Å resolution in the group of the late Herman Watson [28]. Its resolution was improved to 

2.3 Å in our group [26,29]. Both structures were of the holo-enzyme, i.e., the enzyme in presence of the 

cofactor. 

The active site and the nicotinamide-binding site of the two enzymes are very well conserved. This is 

reflected in similar K m (glyceraldehyde-3-phos-phate) values of 0.15 and 0.17 mM for the trypanosomal 

and human enzyme, respectively [61]. Surprisingly, the K m (NAD +) values differ by a factor ten: 0.45 

mM for T. brucei and 0.04 mM for human GAPDH [61]. In view of the conservation of the 

nicotinamide and pyrophosphate binding sites, the substantial difference in K m (NAD +) has to be 

ascribed to the adenosine binding environment. Indeed, some of the residues embracing the adenine ring 

of the cofactor are not identical. In the trypanosomal enzyme the adenine is sandwiched in between a 

Thr in the back and a Met in the front. This Met is replaced by Pro and Phe in the human enzyme 

(Figure 7). A second difference involves the residue flanking the C2 atom of the purine ring. It is a Val 

in the trypanosomal enzyme, and Asn in the human enzyme. These differences apparently account for a 

ten-fold lower affinity for the cofactor. 


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Figure 7 

Comparison of the binding modes of the adenosine moiety of NAD to GAPDH: 

(left) in T.brucei,(right) in the human enzyme. Note the identical hydrogen bonds to 

the purine N6 and the ribosyl hydroxyls. The purine ring is embraced by 

hydrophobic residues that are not conserved. Also, a unique cleft near O2', which we 

called the selectivity cleft, is present in the T.brucei enzyme. (From Ref. 13.Copyright 

1994 by the American Chemical Society.) 

Page 375 

Other differences in the vicinity of the adenosine portion of the NAD + cofactor are prime targets for 

selective inhibitor design. Close to C8 of the adenine ring, the trypanosomal GAPDH exhibits a Leu 

while its human counterpart has a smaller residue, namely Val (Figure 7). Also, the parasite enzyme 

possesses a hydrophobic cleft near the 2' -hydroxyl of the adenosine ribose. This cleft, termed the 

“selectivity cleft” is almost absent in the human enzyme due to a different local backbone conformation 

and the presence of the Ile37 side chain. In conclusion, the adenosine binding region looks like an 

excellent target for selective inhibitor design. 

It is exciting that the residues responsible for binding adenosine in T. brucei GAPDH are identical to 

their counterparts in glycosomal GAPDH of Leishmania mexicana, another trypanosomatid [31]. L. 

mexicana is one of the most common species of Leishmania throughout Central and South America and 

the southern United States. In humans it hides as amastigotes in the macrophages and causes hideous 

skin lesions. Together with about twenty other species of Leishmania these parasites infect about twelve 

million people annually [63]. Though they are less dependent on glycolysis than T. brucei there is 

evidence that stibogluconate, a well-known drug for treating leishmaniasis, specifically inhibits 

glycolysis in these parasites [64]. Therefore, we decided to study GAPDH of L. mexicana in parallel 

with the T. brucei enzyme. Its structure 


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Page 376 

was recently solved at 2.8 Å resolution in our lab [30]. The kinetic parameters of the two enzymes are 

virtually identical: K m (glyceraldehyde-3-phosphate) = 0.13 mM and K m (NAD +) = 0.41 mM for the L. 

mexicana enzyme [62]. As expected, the structures of the two parasite enzymes are very similar: the rms 

deviation for all backbone atoms is 0.7 Å and the adenosine binding environment is perfectly 

superimposable except for Asn39 of T. brucei GAPDH, which is a Ser in the L. mexicana enzyme. In 

this way drug design for one disease may have implications for another one. 

C. Phosphoglycerate Kinase (PGK) 

A monomeric enzyme, PGK transfers the acylated phosphoryl group from 1,3- bisphosphoglycerate to 

ADP, thus forming 3-phosphoglycerate and ATP. The enzyme uses a metal ion as a cofactor, namely 

Mg 2+ [65]. The PGK enzyme from T. brucei has been overexpressed in E. coli [66]. Human PGK is not 

Figure 8 

Steroview of B.stearothermophilus PGK [69] in complex with ADP bound to the 

C-terminal domain. To illustrate the sugar binding site, the 3-phosphoglycerate has 

been added to the figure based on the crystal structure of pig muscle PGK [32].From 

the distance between the two substrates it is obvious that during catalysis a 

hinge-bending motion between the domains of the protein has to occur to bring the 

substrates together. 


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Table 3 Residues Involved in ADP Binding in Glycosomal and Human PGK 

T.brucei Human_1 a Human_2 b In contact with ADP moiety 

Ala 242 Gly 238 Gly 238 Adenine 

Tyr 245 Phe 241 Tyr 241 Adenine 

Lys 259 Leu 256 Leu 256 Adenine 

Ala 314 Gly 309 Gly 309 Adenine 

Ser 378 Thr 375 Thr 375 β-phosphate 

a Somatic PGK [67]. 

b PGK in spermatogenic cells [68]. 

Page 377 

commercially available. However, its sequence has been determined [67] and appears to be 97% 

identical to horse and pig PGK. For completeness it should be mentioned that there is a second human 

PGK in testis tissue that is 87% identical to the somatic enzyme [68]. 

The crystal structures of the apo-enzyme from horse [31] and of the binary complex between pig PGK 

and its substrate [32] (Figure 8) are available from the Protein Databank. The substrate was found to 

bind to the N-terminal domain of the enzyme. The binding site for ADP is known from the structure of 

its binary complex with PGK from B. stearothermophilus [69]. It resides in the Cterminal domain. Since 

the substrate and ADP binding sites are 10 Å apart, a hinge-bending motion between the two domains 

has been postulated to occur during catalysis [70]. 

Kinetically glycosomal PGK from T. brucei and mammalian PGK are very similar: the K m values for 

ATP are 0.29 and 0.46 and mM, respectively; the K m values for 3-phosphoglycerate are 1.62 and 0.62 

mM, respectively (due to the unavailability of the human enzyme the rabbit muscle enzyme was used as 

a substitute) [71]. The residues responsible for binding the substrate [32] are identical between human 

and glycosomal PGK [72]. However, five of the residues involved in the binding of ADP differ between 

the two enzymes (Table 3). Apparently, the biggest difference between human PGK and the parasite 

enzyme is the charged residue Lys259, which has the apolar Leu256 as a human counterpart. Molecules 

that bind at the ADP binding site and specifically recognize Lys259 might therefore be good starting 

points for drug design. 

III. Search For New Leads 

A. Triosephosphate Isomerase: The Crystallographic Cocktail Soak Approach 


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Because there are no known leads that bind to the selectivity region of TIM, the design of selective 

inhibitors is an exercise in de novo ligand design. We tried to 


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Page 378 

design such molecules on the basis of the trypanosomal TIM structure by a linked-fragment approach 

[57]. In that strategy small building blocks are designed to be complementary to the targeted surface of a 

protein. Such fragments can then be synthesized or purchased, tested for their effect on enzyme kinetics 

and for their binding mode by crystallography. Promising fragments are then linked together into larger 

molecules. The idea behind this stepwise approach is to obtain early experimental feedback in the drugdesign 

cycle. 

The crystallographic follow-up of our linked-fragment approach design for trypanosomal TIM was 

disappointing. Two designed fragments were soaked into a crystal of trypanosomal TIM, namely 4hydroxy-2-butanone 

and D-asparagine. Despite high concentrations of these molecules in the mother 

liquor, 220mM and 30 mM respectively, no convincing electron density could be seen in difference 

Fourier maps calculated between 10.0-2.8 Å [72]. Common to both molecules is that they are fairly 

polar, rather flexible, and were expected to displace crystallographically observed water molecules. 

Apparently, de novo design of tightly binding small ligands is far from trivial. 

We also tried to find new leads by a completely experimental approach. For that purpose the 

crystallographic cocktail soak (CCS) approach was developed. In this method cocktails of fine 

chemicals are soaked into a crystal in the hope of finding crystallographic evidence of binding for one of 

the molecules from the cocktail. The identification of such a molecule might not be clear immediately 

because several molecules in the cocktail might be compatible 

with the shape of the electron density, especially if the resolution is not very high. An outcome would be 

provided by a dichotomic approach (Figure 9), in which the crystallographic soaking experiment is 

repeated with ever smaller subcocktails of the original one. For example, if a ligand shows up from a 

cocktail soak of 32 compounds, a second experiment should be done with only half of the compounds. If 

the ligand fails to show up, one knows that it is one of the alternative 16 compounds. After at most six 

experiments the identity of the ligand is known. One might like to think of the CCS approach as the 

experimental analog of the computational methods in programs like GRID [73] or MCSS [74], but with 

thirty-two compounds at a time. 

Figure 9 

Dichotomic search for unknown ligand from 

cocktail 1. The number of compounds in the sub 

cocktail is indicated by n, and the 

interpretation about the presence of compound 

X in the subcocktail is given as Y/?/N. 


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Page 379 

For trypanosomal TIM we experimented with three different cocktails of 32 compounds (Table 4). 

Molecules were chosen in such a way that they would be compatible, soluble, cheap, and as varied as 

possible. Each compound was present at a concentration of 1 mM. The final cocktail solutions were 

clear and devoid of precipitate. Since this was a pilot experiment both subcocktails were checked at each 

stage of the dichotomic strategy. Only the soak with cocktail 1 revealed electron density that could not 

be accounted for by water molecules, hereafter called peak X. The soaks with cocktails 2 and 3 led to 

featureless difference Fourier maps. The quality of the data and refinement can be inspected from Table 

5, while Figure 9 illustrates the dichotomic search to identify peak X. An oxidized molecule of DTT, 

identified in the high-resolution structure of the native TIM crystals [24], served as an internal reference 

to judge the quality of the data and the noise level in the final difference Fourier maps. 

Peak X was found near His95 of the second subunit of the enzyme, i.e., the subunit where the flexible 

loop adopts the closed conformation in this crystal form. Its signal was somewhat weaker than that of 

DTT. The same density showed up when crystals were soaked with subcocktail 1B but not with 1A, 

narrowing down the list of potential ligands to sixteen compounds. However, the next round of the 

dichotomic search led to a problem that has not been solved thus far. Peaks of roughly the same shape as 

the original peak X appeared with both subcocktails 1BA and 1BB. Several strategies were followed to 

improved the quality of the maps. First, the model was further refined with all data while a bulk solvent 

scattering correction [75] was incorporated. Second, a variety of maps were calculated: (|F o|-|F c|) e iαc, 

(2|Fo|-|Fc|) eiαc, (3|Fo|-2|Fc|) eiαc and (|Fo|-|Fo,native|)eiαc. Third, all maps were SIGMAA-weighted [76]. 

Since the shape of peak X varied substantially between the different maps it can be tentatively 

concluded that peak X did not originate from the presence of a compound but was noise. The lesson of 

this experiment seems to be that the crystallographic cocktail soaking approach should only be tried 

when high- resolution data can be obtained, probably better than 1.8 Å resolution. 

B. Glyceraldehyde-3-Phosphate Dehydrogenase: Docking 

In order to discover new ligands that would block GAPDH of T. brucei by occupying the adenosine 

binding region we used the program DOCK [77], version 3.5. This program characterizes a binding site 

by filling it with a set of overlapping spheres. The centers of these generated spheres constitute an 

irregular grid, called a “graph” by mathematicians. Docking of a ligand then consists of matching 

subsets of ligand interatomic distances onto subsets of the receptor graph. Finally, the quality of the fit 

between a docked ligand and the receptor is evaluated. Within DOCK 3.5 three methods are available 

for this evaluation: contact scoring, which measures shape complementarity; force-field scoring, which 

is an estimate of the enthalpy of the intermolecular interaction; and elec- 


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Table 5 Crystallographic Cocktail Soaking Experiments of Trypanosomal TIM Crystals a 

res DTT X 

C a(Å) b(Å) c(Å) m(°) (Å) R-sym Compl R (σ) (σ) 

1 112.7(1) 97.7(1) 46.7(1) 0.7 2.85 0.044 0.97 0.136 5.0 3.5 

1A 112.6(1) 97.6(1) 46.8(1) 0.9 2.50 0.099 0.96 0.173 7.0 2.0 

1B 112.7(2) 97.5(3) 46.8(2) 0.7 2.30 0.044 0.88 0.172 5.0 4.0 

1BA 112.6(2) 97.9(2) 46.7(2) 0.7 2.40 0.051 0.96 0.202 7.8 3.0 

1BB 112.6(1) 97.8(1) 46.7(1) 0.7 2.30 0.034 0.90 0.203 5.0 3.0 

2 112.9(1) 97.8(1) 46.7(1) 0.9 2.40 0.060 0.92 0.172 5.0 - 

3 112.9(1) 97.7(3) 46.7(2) 0.7 2.40 0.057 0.93 0.170 4.0 - 

a The following data are tabulated column-wise: C = cocktail; a, b, c = cell parameters of the P212 12 1 crystals; m = 

mosaicity; res = resolution; R-sym = agreement between symmetry-related reflections; Compl = completeness; R = 

agreement between data and model; DTT = signal of oxidized DTT in the final difference Fourier map. DTT is 

present in the mother liquor but not incorporated in the model. X = signal of peak X in the final difference Fourier 

map. Maps were calculated with data between 10.0 Å and the high resolution limit. 

trostatic scoring, where the linearized Poisson-Boltzmann equation is solved [78]. We report here on 

docking experiments to identify GAPDH inhibitors from the Available Chemicals Directory-3D 93 

(ACD) [80]. 

Force-field scoring and electrostatic scoring require the assignment of partial atomic charges. 

Unfortunately, such charges are not available in the ACD, mainly because there is no consensus on a 

method to calculate them. Since the number of molecules in the ACD is very large, about 73,000 in 

1993, we opted for the charge-equilibration algorithm developed by Rappé and Goddard [81] as 

implemented in the BIOGRAF program [82]. This method leads to charges that are in excellent 

agreement with experimental dipole moments and with atomic charges obtained from electrostatic 

potentials of accurate ab initio calculations. A script was written that processed the entire ACD 

automatically on an R4000 processor in about two days. Charges of the protein atoms were assigned 

from the table of AMBER-derived charges provided with DOCK 3.5. 


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Because there are tens of parameters that can be varied in the program there is no such a thing as the 

DOCK run for a given protein target. We chose to perform two parallel runs that differ in receptor 

description. In run 1 the DOCK sphere center description was used while in run 2 the atomic coordinates 

of 2'- deoxy-2'-(3-methoxybenzamido)adenosine, a designed selective inhibitor of T. brucei GAPDH 

(see Section IV), were picked to describe the receptor site. For each run the same program parameters 

were chosen (Table 6) and the ACD database was split up in batches of 10,000 molecules. The 

computation was done on an Indigo2 workstation with an R4400 processor operating at 175 MHz. It 

took 3 h 19 min of CPU time for run 1 and 34 h 48 min for run 2. The ten-fold time difference between 

the two runs originates from the different number of 


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Table 6 Parameters Chosen for the DOCK Runs with T. brucei GAPDH a 

Program Variable Value Program Variable Value 

SPHGEN dentag X DOCK distance_tolerance 1.0 

dotlim 0.0 nodes_maximum 4 

radmax 5.0 nodes_minimum 4 

radmin 1.0 ligand_binsize 0.4 

DISTMAP polcon 2.3 ligand_overlap 0.1 

ccon 2.8 receptor_binsize 0.8 

discut 4.5 receptor_overlap 0.2 

perang 3 atom_minimum 5 

a Variables as defined in the DOCK 3.5 manual and discussed in References 79 and 83. 

centers to describe the receptor, namely 20 for run 1 and 34 for run 2. According to theory [83], the 

difference should scale as 34 4/20 4 = 8.3, which fits our observation. Since it is well known that scores 

exhibit poor correlation with real affinities [84] we decided to subject the 200 best-scoring ligands of 

each batch to inspection on the graphics. Scanning through the 3200 compounds required about ten 

days. Eventually, sixteen compounds were selected for purchase on 

Table 7 Parasite GAPDH Inhibitors Discovered with DOCK 

Contact Delphi 

Inhibitor IC 50 (mM) Score Rank Score Rank 

Run 1 

2-Guanidinobezimidazole 1.2 97 1432 1.01 1427 

2-Benzimidazoylurea 1.8 102 958 0.27 1241 

4-Nitrophenyl sulfone 2.8 106 608 -0.46 370 

Tryptophan 6.0 128 32 -0.48 357 


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5-Methoxytryptamine 19.0 92 1495 -0.32 488 

Ephedrine 25.0 99 1315 -0.22 590 

Epinephrine >7 a 102 989 -3.09 27 

Aspartic acid dimethyl ester >10 87 1551 -1.66 146 

3-Amino-L-tyrosine >11 a 102 969 -0.14 703 

1,3-Diphenylguanidine >11 a 112 301 -1.28 209 

Octopamine >50 a 106 660 -2.03 83 

Run 2 

2-Nitrobenzoic acid hydrazide 4.4 132 900 0.11 1112 

Dopamine 12.0 143 241 -2.02 156 

Norepinephrine >5.4 a 151 84 -2.09 137 

4'-Amino-N-methyl acetanilide >7.8 a 131 1027 0.04 1021 

L-histidinol >9.3 142 300 0.13 1156 

a Could not be tested at higher concentrations due to solubility problems. 


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Figure 10 

Binding mode of 2-guanidinobenzimidazole to T.brucei GAPDH as predicted by 

DOCK. The benzimidazole moiety occupies roughly the position occupied by adenine 

in the holo-enzyme, whereas the guanidino group a salt bridge to Asp37. 

the basis of structural rigidity, chemical stability, solubility, and electrostatic complementarity. The 

latter property was evaluated with the program DELPHI [85]. 

Page 383 

Each of the sixteen compounds was tested for GAPDH inhibition (Table 7). Half of them were inactive 

while the other ones showed inhibition in a range between 1.2 and 25 mM. Unfortunately, there appears 

to be no correlation between the DOCK scores and the IC 50 values. For examples, norepinephrine and 

1,3-diphenylguanidine are inactive while they have a better score than 2- guanidinobenzimidazole 

(Figure 10), the compound with the best IC 50. Also, it appeared that the two different receptor 

descriptions used led to almost completely different lists of compounds. Only 156 molecules occurred in 

both lists of top-scoring molecules. In the modeled binding mode all of the inhibitors occupy roughly the 

same position as the purine ring of NAD in the crystal structure of GAPDH. While the values obtained 

for IC 50 are indicative of poor inhibition, one has to keep in mind that adenosine exhibits an IC 50 of 50 

mM [13]. By using the program DOCK we were able to discover ligands that have a substantially higher 

affinity for GAPDH than the natural ligand. 

C. Phosphoglycerate Kinase: Leads from the Past 

The starting point for drug design in the case of PGK is quite different from TIM or GAPDH because a 

number of nonsubstrate-like inhibitors have been 


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Figure 11 

Two-dimensional structure of SPADNS, a 

micromolar inhibitor of T.brucei PGK. 

Page 384 

discussed in the literature. Suramin inhibits glycosomal PGK of T. brucei with a K i of 8.0 μM [69]. In 

addition a number of yeast PGK inhibitors are known: gallic acid with a K i = 0.4 mM [86], 

hydroxyethylidene biphosphate with a K i = 24 mM [87]. 1,3,6-naphthalenetrisulphonic acid with a K i = 

5.5 mM, and 2-(p- sulphophenylazo)-1,8-dihydroxy-3,6-disulphonic acid, also known as SPADNS 

(Figure 11), with a K i = 126 μM [87]. None of the four yeast PGK inhibitors are potent, but, for 

SPADNS, the binding mode has been further characterized. Studies by Williams et al. [87] have 

demonstrated that SPADNS is directly competitive with both enzyme substrates, 3-phosphoglycerate 

and ATP. Moreover, by 600 MHz 1H-NMR it was shown that SPADNS interacts with the nucleotide 

binding site while the conformation of the enzyme changes substantially [87]. 

Since the four yeast PGK inhibitors are commercially available it was logical to test them for T. brucei 

PGK inhibition. The first three compounds were active in the millimolar range. However, SPADNS 

exhibited a K i of 10.0 μM in these in preliminary tests [88]. Moreover, when assayed against a 

commercially available rabbit muscle PGK, SPADNS had no influence on the enzyme kinetics up to a 

concentration of 250 μM [88]. In conclusion, SPADNS appears to be an excellent lead because of its 

potency and selectivity. Crystallographic experiments to determine its binding mode to T. brucei PGK 

are underway. 

IV. Lead Optimization: Glycosomal Gapdh 

From the selectivity point of view the adenosine binding site of GAPDH is attractive for drug design, as 

we explained in Section II.B. Unfortunately, inhibition studies on T. brucei and L. mexicana GAPDH 

revealed the poor affinity of our natural lead adenosine with IC 50 values of 100 mM and 50 mM, 

respectively. Moreover, adenosine is an “antiselective” lead because the IC 50 


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for human GAPDH is better than for the parasite enzymes, namely 35 mM [13]. Despite the fact that 

these IC 50 values are about ten thousand times higher than what would be considered a lead in the 

pharmaceutical industry we decided to optimize the affinity and selectivity of adenosine. 

Page 385 

Each of the three areas where differences occur between the parasite and the human enzyme are 

hydrophobic. Therefore, we modeled hydrophobic substituents at positions C2, C8, and O2' of adenosine 

under the constraint that they were conformationally compatible with the C2'-endo pucker of the ribose 

sugar. Designing derivatives at O2' was a problem, however. Each of the two ribosyl hydroxyls forms a 

hydrogen bond with the carboxylate of Asp37. Since making direct derivatives of the hydroxyl, such as 

ethers or esters, would deprive the Asp of a hydrogen-bond partner while burying the carboxylate, 

resulting molecules would have a dramatically reduced affinity. Moreover, an alignment of 47 GAPDH 

sequences made it clear that the Asp is highly conserved [89]. An elegant way to overcome this problem 

was to replace the 2' -hydroxyl by a 2'- amino function. Moreover, coupling with carboxylic acids was 

appealing from a synthetic point of view while the conformational properties of the amido-substituted 

system would ensure the correct orientation of substituents into the selectivity cleft. The modeled 

inhibitors were evaluated for the quality of their fit to the protein surface and subsequently synthesized. 

From Table 8 it can be seen that our predictions were successful. The addition of a methyl group at C2 

of the adenine ring, which is close to Val36, increased the affinity for parasite GAPDH by an order of 

magnitude. The effect of a thienyl substitution on C8, targeted to Leu112, was even bigger, namely two 

orders of magnitude. However, both substitutions are only mildly selective (Table 8). As expected, the 

greatest gain in selectivity was obtained by modifying the 2'-position of the ribose, so that the selectivity 

cleft is filled up (Figure 12). The 2'-deoxy-2'-(3-methoxybenzamido) adenosine compound (Figure 13) 

bound at least 48 times better to L. mexicana GAPDH than to the human enzyme. The selectivity versus 

T. brucei GAPDH appeared to be smaller. This has to be ascribed to a difference in residues contacting 

the 3-methoxy moeity. The residue Asn39 of T. brucei GAPDH has a Ser equivalent in the L. mexicana 

Table 8 Inhibition Gains of Designed GAPDH Inhibitors with Respect to Adenosine 

C2-subst C8-subst C2'-subst T. brucei L. mexicana human 

CH 3 H OH 12.5 6.25

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Figure 12 

Predicted binding mode of 2' -deoxy-2'-(3-methoxybenzamido)adenosine to T.brucei 

GAPDH. (From Ref. 13. Copyright 1994 by the American Chemical Society.) 

enzyme. In conclusion, the strategy of burying hydrophobic residues with lipophilic substituents paid 

off. 

Page 386 

Despite the rather poor IC 50 values of our optimized inhibitors, an evaluation of their effect on live 

trypanosomes appeared to be useful. Enzyme inhibitors that are not taken up by the parasites would be 

of no use as a drug. Therefore, the effect of 2-methyl-adenosine, 8-(thien-2-yl)-adenosine and 2'- deoxy- 

2'-(3-methoxybenzamido)adenosine on the growth of T. brucei in cultures, as described by Baltz et al. 

[90], was monitored. At 0.1 mM all compounds inhibited the growth completely, unlike adenosine 

derivatives that were without inhibitory effect against T. brucei GAPDH [91]. Experiments are 

underway to confirm that the growth inhibition is due to blockage of the glycolytic pathway. Also, te 

mechanism of uptake of te inhibitors will be examined because it is now well established that 

trypanosomes possess a unique P 2 purine transporter that they use for uptake of purines from the host 

[15]. The experimental antitrypanosomal drug 5'-{[(Z)-4-amino-2-butenyl}methylamino}-5'deoxyadenosine(MDL 

73811) Figure 13), which is an irreversible S- adenosyl-L-methionine 

decarboxylase inhibitor, is actively taken up through the P2 transporter. Moreover, MDL 73811 is not 

actively transported in the human host, which presumably contributes to the drug's selectivity [19]. It is 

not unthinkable that our inhibitors might use the same transporter because of the nature of their scaffold, 

adenosine. 


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Figure 13 

Two-dimensional structures of 2'-deoxy-2'-(3-methoxybenzamido) 

adenosine (MMBA), a selective T.brucei GAPDH inhibitor and 

MDL 73811, an irreversible 

inhibitor of trypanosomal S-adenosyl 

methionine decarboxylase. 

Page 387 

Finally, we want to point out that pessimism about adenosine derivatives as drugs is not necessarily 

warranted. This doubt stems from the argument that many proteins recognize NAD(P), adenosine, or 

ATP. Cross-reactivity of adenosine with these different proteins and, therefore, toxicity may be 

expected. That this is not necessarily so is evident from the use of adenosine derivatives as antileukemia 

agents. For example, fludarabine, a C2'-epimer of adenosine, exhibits relatively low toxicity [92]. The 

much bigger changes to the adenosine scaffold in our inhibitors may hence lead to a surprisingly high 

overall selectivity. 

V. Conclusions 

Our goal is to discover and design selective inhibitors of trypanosomal glycolsysis. Thusfar, three 

enzymes have been targeted. Whereas little success was obtained with TIM, substantial progress is being 

made with GAPDH and PGK. 

Obstacles encountered during this project were the need for selective inhibitors and the absence of 

potent inhibitors as lead compounds. From our studies to inhibit TIM it appears that it is difficult to 

come up with inhibitors for areas on the protein surface for which no known inhibitors exist. On the 

other hand, lead optimization for GAPDH by over two orders of magnitude in just one cycle of drug 

design was straightforward. Selectivity was obtained by using part of the cofactor as a lead and 

exploiting the hydrophobic patches at the surface of the parasite enzyme. In particular, 2'-deoxy-2' (3methoxy-benzamido)ade- 


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Page 388 

nosine proved to inhibit the parasite more than fifty times better than the human enzyme. Additionally, 

by means of the program DOCK, eight new leads for GAPDH inhibition were found. None of them 

were micromolar inhibitors but all of them were more potent than the natural lead, adenosine. For 

trypanosomal PGK a potent lead compound, SPADNS, was discovered by testing inhibitors that had 

been described as weak inhibitors of yeast PGK. Moreover, this lead had no effect on mammalian PGK 

at concentrations up to twenty-five times higher than that needed for T. brucei inhibition. At present, 

none of our inhibitors is potent enough to consider clinical tests. There is hope, however, since our 

GAPDH inhibitors inhibit the growth of trypanosomes in cultures. 


It is a pleasure to thank the many colleagues and collaborators who have contributed to this project: Paul 

Michels, Veronique Hannaert, Sylvie Allert, and Linda Kohl (Institute for Cellular Pathology in 

Brussels) for cloning and overexpressing trypanosomatid enzymes; Phil Petra (University of 

Washington, Seattle) for helping us out with protein purification protocols; Mia Callens and Fred 

Opperdoes (Institute for Cellular Pathology in Brussels) for enzymology and parasitology; Rik 

Wierenga, Martin Noble, Fred Vellieux, Randy Read, Risto Lapatto, Hillie Groendijk, Tjaard Pijning, 

and Kor Kalk for laying the structural foundation of the project in Groningen and Heidelberg; Cees 

Witmans and the late Alan Horn (University of Groningen), Michèle Willson and Jacques Perié 

(University of Toulouse), Serge Van Calenbergh, Arthur Van Aerschot, and Piet Herdewijn (University 

of Leuven) for synthesizing TIM and GAPDH inhibitors; Kim Simons (University of Washington) for 

going after completely new GAPDH inhibitors; Véronique Mainfroid and Joseph Martial (University of 

Liège) for providing human TIM; Klaus Muml;uller and Klaus Gubernator (Hoffmann-La Roche, Basel) 

for valued modeling advice; and Mike Gelb (University of Washington) for valued discussions. 

Financial support for these investigations has been provided by the World Health Organization, 

Hoffmann-LaRoche in Basel, the Dutch Organization for the Advancement of Science (NWO), the STD 

program of the European Community, the School of Medicine of the University of Washington, and the 

Murdock Charitable Trust. 


We just solved the ternary structure of PGK from Trypanosoma brucei in complex with ADP and 3phosphoglycerate. 

The enzyme is in the closed conformation that has eluded crystallographers for 20 

years. 


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16 

Progress in the Design of Immunomodulators Based on the Structure of 

Interleukin-1 

Glen Spraggon 


Pandi Veerapandian 

Axiom Biotechnologies, Inc., San Diego, California, and 

La Jolla Institute for Experimental Medicine, La Jolla, California 


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The interleukin-1 (IL-1) family of cytokines exhibit both normal and pathological effects in almost 

every tissue and organ system and, as such, have been associated with cells engaged in the immune 

response, inflammatory cells, and cells engaged in development, differentiation, and repair processes 

[1–4]. Interleukin-1 can produce either a direct response on one specific target cell or act as an indirect 

effector molecule, inducing the expression of a variety of genes and synthesis of several proteins such as 

IL-2–IL-8, tumor necrosis factor (TNF), colony-stimulating factors (CSF), platelet-derived factors 

(PDFs), and other cytokines. Pathologically uncontrolled, IL-1 activity can induce disease states and has 

been linked to septic shock, the growth of acute and chronic myelogenous leukemia cells, inflammation 

associated with arthritis and colitis, development of atherosclerotic plaques, insulin-dependent diabetes, 

osteoporosis, parsitemia, and cancer [1,5,6]. Strategies to treat such diseases are being developed and 

usually involve the inhibition of IL-1 synthesis or the blocking of its activity [7]. The therapeutic 

advantage of reducing the activity of IL-1 resides in preventing its deleterious biological effects without 

interfering with homeostasis. In order to achieve such a goal, an understanding of the structure-function 

relationship of the IL-1 family of proteins at the molecular level is important. Such a knowledge could 

lead to the design and development of 


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synthetic agents with the ability to modulate IL-1 responses. Here we review the present knowledge of 

the IL-1 system and the attempts toward the development of its modulators. 

A. Cytokines and Physiological Responses 

Cytokines are multifunctional hormones produced by a variety of cells to carry out a wide range of 

overlapping biological actions including communication within the immune system and between other 

cell types. They are produced by macrophages, endothelial cells, fibroblasts, keratinocytes, T cells, B 

cells, and natural killer cells in response to injury and infection [7]. Upon production they act either 

locally or as systemic intercellular signaling factors and induce the production of other cytokines for 

continuing their communications via cell surface receptors. The cytokines network also has associated 

with it a complementary set of soluble or membrane-bound antagonist or mediator molecules that are 

capable of shutting down these effects [8]. During infection and injury, the leukocytes adhere 

themselves to the endothelium and migrate into tissues. Cells like T-lymphocytes, monocytes, 

macrophages, and neutrophils collect themselves as inflammatory infiltrate. Once within the tissues, 

these cells neutralize the microorganisms or infected cells, secreting other cytokines that can modulate 

the adhesion molecule expression on the endothelium. In addition, chemotactic signals are delivered to 

cells passing in the circulation. Cytokines are also involved in tissue repair, i.e., remodeling of 

connective tissues and revascularization of damaged areas. Soluble mediators are released from the site 

of damage into the circulation to act in an endocrine fashion. The cytokines then control hepatic 

responses to tissue damage. During the regulation of hematopoiesis, cytokines induce the production and 

release of a number of colony-stimulating factors and other interleukins. Thus induced factors are 

responsible for the replacement of leukocytes and erythrocytes that were lost after trauma. 

A large number of cytokine molecules have been characterized [7]. Structurally it appears that these 

cytokines can be classified into the following main groups based on their folding pattern: 4-helix 

bundles, beta-trefoil, beta sandwich, EGF-like, beta cysteine knot, and alpha/beta. Thus far the structures 

of many representatives of each family have been solved by both x-ray crystallography and NMR 

(Table 1). Extensive experimentation has indicated that despite a large degree of structural diversity, 

there is a large degeneracy in the cytokine network and the molecules have a wide range of overlapping 

biological functions. For example, IL-1, TNF, PDGF, and TGFB all exhibit similar biological behavior. 

The structures of the receptors for these molecules have not been forthcoming and only three examples 

of the extracellular domains of the receptors have been reported. They are growth hormone receptor in 

complex 


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Table 1 Known Three-Dimensional Structures of Cytokines 

Brookhaven Structure 

Molecule Type Code Determined by 

Interleukin-1 alpha (IL-1α) Beta trefoil 2ILA X-ray crystallography 

Interleukin-1 beta (IL-1β) Beta trefoil 1ILB, 2ILB, 4ILB, 

5ILB, 6ILB 

Interleukin-1 receptor antagonist 

(IL-1ra) 

Fibroblast growth factor (acidic) 

(aFGF) 

Fibroblast growth factor (basic) 

(bFGF) 

Granulocyte-colony stimulating 

factor (G-CSF) 

X-ray crystallography and 

NMR 

Beta trefoil 1ILR, 1IRP X-ray crystallography and 

NMR 

Beta trefoil 1AFC X-ray crystallography 

Beta trefoil 1BFG X-ray crystallography 

Long-Chain 4 helix 

bundle 

Growth hormone Long-Chain 4 helix 

bundle 

Leukemia inhibitory factor (LIF) Long-Chain 4 helix 

bundle 

Granulocyte-macrophage colony 

stimulating factor (GM-CSF) 

Short-Chain 4 helix 

bundle 

Interleukin-2 (IL-2) Short-Chain 4 helix 

bundle 

Interleukin 4 (IL-4) Short-Chain 4 helix 

bundle 

1RHG, 1GNC X-ray crystallography 

1HGU X-ray crystallography 

1LKI X-ray crystallography 

1CSG, 1GMF X-ray crystallography 

3INK X-ray crystallography 

1BBN, 1ITM, 

2CYK 

NMR 

Interferon gamma (IFN-γ) Dimeric 4 helix bundle 1IKI X-ray crystallography 

Interleukin 10 (IL-10) Dimeric 4 helix bundle 1ILK X-ray crystallography 

Nerve growth factor (NGF) Beta cysteine knot 1BET, 1BTG X-ray crystallography 

Platelet derived growth factor 

(PDGF) 

Beta cysteine knot 1PDG X-ray crystallography 


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Transforming growth factor beta2 

(TGFB2) 

Transforming 

growth factor 

alpha 

(TGFA) 

Tumour 

necrosis 

factor 

(TNF) 

Macrophage inflammatory protein 

1-beta 

Beta cysteine knot 1TFG, 2TGI X-ray crystallography 

Beta EGF-like 2TGF NMR 

Beta sandwich 1TNF X-ray crystallography 

Alpha/Beta 1HUM NMR 

Interleukin 8 (IL-8) Alpha/Beta 1IL8 NMR 

Melanoma growth stimulating 

activator (MGSA) 

Alpha/Beta 1MGG NMR 

Platelet factor 4 Alpha/Beta 1RHP X-ray crystallography 


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with growth hormone, prolactin receptor (PRLR), and tumor necrosis factor with a ligand. In the case of 

the human growth hormone-receptor complex and tumor necrosis factor-receptor complex the 

interaction between the ligand and the receptor is intricate and takes place over a large area. It is likely 

that this will be a characteristic feature of all cytokine-receptor complexes. 

B. Cytokine-Based Therapy 

Certain disease states can occur due to a disregulation of the cytokine network. This can lead to chronic 

stimulation of the immune and inflammatory response and ultimately to disease. Many cytokines have 

been implicated in autoimmune diseases like myasthenia gravis, insulin-dependent diabetes, 

atherosclerosis, systemic lupus erythematosus, and rheumatoid arthritis [1,7,9]. The intimate relationship 

between cytokines and the pathology of disease development and progress can be exploited to provide 

therapeutic benefit. By subtly manipulating the cytokine communication network one can modulate the 

disease process. Understanding of the functional roles of cytokines that mediate the communication 

between them and the factors involved in the immune system's cell-cell interactions forms the 

foundation for cytokine therapies. Such therapeutic agents are now a possibility due to modern 

techniques such as molecular biology, structural biology, high-power computation, combinatorial 

chemistry, and functional screening. The findings from biological techniques in conjunction with the 

structural insights as obtained through protein crystallography and nuclear magnetic resonance form the 

foundation for rational drug design [10–13]. Discovery of IL-1-based therapeutic agents have led to 

promising applications in the treatment of the above mentioned diseases. Design of synthetic adjuvants, 

for exogenous immunomodulation, is also an important factor to be considered in the field of vaccine 

development. In this review we will discuss the available literature on interleukin-1 and the recent 

attempts toward the design of immunomodulators. 

II. The Interleukin-1 Family 

The three molecules of the IL-1 family, interleukin-1α (IL-1α), interleukin-1β (IL-1β), and interleukin-1 

receptor antagonist (IL-1Ra) map to the long arm of chromosome two in the human genome. It appears 

that the family arose via a gene duplication event some 350 million years ago, and the molecules possess 

between 27.5 and 36% sequence identity with each other (Table 2) [1,14,15]. In addition, the genes for 

the two IL-1 receptors IL-1R1 and IL-1RII [16,17], and an IL-1R accessory protein (IL-1RacP), which 

binds to the IL-1, IL-1 receptor complex [18], have been identified. Together, these molecules via their 

differential activity serve primarily to modulate the host defense mechanism. 


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Table 2 RMS Distance of Aligned Cα Moieties of the Three Interleukin-1 Structures 

IL-1β IL-1α IL-1Ra 

IL-1β 0.0 1.8 Å (36.3%) 1.42 Å (30.7%) 

IL-1α 1.8 Å (36.3%) 0.0 1.8 Å (27.5%) 

IL-1Ra 1.42 Å (30.7%) 1.8 Å (27.5%) 0.0 

Number in parentheses referred to sequence identities between the aligned structures. 

A. Expression and Processing of IL-1 

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All three IL-1 molecules, IL-1α, IL-1β, and IL-1Ra are synthesized as 31 kDa precursor molecules 

produced primarily by mononuclear phagocytes. These precursor proteins can be subsequently 

processed to mature 17 kDa molecules. The means whereby this is achieved appears to be different for 

each type of molecule and may help to explain the roles of the three. Both agonist molecules IL-1α and 

IL-1β lack a classical hydrophobic leader sequence and thus must be processed in an alternative way. 

The two can be separated by their biological activity in the precursor form, IL-1α being active in both 

precursor and mature forms while IL-1β produces a biological response only in its processed form. In 

addition, a specific enzyme, Interleukin-1 beta converting enzyme (ICE), cleaves IL-1β to its mature 

form. This enzyme is a cysteine protease whose only known substrate is proIL-1β, which it cleaves at 

Ala 117 [19,20]. The means whereby pro IL-1α is processed is still largely a mystery although it has 

been postulated that a calpain or related protein may perform the task. 

Naturally occurring IL-1Ra is a 22 kDa glycosylated protein [14,15,21– 24] that possesses a signal 

sequence. It is likely that it is processed in the conventional manner as can be concluded by the 

glycosylation states of the molecule not present in the agonists [25]. The LPS-stimulated human blood 

monocytes initially express the gene for IL-1Ra [25]. An alternatively spliced form of IL-1Ra also exists 

(intracellular IL-1Ra), which remains inside the cell presumably to block intracellular IL-1 action [26]. 

Both soluble and intracellular forms of IL-1Ra block IL-1R but do not trigger any biological response. 

B. Receptors and Responses 


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All effects of the IL-1 family are exerted via their receptors, which are present on a variety of different 

cell types. There are two known IL-1 receptors. The first, type I (IL-1RI) is an 80-kDa formation found 

mainly on T cells and fibroblasts; the second, type II (IL-1RII) is present on B cells, monocytes, 

neutrophils, and heptoma cells and is approximately 60 kDa in size. From sequence analysis these 

molecules are believed to belong to the Ig-superfamily, the extracellular portion of the receptors 

consisting of three immunoglobulin-like domains of approximately 100 residues each [27,28]. The IL- 

1RI receptor 


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Figure 1 

Schematic representations of the members of the IL-1 family. 


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has a single membrane-spanning region and a cytoplasmic region of 213 amino acids. The IL-1RII 

receptor has a single membrane-spanning region and a cytoplasmic region of 29 amino acids. 

Page 401 

All three IL-1 molecules bind with a high affinity to the receptor IL-1RI. However only IL-1α and IL- 

1β produce a detectable response [29]. The IL-1Ra molecule completely blocks the binding of the 

former two molecules without inducing any signal transduction events. The second IL-1 receptor, IL- 

1RII, similarly binds IL-1 tightly but does not produce any signal [30]. The soluble portion of IL-1RI 

binds to IL-1Ra, IL-1α, and IL-1β in decreasing order of affinity whereas the soluble portion of IL-1RII 

binds IL-1β, proIL-1β, IL-1α, and IL-1Ra, in that order. It therefore appears that both IL-1Ra and IL- 

1RII take part in modulation of the agonist molecules, an argument supported by the finding that soluble 

IL-1RII binds IL-1β with a similar affinity to the cell-associated receptor; whereas, the affinity of IL- 

1Ra to such a soluble receptor is some 2000 times less [31,32,33]. Greenfeder, et al., [18] have 

identified a new molecule, the IL-1 accessory protein IL-1RacP. The complex of IL-1 and IL-1 receptor 

binds to IL-1RacP and together they induce the signal [18]. Greenfeder also observed that antibodies to 

IL-1RI and to IL-1RacP block IL-1 binding and activity. From sequence analysis it appears that the 

cytoplasmic domains of IL-1R and IL-1RacP contain the same amino acid domains commonly found in 

the members of the GTPase family of proteins [34]. It has been proposed that such a complexation may 

lead to a closer proximity of these cytoplasmic domains and thus facilitate signal transduction. A scheme 

showing functional relationships between molecules that make up the IL-1 system—IL-1α, IL-1β, IL- 

1Ra, IL-1RI, IL-1RII, and related receptors—is shown in Figure 1. 

C. Autoantibodies of IL-1 

In addition to these molecules, naturally occurring neutralizing autoantibodies of IgG type to IL-1α have 

been identified. These have been detected in serum isolated from human donors. [35,36]. These 

antibodies bind to both proIL-1α and 17-kDa IL-1α [37] and completely prevent the binding of IL-1α to 

type-I cell surface receptors [38]. Patients with autoimmune diseases have higher populations of these 

antibodies [39]. 

III. Three-Dimensional Structural Information 

In an effort to understand the natural and interactions of the IL-1 family, various laboratories have 

undertaken the task of elucidating the three-dimensional structure of the molecules. To the present this 

has resulted in the structures of all three members of the IL-1 family being solved independently by both 

x-ray 


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crystallography and heteronuclear NMR spectroscopy [40–49]. In addition to these, the structure of IL- 

1β converting enzyme has been determined [50]. The molecule is an oligomeric cysteine protease 

consisting of 10- and 20-kDa subunits. To date proIL-1β is its only physiological substrate. As 

processing is essential for IL-1β extracellular transport and activity, inhibitors of ICE could provide a 

method to block the production of the active form of IL-1β. Although the elucidation of these structures 

have been important in determining various differences among the individual proteins, perhaps a more 

important result would be the elucidation of the structures of the two interleukin-1 receptor molecules, 

either individually or in complex with their substrates. This information could provide a means to draw 

together the present structural and biochemical knowledge into a coherent picture of the agonistic 

activity of IL-1α and IL-1β as opposed to the antagonist effect of IL-1Ra. The structures should also 

provide a foundation upon which structure-based drug design could proceed. To date no crystals or 

structural reports for the receptors have been published. 

A. The β-Trefoil Fold 

Structurally IL-1 exhibits a unique fold, known as a β-trefoil fold. Each IL-1 molecule show the 

characteristic β-trefoil fold and small deviations of the back-bone despite the relatively low sequence 

identity (Table 2). Figure 2 shows the structural alignment of the IL-1 molecules. The β-trefoil fold was 

first observed in Kunitz-type soybean trypsin inhibitor [51]. The fold has since been identified numerous 

times in the Kunitz family of protease inhibitors, the interleukin-1 system molecules, and the acidic and 

basic fibroblast growth factors [52]. Although these proteins have diverse biological function and low 

sequence identity to each other, all appear to have a common structural core. Each one, however, binds 

to its specific receptor with high affinity. The present known examples of proteins with such a fold are 

composed of between 125 and 170 amino acids. The overall fold itself is an antiparallel β barrel 

consisting of six two-stranded hairpins. Three of these form a barrel structure (strands denoted β1, β4, 

β5, β8, β9, and β12) while the other three are in a triangular array that caps the barrel. The arrangement 

of these moieties is such as to give the molecule a pseudo-three-fold axis [52]. Each fragment 

contributes one pair of antiparallel beta strands to the barrel and one pair to the cap of the barrel, thus 

forming a so called “open” and “closed” end to the structure [42]. 

B. Three-Dimensional Structure of IL-1β 

The structure of IL-1β was the first of the IL-1 family to be solved and has been solved independently 

five times: four times by x-ray crystallography [40– 


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Figure 2 

Structural alignment of IL-1α, IL-1β, and IL-1Ra. Residue numbering is taken from IL-1β. Residues bordered in black are conserved 

over the three molecules while those in gray constitute a conservative substitution. Arrows indicate sheet region, and the cylinders 

indicate helix. Figure produced by Stamp [109] and Alscript [110]. 


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Figure3 

(a) Stereo diagram of the secondary structural elements of IL-1β. Produced by Molscript [106]. 

(b) Stereo plot of all the atoms of IL-1β viewed parallel to the axis of the barrel. 

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42,44], and once by NMR [43]. The four crystal structures, each to 2.0 Å, were all solved in the same 

space group, P4 3, each structure being in relatively good agreement with the others [53]. As pointed out 

previously, the molecule adopts a β-trefoil fold with about 65% of the molecule in beta sheet and 35% in 

random coil/turn (Figure 3a). The IL-1β molecule resembles a conical barrel with a shallow open face 

on one end and a closed face on the other. The length of the long tubular core of the molecule is about 

23 Å. Twelve antiparallel β 


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strands constitute the secondary structural elements of IL-1β. Three pairs of β strands (one pair from 

each of the fragments) form the six-stranded barrel and the other three pairs cover one end of the barrel, 

referred to as the “closed end”. The amino and carboxy termini are close to each other at the “open end” 

of the barrel (Figure 3b). The overall structure of the molecule consists of three similar fragments (F1, 

F2, F3) related by a pseudo-3-fold symmetry, with each fragment having a βββLβ motif. Residues 1 to 

52 (F1), 53 to 107 (F2), and 108 to 153 (F3) form these fragments. These βββLβ motifs can be 

superposed to display structural similarities. 

The core of the molecule consists entirely of hydrophobic residues, two-thirds of which are leucines and 

phenylalanines—a feature that seems to be essential to maintain the structural integrity of the molecule. 

Most of the aromatic groups have their planes aligned along the barrel axis and both ends of the barrel 

have concentrations of exposed polar residues that may be involved in binding interactions with the 

receptor (see below). No alpha-helical structure is observed in IL-1β although one short region of 3 10 

helix is observed between residues Gln34 and Gln38. Two of the β hairpins are in the open end and 

three are at the closed end. Residues 18 to 28, 69 to 82, and 122 to 135 form the three β hairpins at the 

closed end where three strands (residues 24–28, 78–82, and 129–135) are in close proximity and form 

three sides of a triangle, covering this end of the barrel. 

C. Open End of the Barrel 

Analysis of the surface of IL-1 reveals an epitope consisting of many polar residues widely spaced, 

approximately in an annular fashion, around the open end of the barrel (Figure 3b). It has a broad 

surface area containing many polar residues [42]. Residues from six β-turns [type I, β turn 1 (T1=11- 

17); type III, β turn 3 (T3=33-36); type I', β turn 6 (T6=52-55); type I, β turn 7 (T7=62-65); type I, β 

turn 9 (T9=86-89); type I', β turn 10 (T10=106-109)] are present in this open end. There is a β bulge 

between strands β4 and β5 in one of the hairpins; this bulge may play a key role in the formation of the 

putative binding surface. The β bulge in IL-1 is of the “wide” type stabilized by multiple interactions, 

whose conformation is such as to place the side chains of polar residues (Glu51, Asn53, Asp54) in the 

proposed binding surface, fanning out in the direction of the open end of the barrel. Multiple interactions 

serve to stabilize the strained conformation of the loop, between residue 86 and 94, presenting the side 

chains of Asp86, Lys88, Asn89, and Lys93 at the open end of the barrel. In well-defined residues having 

abnormal φ, ψ values, strained conformations often may be related to functional properties and are 

observed either in the active site or in the region responsible for its activity. The reason for having such 

strains in the loop at this open end may be to place these charged side chains in the putative receptor 

binding surface. There is a short 3 10-helix in the region between β3 and 


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β4 (residue Gly33 and Gln39). By such a conformation, residues Gln32, Gln34, and Glu37 have their 

side chains in the proposed binding surface. A β turn between 62 and 65 is folded in such a way that the 

charged groups of the residues Lys63, Glu64, and Lys65 present themselves on the proposed binding 

surface. Conformational arrangement via saltbridge scaffolds and other multiple hydrogen-bonding 

interactions stabilize the residue 102 to 113 loop, and orient the side chains of Lys103, Glu105, Asn107, 

and Asn108 in the proposed surface at the open end of the barrel. Similar arrangement places the side 

chains Arg11 and Gln15 in the proposed epitope. The amino and carboxy termini form a part of the 

proposed epitope, contributing the polar side chains of Arg4, Ser5, Asn7, Gln149, Ser152, and Ser153. 

Based on extensive analysis, we found that the structural elements like electrostatic and hydrogenbonding 

interactions in the loops between strands allow the polypeptide to adopt a conformation that 

enables an unusual concentration of polar and charged groups to be presented at the open end of the 

barrel. Such a cluster of charged residues around an area that is almost perpendicular to the barrel axis 

forms a hydrophilic patch with which IL-1 might bind to the receptor [42]. The core of the barrel, 

though probably not involved in binding, must nevertheless be important to the function of IL-1 because 

mutations within it reduce activity but not binding. We hypothesized that binding and cell proliferation 

through signal transduction involve separate regions of the IL-1 molecule; the surface polar loops are 

required for the binding and the core of the barrel is required for the physiological response. 

D. Three-Dimensional Structure of IL-1α 

The structure of IL-1α has been determined by x-ray crystallography [45] to a resolution of 2.7 Å in 

space group P2 1. Its general fold is very similar to that of IL-1β, having the same central β barrel along 

with the adjoining loops (Figure 4a, b). The overall rms distance between 133 aligned Cα positions of IL- 

1β and IL-1α is 1.8 Å (Figure 5). The major difference between the two molecules is an N-terminal 

extension of 8 residues beyond the N-terminus of IL-1β. This projection forms a short β strand (residues 

6-10), the presence of which positions the N-terminus of IL-1α in an alternative conformation. It has 

been postulated that this conformation is responsible for the differences in binding of precursor IL-1α 

and β: while precursor and mature IL-1α bind to IL-1RI and elicit response, only processed IL-1β binds 

with the receptor. This suggests that the N-terminal region of IL-1 probably plays a role in receptor 

binding. Extra residues in the alternate conformation of immature IL-1β serve to inhibit the receptor 

binding and thereby the biological activity. In contrast to IL-1β, IL-1α incorporates two other secondary 

structural elements: a short strand (residues 97–99) and about two turns of a 3 10 helix (residues 

101–105), neither have been shown to be important in the function of the molecule. 


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Figure 4 

(a) Stereo diagram of the secondary structural elements of IL-1α. Produced by Molscript [106]. 

(b) Stereo plot of all the atoms of IL-1α viewed parallel to the axis of the barrel. 

E. Three-Dimensional Structure of IL-1Ra 

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The x-ray structure of IL-1Ra was first reported in 1994 [47] and has since been solved by x-ray 

crystallography and NMR by several different laboratories [46–49]. Again, this molecule possesses the 

characteristic trefoil fold (Figure 6a) and similar tertiary structures (Figure 6b). The structure is similar 

to that of IL-1α and IL-1β with an rms deviation of 1.8 and 1.42 Å, respectively, when considering their 

Cα positions (Figure 7a). Structural superposition of these three molecules shows a common trefoil core 

(Figure 7b). In the absence of receptor substrate complexes, the comparison of all three structures 

combined with mutational studies (see below) is invaluable in obtaining a model of the regions 


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Figure5 

Superposition of the Cα atoms of IL-1α and β. Thinner line represents IL-1α 

and the thicker line is that of IL-1β. 


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Figure 6 

(a) Stereo diagram of the secondary structural elements of IL-1Ra. Produced 

by Molscript [106]. (b) Stereo plot of all the atoms of IL-1Ra viewed parallel to the axis 

of the barrel. 

involved in binding and exertion of biological effect in the IL-1 system. Similarity between the three 

molecules has been observed mainly in the β strands. One notable region of 


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difference between IL-1Ra and IL-1 agonists has been observed on the side of the beta barrel on site B 

of the receptor binding site (see below). The Cα positions in this region (residues 84–94 in loop β7–β8) 

differ by 9.1 Å [26] in comparison with IL-1β. This region also contains Asn84, which is the site for at 

least two forms of N-linked glycosylation that occur in IL-1Ra in addition to the nonglycosylated form. 

Mutagenesis experiments have also 


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Figure 7 

(a) Superposition of the Cα atoms of IL-1α, IL-1β, and IL-1Ra. (b) A 

common trefoil core based on the structural superposition of all the three IL-1 

molecules. 

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identified this region and the surrounding area in IL-1β with a large receptor binding epitope 

encompassing the N and C termini of the protein as well as loops β4–β5 and β9–β10. The other 

postulated binding epitope (epitope A) is structurally conserved in IL-1Ra. Other differences in structure 

not related to any implicated biological structure are an extended 3 10 helix in residues 92–99 in IL-1Ra 

as opposed to a type-1 β turn found in IL-1β. 

F. Three-Dimensional Structure of Interleukin-1 Beta Converting Enzyme (ICE) 

As mentioned above, the processing of the different members of the IL-1 family is important to 

discovering their function and may also provide clues in the 


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design of inhibitors. The structure of ICE has been solved by x-ray crystallography to 2.6 Å in complex 

with an acetyl-Tyr-Val-Asp-H tetra-peptide [50]. Figure 8a illustrates the tertiary structure of the 

heterodimer with a dimension of about 45 Å × 35 Å × 25 Å. The molecule itself is oligomeric and 

contains two subunits, p20 (residues 120–297) and p10 (317–404) of relative molecular weight 20 kDa 

and 10 kDa, respectively. The two subunits form an intimately connected heterodimer. The core of ICE 

is a six-stranded β sheet containing 5 parallel strands and one antiparallel strand. The core is bounded by 

six alpha helices that lie parallel to the beta sheets. Physiologically ICE occurs as a (p20) 2(p10) 2 

tetramer. The molecule is a cysteine protease, which has a unique preference among the mammalian 

proteases for cleaving bonds. It has only one known with aspartic acid adjacent and N-terminal to the P 1 

scissile bond. It has only one known physiological substrate, proIL-1β, which it cleaves to active IL-1β. 

The struc- 

Figure 8 

(a) Stereo diagrams of the heterodimer structure of Interleukin-1 Converting 

Enzyme (ICE). (b) The tetrapeptide inhibitor (Asp-Ala-Val-Tyr) covalently 

bound to Cys285 in the active site. Tetrapeptied shown in black, p20 subunit in 

dark gray, and p10 subunit in light gray. Produced by Molscript [106]. 


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ture is unlike other cysteine proteases such as papain and appears to have only one known possible 

homologue, CED-3 protein [54]. The crystal structure with the tetrapeptide (acetyl-Tyr-Val-Ala-Asp-H) 

substrate in the binding pocket (Figure 8b), coupled with the results obtained by the mutational 

experiments, has provided a detailed understanding of the enzyme mechanism and substrate bonding of 

the molecule. Structural analysis has pinpointed a catalytic diad of residues Cys285 and His237 

responsible for the catalytic activity of the molecule: Cys285 acts as an active-site nucleophile while the 

imidazole ring of His237 participates in destabilizing the cysteine Oγ hydrogen. Although both of above 

these residues are contained in the p20 subunit, the p10 subunit is essential for the maintenance of a 

binding pocket (and thus the specificity of the molecule), providing binding sites S2 to S4 (residues 

338–341) and jointly contributing, with subunit p20, the S1 (Arg179–Arg341) site. The uniqueness and 

substrate specificity of ICE make it an ideal target for small molecules to block the production of 

mature, active IL-1β. Since the substrate peptide sequence is known (Tyr-Val-Ala-Asp), it has been a 

starting point to develop peptidomimetics to inhibit ICE. 

IV. Therapeutic Strategies 

A. Manipulating the IL-1 System 

Since IL-1 is an important mediator of human disease processes, modulating its activity or completely 

inhibiting its synthesis may be of therapeutic benefit. Blake and Henderson [7] reviewed and portrayed a 

general strategy to interfere with the cytokine at any one of the following stages: induction or initiation 

of gene expression–transcription, RNA processing and translation in IL-1 production, folding, release 

and secretion of extracellular protein, IL-1 in circulation, IL-1 binding to its cell surface receptors, signal 

transduction and resulting activities (Figure 9). All of the above approaches hold promise for the future. 

The wealth of structural information on IL-1 combined with extensive site-directed mutagenesis studies 

on the molecules have helped to build up a picture of the regions involved in biological function. A 

schematic representation of the present-day efforts to design efficient antagonists of IL-1 its shown in 

Figure 10. 

B. Site-Directed Mutants 

Extensive mutagenesis studies have provided information related to the structural integrity, receptor 

binding region, and residues that are important for IL-1 function. Site-directed mutagenesis (SDM) can 

create a single-site mutant and its receptor binding and bioactivity values can be calculated. The results 


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Figure 9 

Points of intervention for IL-1-based immunomodulation. 

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obtained from such studies can be imported into the available 3-dimensional structures. A combination 

of such structural insights coupled with the bioassay results provide clues leading to IL-1 functional 

information. In a previous structural report on IL-1β, we summarized the SDM results and identified a 

plausible receptor-binding epitope of interleukin-1 [42]. As mentioned before, electrostatic and 

hydrogen-bonding interactions in the loops between strands allow the polypeptide to adopt a 

conformation that enables an unusual concentration of polar and charged groups to be presented at the 

open end of the barrel. This cluster of charged residues forms an epitope with which IL-1 might bind to 

the receptor. Following our proposal, many groups have supported this hypothesis by employing 

mutagenesis studies. For a list of mutants and their activity results refer to References 42, 56, 59, and 61. 

Ju and coworkers are employing SDM to characterize interleukin-1 [55,56,58,61]. Substitution of Lys 

for the Asp145 of IL-β (D145K) greatly reduced agonist activity, while retaining 100% binding to the IL- 

1RI [56]. Based on the sequence alignment of IL-1β with IL-1Ra, they selected Lys145 of IL-1Ra for 

mutagenesis and converted it to an aspartic acid. This mutant analog (IL-1Ra K145D) maintained 

receptor binding and gained partial agonist activity [56]. Following this study, Ju and coworkers selected 

five other amino acid residues in IL-1Ra for further analysis because the side chains of these residues 

appear to be in close proximity to Lys145 in IL-1Ra [61]. Mutations were made at Val18, Thr108, 

Cys116, Cys122, and Tyr147, usually by a replacement with the corresponding amino acid of IL-1β at 

each position. None of these muta- 


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Figure 10 

Schematic diagram showing the IL-1-based therapeutic molecules. 

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tions provided enough information related to the structure-activity relationship. The mutant IL-1Ra 

K145D was mutated further to V18S, T108K, C116F, C122S, C122A, Y147T, Y147G, H54P, and a 

H54I. Of these, K145D + T108K showed a 2-fold decrease in IL-1RI binding and a 3-fold decrease in 

bioactivity compared to the IL-1Ra K145D analog. The K145D + C116F combination resulted in the 

complete loss of bioactivity, whereas full receptor-binding activity was maintained. The observation that 

receptor-binding activity is preserved indicates that the binding site is not altered that much. Structural 

alignment 


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indicates that Cys116 in IL-1Ra is in a homologous position with Phe117 in IL- 1β. The K145D + 

Y147T analog lost all detectabled activity (both binding and bioactivity), whereas the Y147G analog 

lost all bioactivity but retained 100% binding. These data suggest that Tyr147 is important for 

bioactivity of IL-1Ra K145D. 

C. Insertion of β Bulge 

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A region of charged amino acids (Gln48 to Asn53, a β bulge) positioned between β strands 4 and 5 has 

been implicated in IL-1β binding to its receptor and its immunostimulatory properties [42, 64–67]. The 

β-bulge residues form a protrusion on the edge of the open end of the β barrel. Evidence for this patch of 

amino acids involved in the function of this molecule comes from mutagenesis studies in which 

deletions or substitutions of residues in this region reduced IL- 1β agonist activity without affecting 

receptor binding [62,63]. Simoncsits et al. have shown that deletion of amino acids 52–54 (SND) in IL- 

1β reduces IL-1RI binding by 10 fold and biological activity by 1000 fold [63]. Also, studies indicate 

that a synthetic peptide derived from IL-1β (VQGEESNDK), which contains these six β bulge amino 

acids, has immunostimulatory but no inflammatory effects normally associated with IL-1 [64–66]. The 

insertion of VQGEESNDK into recombinant human ferritin H chain and recombinant flagellin from 

Salmonella muenchen increased the immunogenicity of these antigens in mice [67]. 

Greenfeder et al. inserted this β-bulge region into IL-1Ra K145D either after Ile51 of after Pro53 [61]. 

The insertion of the β bulge (QGEESN) after either position 51 or 53 of IL-1Ra K145D resulted in 

analogs that retained full IL-1RI binding and increased bioactivity by 3–4 fold. Aslo they tried to obtain 

the analogs of the QGEESN insertion in the absence of the K145D mutation either after amino acid 51 

or 53 of IL-1Ra. None of the plasmid clones with the insertion at position 51 of IL-1Ra produced the 

appropriate protein, whereas they were able to isolate clones with the insertion at position 53. Based on 

these results, Greenfeder et al. suggest that in the first case the insertion interfered in the proper folding 

of the protein, whereas the second mutant folded properly and exhibited only 10 to 20% of the IL-1RI 

binding activity. The mutants with K145D + QGEESN insertion after Ile51 of IL-1Ra showed an 

increase in bioactivity in the range of 3–8 fold. The triple mutant IL-1Ra K145D/H54P/QGEESN 

showed a higher bioactivity, and based on their mutagenesis study, Greenfeder et al. suggest that this 

increase in activity may be due to the introduction of Pro and not the removal of His at position 54 in the 

K145D mutant of IL-1Ra. The cumulative effects of these three mutations are also interesting since their 

positions on the IL-1Ra protein appear to be spatially separated. The residues Ile51 and His54 are 

located on the open face of the β barrel of IL-1Ra, whereas Lys145 is located away from the open barrel 

end. In IL-1β, the same relative 


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positions of the β bulge and Asp145 are observed [42] with the two regions separated by the known IL- 

1RI binding site. 

We have compiled the available site-directed mutants of IL-1β and sorted them according to the 

following four categories: (1) mutants that shows significantly higher agonistic activity, A1T, P2M, 

S5R, N89G, K92R, and K93R; (2) mutants that show significantly higher antagonistic activity, R4E, 

C8S, T9G, T9Q, T9E, L10T,C,S,A, C71X, R11G, K93M, M95R, E96Q, K103Q, and D145K;, (3) 

mutants that show significantly higher binding, A1T + P2M, S5R, T9L, T9W, P87S, P87H, K88V,G,L, 

N89R, E96Q, K103Q, G, C and M148A; and (4) mutants that show significantly lower binding, 

R4A,K,D, L6A, T9E, L10N,T,C,S,A, H30R, M44S, F46D, A, 156A, V58A, K92E, K93L,A,F,S,E,Q,L, 

K103S, and E105S,K. These four types of mutants are shown in Figure 11a–d. 

Taken together, this information supports our earlier proposal of the receptor-binding epitope. This was 

further characterized as functional sites A and B of interleukin-1. The first site, Area A is structurally 

conserved in all three molecules and contains residues Arg11, His30, and Asp145 in IL-1β Asn17, 

Ala36, and Asp147 in IL-1α; and Trp16, Tyr34, and Lys145 in IL-1Ra. Present in both active IL-1 

molecules, Asp145 has been recognized as an important residue in IL-1 binding. Area B has also been 

identified in both IL-1α and IL-1β it contains a large hydrophilic ridge around solvent-accessible 

hydrophobic residues. In-IL-1β, this region contains the 7-residue hydrophilic ridge (Arg4, Gln48, 

Glu51, Asn53, Lys93, Glu105, Asn108) around 5 hydrophobic solvent-accessible residues (Leu6, 

Val47, Ile56, Leu110, Val151). Figure 12 shows a surface presentation of the proposed receptor-binding 

epitope. In IL-1α, the 7-residue hydrophilic ridge has been identified with residues (Arg12, Ile14, 

Asp60, Asp61, Ile64, Lys96, and Trp109, which when mutated resulted in significant loss of binding to 

the receptor. Area B is structurally conserved in IL-1β and in IL-1α, but is lacking in IL-1Ra. For this 

reason Area A has been proposed as the binding region while Area B has been proposed as the 

triggering region. 

D. Peptide Fragments 

Peptide-based IL-1 antagonists have been derived from the primary sequence of IL-1α or IL-1β. 

Stepwise synthesis of a series of peptides from amino-terminal to carboxy-terminal regions did not 

provide any satisfactory results [68]. Polypeptide fragments of IL-1β, termed somnogeneic peptides, 

induced sleep in mammals [69], 61.5% NREMS at a dose of 20 ng. The numbering of these peptides in 

proIL-1 are 178–207, 199–225, 208–240 and that of mature IL-1β are 62–91, 83–109, and 92–124 (seq 

92–124=KKKMEKRFVFNKIENNKLEFESAQFPNWYIST). Monsanto company has identified a 

peptide fragment of IL-1β (41–70) exhibiting inhibitory effects for IL-1β and IL-1β, but not TNF-α, at 5 

nM (5 ng/mL). Peptide 56–70 is a 


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Figure 11 

Functional residues of IL-1β, identified by the 

site-directed mutagenesis results. Produced by 

Molscript [106]. 


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Figure 12 

The open end of IL-1β, shown as a surface. The positions of some of the 

residues that have been subjected to site-directed mutagenesis studies have 

been marked. Produced using the program GRASP [108]. 

Page 418 

weak agonist. Peptide 47–55 (VQGEESNDK) has been identified as an activator of T cells. It also 

stimulates glycosaminoglycan synthesis, excites antitumor activity in vivo, and lacks proinflammatory 

and pyrogenic activities [70]. Later a shorter segment (49–53 = GEESN) with higher activity than 47–55 

has been identified. Peptides based on IL-1α and IL-1β sequences were claimed to induce production of 

prostaglandin E2 but actually maintain other biological activities. Few other peptides or peptidecontaining 

epitopes were identified by using neutralizing antibodies. Labriola-Tompkins and his 

colleagues have reported obtaining epitopes for neutralizing antibodies by fractionation of a goat 

polyclonal antiserum over columns containing individual immobilized synthetic 


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Figure 13 

The identified peptide fragments. Peptide fragments are shown as striped 

coil, with residues at the start and end of the peptides are numbered. Produced by 

Molscript [106], modified by R. Esneuf. 

peptides derived from an IL-1α sequence [58]. Their work resulted in 4 peptide regions with residue 

numbers 4–12, 44–63, 64–88, and 89–105. Peptides 47–55, 81–99, 92–124, 121–153 in the 3dimensional 

structure of IL-1β are shown in Figure 13. 

E. Other Strategies 

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Biochemical and structural knowledge has opened many pathways for the development of novel 

therapeutics. Such strategies include inducer blockers, nucleotide intercalators, antisense RNAs, and 

other novel molecular mimics. It is known that potent inducers such as lipopolysaccharides, c5a, and 

integrins bind to IL-1-producing cells and induce the over expression of IL-1. Such an induction can be 

interrupted by raising the level of neutralizing monoclonal antibodies against the inducers. Alternatively, 

one can design ligands that can bind to the inducer receptors, which leads to the inhibition of the IL-1 

synthesis process. Transcription factors bind to specific DNA sequences and stimulate gene 

transcription. Controlling such a specific gene transcription can be achieved by a number of means. 

Intercalators or fragments of the same DNA sequences as those bound by the transcription factors can 

selectively inhibit transcription. Double-stranded oligonucleotides, having the same consensus sequence, 

could complete with the transcription factor for binding to the promoter region. Antisense RNA, which 

when introduced into eukaryotic cells induces sequence-specific inhibition of target gene expression, 

can be used. The 


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antisense strand hybridizes the complementary mRNA to form a double-stranded helix thereby 

achieving the inhibition of the gene products. Antisense oligo-deoxynucleotide derivatives have been 

shown to inhibit species-specific fibroblast PGE 2 synthesis stimulated by IL-1. These approaches 

generated much interest and many laboratories are pursuing the design of compounds based on antisense 

nucleotides, triple-helical transcription inhibitors, aptamers, and other novel nucleic acid derivatives. 

V. Neutralizing Soluble IL-1 in Circulation 

Once IL-1 is released into the extracellular fluid, the following molecules can be employed to 

manipulate its activity: soluble receptors, IL-1Ra, IL-1 neutralizing antibodies, IL-1-specific binding 

proteins, high-affinity small molecules (Figure 10). 

A. Soluble IL-1 Receptors 

The most effective neutralizer of a cytokine is likely to be its receptor. Some viruses have been reported 

to use an IL-1 receptor mimic to evade the immune system [72]. Natural shedding of cytokine receptors 

is a common occurrence and may form part of a normal homeostatic regulatory system, and there is a 

high potential for the use of such soluble forms as therapeutic agents. The binding affinity of the mature 

forms of the interleukin-1 molecules and their receptors have been reported [8,31–33,71]. These studies 

show that the affinity of both the membrane-bound and soluble forms of human IL-1RI for the mature 

forms of human IL-1α, IL-1β, and IL-1Ra are approximately the same. In contrast to IL-1RI, IL-1RII 

binds IL-1βpreferentially. Based on the binding-affinity studies one can infer that soluble IL-1RI is a 

better inhibitor of IL-1α than IL-1β and soluble IL-1RII is a better inhibitor of IL-1β. Both soluble 

receptors, at sufficiently high concentrations, will completely block the binding of both IL-1 forms to 

cells. Dower and coworkers [8] have studied the real-time binding of human IL-1α, IL-1β, and IL-1Ra 

to human soluble IL-1RI and IL- 1RII. It seems that the binding of IL-1Ra to IL-1RI is essentially 

irreversible, whereas its binding to IL-1RII is rapidly reversible. In contrast, IL-1RII binds IL-1β 

irreversibly [8,1]. This indicates that the IL-1RII can be used as a high-affinity antagonist of IL-1β. 

Dower further suggest in his studies that the soluble receptors have several potential advantages over 

anticytokine antibodies due to the fact that they have much higher affinities (100 to 1000 fold) and 

should not be recognized by the immune system. Even though soluble receptors have high-binding 

affinity they are difficult to synthesize in large quantities and they have a low half-life. 


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B. Interleukin-1 Receptor Antagonist 

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Over the past 5–10 years a number of inhibitors of IL-1 and TNF have been found in biological fluids 

and cell-culture supernatants. Interleukin-1 receptor antagonist was the first protein receptor antagonist 

to be described. Recombinant IL-1Ra has been shown to block the activity of IL-1α and IL-1β both in 

vitro and in vivo in animal models by binding to both type I and type II IL-1 receptors without 

demonstrable agonist activity. These effects have been extensively studied. However clinical trails 

carries out by Synergen on human subjects provided a negative support for the antagonist behavior of IL- 

1Ra for sepsis. It may be that the system took an alternative route by inducing TNF in excessively high 

levels thus achieving signal transduction. Therefore neutralizing TNF in combination with IL-1 

inhibition could be an alternate procedure. Even though IL-1Ra exhibits very high binding affinity it is 

rather a poor inhibitor of IL-1 action in vivo. It must be present at greater than a 100-fold molar excess 

over either agonist form to block action, and large doses are required to block IL-1-mediated effects in 

vivo [1]. Burger and Dayer suggest that the simultaneous use of IL-1Ra and IL-1RII might be beneficial, 

since this mixture—contrary to the use of IL-1Ra alone—completely abolished the production of 

interstitial collagenase in the inflammatory pathway [73]. 

C. Monoclonal Antibodies 

Chimarized or humanized neutralizing monoclonal antibodies for IL-1 can be used as IL-1 can be used 

as IL-1 antagonists. Otsuka Pharmaceuticals has developed a monoclonal antibody (IgG1 kappa) against 

IL-1β. It can be used in the immunoassay method for the selective detection of human IL-1β and also to 

determine the biological activity of IL-1β. Their patent (EP 0-364-778) also covers the use of such an 

antibody against IL-1β. when it is abnormally produced in disease states. Another patent of Otsuka 

Pharmaceuticals (EP 0- 408-859-A2) relates to an antibody and its application to inflammatory 

processes. This antibody is directed to a specific antigen on activated human endothelial cells (1E7/2G7) 

and blocks the binding of white blood cells causing inflammatory responses. Based on the antibody 

complementarity-determining region low-molecular-weight nonpeptide mimetics can be developed. This 

attempt might result in low-molecular-weight, orally active compounds. 

D. Low-Molecular-Weight Antagonists 

Low-molecular-weight antagonists are attractive due to their low cost and bioavailability. Available 

literature indicates that many laboratories are attempting to create immunomodulators of small synthetic 

molecules, peptidomimetics, bacterial cell-wall components, macrocycles, corticosteriods, and others 

[74]. 


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Figure 14 

2-Dimensional structures of small molecules that exhibit IL-1 modulating 

activities. They are tenidap, ciprofloxacin, 3-Deazaadenosine, (SK&F 86002), 

E5110, DMARDs (Chloroquine, Auranofin, Sodium aurothiomalate and 

Dexamethasone) tiaprofenic acid, dexamethasone, tricyclic-ylidene-acetic 

acid and its derivative, Probucol, eicosapentenoic acid + docosahexenoic, 

pentoxifylline, Denbufylline, and Romazarit (Ro-31-3948). 


Page 422

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Page 423 

American Home Products has patented molecules ranging from substituted quinoline, piperidine, and 

naphthpyridine compounds as immunomodulators. Ciprofloxacin, a quinolone antibiotic, reduced the 

extracellular IL-1 activity in human monocytes and delayed the peak production of IL-1α IL-β by 24 h 

and decreased total IL-1β production, but did not change total IL-1α production [75,76]. 

Tenidap, an antiarthritic drug, has shown efficacy both in rheumatoid arthritis and osteoarthritis [77,78]. 

It is a known inhibitor of 5-lipoxygenase and cyclooxygenase (5-LO/CO) and in vitro studies indicate 

that it also inhibits the synthesis of mature IL-1 and pro IL-1 [77,79]. Kadin reports analogs of Tenidap 

as antiinflammatory agents and analgesics [80]. An antiarthritic molecule, 3- Deazaadenosine, has been 

demonstrated to inhibit IL-1 production by LPS- stimulated human PBMCs acting at the level of RNA 

synthesis and also by blocking the effects of IL-1α on EL4 cells and induction of PGE 2 release by 

human fibroblast [81,82]. Another 5-LO/CO inhibitor (SK&F 86002) has been reported to inhibit the 

synthesis of IL-1 in human monocytes and human synovial cells in a dose-dependent manner [83,84]. 

Analogs of this compound 


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Figure 14 

(Continued) 

have been patented as IL-1 inhibitors [85–87]. In human monocytes, E-5110 is a dual 5-LO/CO 

inhibitor found to reduce extra- and intracellular IL-1 activity induced by LPS in a dose-dependent 

manner. This compound also inhibits the IL-1 generation induced by antigen-antibody complexes, 

zymosan, and silica particles [88]. 


Page 424

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Page 425 

Antinflammatory DMARDs such as chloroquine, auranofin, sodium aurothiomalate, and dexamethasone 

have been shown to inhibit IL-1 synthesis [89]. Analogs of these compounds have exhibited potent 

inhibition of IL-1α- induced cartilage resorption [90]. Elevated collagenase and proteoglycanase levels 

caused by IL-1 in human cartilage were found to be reduced by tiapro- 


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Figure 14 

(Continued) 

Page 426 

fenic acid and dexamethasone [91]. A patent report from the National Institutes of Health describes a 

method of treating diseases associated with elevated levels of interleukin-1. Rosenthal, as the inventor of 

this patent, describes a method for inhibiting the release of IL-1 from IL-1-producing cells by 

administering a therapeutically effective amount of an aromatic diamidine (WO9115201-A). 


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Page 427 

These aromatic diamidines inhibit IL-1 production and also block IL-6 and TNF. Imidazoline blocks IL- 

1 and TNF and is less toxic to the cell with an in vitro LD 50 >>10 -4. Examples of these compounds are 

1,5-bis(4-amidophenoxy) pentane (pentamidine), in the form of pentamidine isothionate, and an 

imidazoline in the form of 1,5-di(4-imidazolinophenoxy)pentane. Tricycli- cylidene-acetic acid [92] and 

its 2-chloro derivative [93] were found to be inhibitors of IL-1 release, claiming clinical improvement in 

patients with psoriasis, periodontal disease, and Alzheimer's disease. In vitro this compound blocks the 

synthesis of prostaglandins and inhibits the release of IL-1α and IL-1β from human monocytes and 

murine macrophages. 

Probucol, a hypocholesterolemic drug that possesses antioxidant activity, inhibits the ex vivo release of 

IL-1 from LPS-stimulated macrophages of mice pretreated orally with 100 mg/kg/day of this compound 

[94,95]. This compound has been shown to inhibit LPS-induced zinc-lowering effect, is cited as direct 

evidence for the inhibition of IL-1 release, and may be useful candidate for the treatment of 

atherosclerosis [95,96]. An amino-dithiol-one derivative (RP 54745) blocked the proliferative action of 

IL-1β on murine thymocytes in vitro and also inhibited the production of IL-1 in mouse peritoneal 

macrophages in vitro and in vivo. The compound RP 54745 selectively inhibited the expression of IL- 

1α and IL-1β mRNA while TNFα mRNA was unaffected [97, 98]. 

Administration of a cocktail containing eicosapentenoic acid and docosahexenoic acid to volunteers for 

up to 6 weeks, resulted in a significant depression in IL-1β (61%), IL-1α (39%), and TNF (40%) 

synthesis. These levels returned to normal after a few weeks [99]. In vitro studies indicate that 

Pentoxifylline can block the effects of IL-1 and TNF on neutrophils [100]. It is a phosphodiesterase 

(PDE) inhibitor that causes increased capillary blood flow by decreasing blood viscocity and is used 

clinically in chronic occlusive arterial disease of the limbs with intermittent claudication. Denbufylline, 

a closely related xanthine, has been patented as a functional inhibitor of cytokines and exhibits a similar 

profile to Pentoxifylline [101]. Romazarit (Ro-31-3948) derived from oxazole and isoxazole propionic 

acids has been shown to block IL- 1-induced activation of human fibroblasts in vitro and in animal 

models reduces inflammation [102,103,104]. By using a spontaneous autoimmune MRL/lpr mouse 

model, a significant efficacy was shown [105]. Two-dimensional structures of some of these molecules 

are shown in Figure 14. 

Even though the above mentioned small molecules exhibit IL-1 inhibition none of them were discovered 

based on defined functional or structural aspects. An understanding of the three-dimensional structure of 

IL-1s and their receptors, by themselves or in complexes, will form a very strong foundation for 

structure-based design of more specific and potent IL-1-based immunomodulators. 


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VI. Conclusion 

Page 428 

The design of novel compounds to inhibit or manipulate the IL-1 system remains a daunting task. At this 

time, the design of immunomodulators for the IL-1 system is still in its infancy and has largely been 

confined to the use of whole or fragmented proteins or the identification of nonspecific small molecules. 

In addition, newer approaches have also been initiated and these include the use of antisense 

oligonucleotides, small molecules designed to compete with IL-1's binding to its receptor, ICE 

inhibitors, and intracellular signaling inhibitors. All such strategies show promise. 

Structure-based design has not been explicitly used in the design of agonists and antagonists of IL-1. But 

as of now we have the structures of IL-1α, IL-1β, and IL-1Ra. A new insight may be forthcoming once 

the complex crystallographic structure of one of the interleukin-1 molecules and its corresponding 

receptor molecule is available. This structural information, coupled with the anticipated IL-1 + IL-1R 

complex structure, will form the foundation for rational design of inhibitors with improved selectivity 

for the treatment of various IL-1-mediated diseases. 

Acknowledgements 

Our special thanks to Professor Russell Doolittle for his encouragement and support, and also to Dr. 

Mitch Lewis for providing us with the very high-resolution coordinates of IL-1α. We gratefully 

acknowledge San Diego Super Computing Center for their assistance and support in providing valuable 

software and high-power computing time. We thank Dr. Donald Kyle for his valuable comments. We 

also thank Dr. Per Kraulis for providing us the latest version of MOLSCRIPT, Dr. Anthony Nicholls for 

the program GRASP, Dr. Rob Russell for the structural alignment, and Professor Lynn Ten Eyck and 

Dr. Jerry Greenberg for their help. 

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107. Merrit EM, M RASTER 3D version 2.0: a program for photorealistic molecular graphics. Acta 

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thermodynamic properties of hydrocarbons. Proteins 1991; 11:281–296. 

109. Russell RB, Barton CJ, Proteins, 1992; 14:309–323. 

110. Barton CJ, Protein Engineering, 1989; 6:37–40. 


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17 

Structure and Functional Studies of Interferon: A Solid Foundation for 

Rational Drug Design 

Michael A. Jarpe 

Cambridge NeuroScience, Inc., Cambridge, Massachusetts 

Carol H. Pontzer 

University of Maryland, College Park, Maryland 

Brian E. Szente * and Howard M. Johnson 

University of Florida, Gainesville, Florida 


The interferons (IFNs) were discovered in 1957 by Isaacs and Lindenman when they observed that a 

substance secreted by virally infected cells could protect other cells from viral infection [1a]. They 

called this substance interferon and found that it was a protein that caused uninfected cells to produce 

other proteins that made them resistant. 

Page 435 

Researchers since then have been finding a growing family of structurally related molecules: the 

interferons. Through the years, the interferons have been given many different names including immune, 

fibroblast, leukocyte, Type I, and Type II interferons. The recognized nomenclature includes alpha, beta, 

omega, tau, and gamma (α, β, ω, τ, and γ) interferons. Alpha, beta, omega, and tau all belong to the 

similar Type I subclass. Gamma is the sole member of the Type II or immune interferon class. The Type 

I interferons all share a greater sequence homology to each other than they do to IFN-γ (for a recent 

general review of the IFNs, see Reference 1b). 

The IFNs exert their actions on cells via cell surface receptors. Type I IFNs share the IFN Type I 

receptor (IFN-R1) while IFN-γ has its own unique Type II receptor. The signal transduction pathways of 

Type I and Type II 

* Current affiliation: Brigham and Women's Hospital, Boston, Massachusetts. 


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Figure 1 

Interferon activity. 

Page 436 

receptor activation are similar. These pathways involve ligand and receptor binding followed by the 

activation of tyrosine kinases and the phosphorylation of various proteins and their subsequent 

interaction with transcription elements on DNA. The two receptor activation pathways differ at the level 

of ligand binding. Type I IFNs bind their receptor as a complex of a single ligand, ligand binding 

element, and an accessory molecule. The Type II binding event occurs as a dimer of IFN-γ binding to 

two identical receptor molecules leading to receptor dimerization and activation. 

The IFNs of all subclasses posses antiviral activity. Additionally, they produce cellular responses that 

are distinct from antiviral activity, including antiproliferative and immunomodulatory activities (Figure 

1). These activities have led to an interest in their use as potential therapeutics to combat viral disease, 

cancer, and autoimmune disease. Currently, the IFNs have a worldwide market in excess of 2 billion 

dollars annually. There are six FDA-approved indications in the United States with several more in 

clinical trials (Table 1). In fact, two of the top-ten grossing biotechnology-based drugs on the U.S. 

market are IFNs. Intron A is an IFN-α used for immune protection and has an annual U.S. market of 

$570 million. Roferon-A is another IFN-α used for hairy-cell leukemia and Kaposi's sarcoma with an 

annual market of $170 million. These sales are despite profound negative side effects associated with 

IFN treatment. High doses are required to achieve positive clinical results and can lead to severe flu-like 

symptoms including nausea, vomiting, and fever. These side effects can cause patients to drop out of 

treatment before beneficial effects are seen. Another drawback of IFNs as drugs is that they require 

parenteral delivery. The IFNs are protein drugs that must be administered by injection and 


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Table 1 Approval Indications for IFNs 

FDA Approved Clinical Trials 

IFN-α Chronic hepatitis HIV infection 

Kaposi's sarcoma Colon tumors 

Genital warts (papillomavirus) Kidney tumors 

Bladder cancer 

Hairy cell leukemia Malignant melanoma 

Non-Hodgkin's lymphoma 

Chronic myelogenous leukemia 

Throat wartz (papillomavirus) 

IFN-β Relapsing remitting multiple sclerosis Basal cell carcinoma 

IFN-γ Chronic granulomatous disease Kidney tumors 

Leishmaniasis 

cannot be given orally. For many of the clinical indications, treatments of many months are needed 

requiring repeat injections. 

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These drawbacks, coupled with the market value of IFN-related treatments, now and in the future, have 

created an interest in producing second-generation molecules that can mimic IFN activity. These 

“mimetics” could potentially have greater specificity with fewer side effects. They may also have the 

advantages of reduced manufacturing costs and more versatile delivery. The design of mimetics can be 

achieved through structure-based drug design methodologies that are currently being developed. 

However, in order to apply structure-based drug design to a protein, a solid understanding of the 

structure/function relationship is needed. A three-dimensional structure, taken alone, gives little insight 

into the activity of a protein. Structure/function studies must be done for the full potential of structurebased 

drug design to be realized. 


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Structure/function studies can take a variety of forms and use a number of techniques including the use 

of molecular biology, synthetic peptides, and antibodies, or combinations of these methods. Molecular 

biology is a powerful tool for structure/function analysis. Mutagenesis of cDNAs to produce mutant 

proteins with point mutations, truncations, or deletions can identify functional sites. One drawback to 

this approach, especially with large proteins, is the proverbial “needle in a haystack” problem. One has 

difficulty determining where to begin placing mutations. The synthetic peptide approach can be equally 

as powerful. One can synthesize individual domains or segments of proteins and test them for agonist or 

antagonist activities thereby identifying functional domains. Synthetic peptides can be used to map the 

epitope specificity of antibodies that block the activity of a protein. Peptides can also be used to produce 

monospecific antisera to a defined region of a protein. The antibody approach has also proved quite 

useful in determining functional sites of 


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proteins. One potential disadvantage to using antibodies is the possibility of over interpreting the 

blocking results because of steric hindrance. A large antibody molecule may inhibit function through 

binding to a distant site and covering up a functional site. These approaches have all been used with 

success on a variety of proteins, but are best used in combination. For example, the information obtained 

from synthetic peptides and antibodies can significantly narrow down the region for site-directed 

mutagenesis studies. The entire sequence is narrowed to a segment, which reduces the size of the 

“haystack” in which the needle is hidden. 

Even though these approaches are powerful methods for determining functional sites on proteins, they 

are limited if not coupled with some form of structural determination. As Figure 2 illustrates, molecular 

biology and synthetic peptide/antibody approaches are not only interdependent, they are tied in with 

structural determination. Structural determination methods can take many forms, from the classic x-ray 

crystallography and NMR for three-dimensional determination, to two-dimensional methods such as 

circular dichroism and Fourier Transformed Infrared Spectroscopy, to predictive methods and modeling. 

A structural analysis is crucial to the interpretation of experimental results obtained from mutational and 

synthetic peptide/antibody techniques. 

Figure 2 

Flow diagram of structure/function studies. 


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Table 2 Three-Dimensional Structure Studies of the IFNs 

Type of Study Reference 

X-ray studies 

IFN-β 

IFN-γ human 

IFN-γ bovine 

IFN-γ rabbit 

IFN-γ human + receptor 

NMR studies 

Models 

IFN-γ human 

IFN-γ mouse N-terminal peptide (1–39) 

IFN-α2a 

IFN-α8 

IFN-τ sheep 

2 

3 

C.T. Samudzi, J.R. Rubin, unpublished data 

4 

5 

6 

7 

8 

9 

10 

T. Senda, S.I. Saitoh, Y. Mitsui, J.Li, and R.M. Roberts, 

unpublished data 

Note: This is not meant to be an exhaustive list of all structural studies of the IFNs. It only highlights some of the 

three-dimensional studies that have been conducted. 

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While there are no hard-and-fast rules for conducting structure/function studies, the approaches taken for 

studying the IFNs can be used to illustrate some of the methods that have been successful. Over the 

years, a large body of work has accumulated on the IFNs, including a number of structural studies. Table 

2 summarizes some of the studies exploring the three-dimensional structure of the IFNs. The following 

sections review some of the structure/function studies that have begun to elucidate important features of 

IFN activity and form a basis for future rational drug design. 


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II. Type IFNs 

A great deal of structure/function analysis has been done on the Type I IFNs. One Type I IFN in 

particular, IFN-τ has received attention recently because of its lower cytotoxicity compared to the IFNαs. 

Structure/function studies have concentrated on comparing IFN-τ with IFN-α. Therefore, IFN-τ 

provides an excellent example of structure/function studies of the Type I IFNs. 

First isolated from the conceptuses of sheep, IFN-τ is the major conceptus secretory protein responsible 

for signaling maternal recognition of pregnancy in ruminants [11]. it is produced in large quantities (200 

μg in 30 h from a day 16 conceptus culture). The protein was purified using a combination of anion 

exchange and molecular sieve chromatography. 


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A. IFN-τ Synthetic Peptide Studies 

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A sheep blastocyst library was screened with a probe based on the N-terminal sequence of the IFN-τ 

protein and the cDNA obtained (Table 3). Surprisingly, it exhibited 45–55% homology with various 

IFNs from human, mouse, rat, and pig and 70% homology with bovine IFN-ω [12]. It shared both 

molecular weight (19 kDa) and pI (5.4–5.6) with IFN-αs, while its length, 172 amino acids, was 

equivalent to the IFN-ωs. In competition studies, IFN-τ was found to compete with IFNs α, β, and ω for 

binding to the Type I IFN receptor [13]. In contrast, IFN-τ exhibited several unique properties such as 

its reproductive function, its poor inducibility by virus, and its apparent reduced cytotoxicity. Thus, IFNτ 

conceptus protein appears to be a novel IFN. 

Structural studies began with production of overlapping synthetic peptides, each 30–35 amino acids in 

length, corresponding to the entire 


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sequence of the molecule [14]. The peptides were used in competition assays with the native molecule. 

Peptide inhibition of a particular function would implicate the region of the molecule that it represented 

in the elicitation of that function. The effect of the IFN-τ peptides on the antiviral activity of ovine IFNτ 

was examined in a dose/response assay using Madin Darby bovine kidney (MDBK) cells challenged 

with vesicular stomatitis virus. The carboxy-terminal peptide oIFN-τ(139–172) was found to be the most 

effective inhibitor of antiviral activity. Three additional peptides, oIFN-τ(1–37), (62–92), and 

(119–150), also reduced IFN-τ antiviral activity. This suggested that multiple regions of the IFN-τ 

molecule interact with the Type I IFN receptor and elicit antiviral activity. These regions are underlined 

in Table 3. The data were consistent with studies of antiviral activity and receptor binding with IFN-α 

analogs demonstrating that 3 distinct sites, located in the amino-terminal, internal, and carboxy-terminal 

regions of the molecule, influenced human IFN-α activity [15]. 

To verify functional results using synthetic peptides, antipeptide antisera were produced [14]. All 

antipeptide antisera were reactive with the native molecule. Interestingly, antisera titers correlated with 

the hydropathic index of the peptide, rather than with the predicted surface accessibility of the specific 

region in the 3-D configuration. Consistent with the peptide studies, antisera against the same four 

regions of the molecule inhibited IFN-τ activity while antisera to other regions did not. 

Since IFN-τ and IFN-α bind to the same receptor, the ability of the IFN-τ synthetic peptides to block 

both bovine and human IFN-α was examined. Interestingly, only three of the four inhibitory peptides 

were effective competitors of IFN-α. Cross-inhibition of IFN-α by the internal and carboxy-terminal 

peptides was observed and suggested that these residues may adopt a similar conformation in both 

molecules and bind to a common site on the receptor. The aminoterminal peptide failed to reduce IFN-α 

function entirely. Thus, either the IFN-α amino-terminus has a much higher affinity for receptor or the 

IFN-τ aminoterminus binds a unique site on the receptor complex that may be associated with its unique 

properties. As expected, none of the peptides blocked the antiviral activity of IFN-τ, which interacts 

with a different receptor. 

Next, it was determined whether the same active regions of IFN-τ were involved in additional systems. 

The Type I IFN receptor on cells has been reported to be somewhat more promiscuous than on other 

cell types [16]; therefore, vesicular stomatitis challenge of Fc-9 cells was performed. Only the carboxyterminal 

peptide inhibited IFN-τ activity in this system [17]. This suggested that it was the carboxyterminus 

that was crucial to receptor interaction. In studies examining IFN-τ-treated feline 

immunodeficiency virus infected FeT-1 cells and human immunodeficiency virus-infected peripheral 


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blood lymphocytes, peptide inhibition of IFN-τ antiretroviral activity implicated both the amino- and 

carboxy-termini as functionally important [17]. 

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The structural basis of the antiproliferative activity of IFN-τ was also investigated. While multiple 

regions were again involved in IFN-τ antiproliferative activity, it was the area adjacent to the carboxy 

terminus, rather than the carboxy-terminus itself, which was the most crucial for antiproliferative 

activity, inhibiting cell division by blocking entry into the S phase of the cell cycle [18]. Since, for a 

particular IFN-α subtype, antiviral potency does not necessarily correlate with antiproliferative potency, 

localization of these functions in different domains of the molecule is not unexpected [19]. Within all 

known IFN-αs, the 8 amino acids from 139 to 147 are highly conserved. These residues are contained in 

both carboxy-terminal peptides, but while they may be involved in antiviral activity, they do not appear 

to be solely responsible for antiproliferative activity since the two peptides are not equivalent inhibitors 

of IFN-τ antiproliferative activity. This observation is consistent with inhibition of antiviral activity but 

not antiproliferative activity by a monoclonal antibody in this conserved region in human IFN-α and 

with the requirement for tyrosine at position 123 for human IFN-α 1 antiproliferative activity [20,21]. It 

has also been reported that mutations around Arg33 affected both antiviral and antiproliferative activity 

of human IFN-α 4 on human cells [22], while the amino-terminus did not appear to be as important in 

IFN-τ antiproliferative activity on bovine cells. 

B. IFN-τ Monoclonal Antibodies 

Another approach to structure/function analysis of IFN-τ involved generation of anti-IFN-τ monoclonal 

antibodies. Four monoclonal antibodies were produced that reacted with the native IFN-τ protein. They 

were epitope mapped using the available IFN-τ peptides. Two of the antibodies were directed against the 

carboxy-terminus of the molecule, one against a region adjacent to the aminoterminus, and the final one 

appeared to react with a conformational, rather than a linear determinant (C. Pontzer, unpublished data). 

When these antibodies were used as competitors in binding assays, all four inhibited IFN-τ binding to 

the Type I IFN receptor on MDBK cells. That anti-IFN-τ carboxy-terminal antibodies would inhibit 

binding is not unexpected, but the inhibitory activity of the monoclonal antibodies directed against the 

more amino-terminal region was not anticipated. There is the caveat that warns that results using 

monoclonal antibodies to delineate function sites must be interpreted with caution since their size may 

cause significant steric hindrance. 

To proceed further with structural studies of IFN-τ, access to larger quantities of pure protein was 

required. The obvious route to this end entailed production of recombinant protein. A synthetic gene for 

IFN-τ was designed 


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that allowed for optimal expression in both bacterial and yeast systems [23]. In addition, restriction sites 

were incorporated at intervals throughout the length of the sequence to allow for cassette mutagenesis. 

Using the Pichia pastoris expression system, 50 mg of purified IFN-τ were obtained from a one-liter 

culture. 

C. IFN-τ Binding and Signal Transduction 

Detailed receptor-binding studies were performed comparing recombinant human IFN-α and IFN-τ 

[24]. The K d of 125I-IFN-τ and 125I-IFN-αA for receptor on MDBK cells was 3.9 × 10 -10 M and 4.45 × 

10 -11 M, respectively. Consistent with the higher binding affinity, IFN-αA was several fold more 

effective than IFN-τ as a competitive inhibitor. Functionally, the two IFNs had similar specific antiviral 

activities, but IFN-τ was 30 fold less toxic to MDBK cells at high concentrations. Phosphorylation of the 

signal transduction proteins, Tyk2, Stat1a, and Stat2 did not appear to be involved in the cellular toxicity 

associated with IFN-α relative to IFN-τ. Excess IFN-τ did not block the cytotoxicity of IFN-αA, 

suggesting that they recognize the receptor differently. While maximal IFN antiviral activity required 

only fractional receptor occupancy, toxicity was associated with maximal occupancy. Thus, “spare” 

receptors may exist with respect to certain biological properties, and IFNs may induce a concentrationdependent 

selective association of receptor subunits. 

D. Structural Biology of IFN-τ 

In order to better interpret the information derived from the above studies, an understanding of the 3-D 

structure of IFN-τ is required. Prior to resolution of the crystal structure, modeling techniques were 

employed for structural predictions [10]. For IFN-τ, the homology it shares with the other IFNs can be 

exploited. Since the x-ray coordinates for IFN-β are known [2] (Figure 3), it was used as a template for 

predicting the topology of IFN-τ. When the sequences of IFN-τ and IFN-β are aligned, the overall 

homology is approximately 30%. When residues are compared on the basis of conservative 

substitutions, the similarity rises to about 50%, and if only the location of hydrophobic residues is 

compared, the similarity is approximately 75%. This is important because hydrophobicity is thought to 

be a critical factor in driving protein folding. The interferons IFN-β, IFN-α-2, and several other 

cytokines including IL-2, IL-4, growth hormone, and GM-CSF belong to a family in which all share a 

four-helix bundle structural motif. Four-helix bundles exhibit a characteristic apolar periodicity in the α 

helices where every third or fourth residue is apolar, forming a hydrophobic strip down one side of the 

helix, which facilitates packing. The aligned helical regions of IFN-τ show the same apolar periodicity, 

suggesting a four-helix bundle motif. 


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Figure 3 

Stereo view of IFN-β crystal structure [2]. 

Page 444 

The structure of IFN-τ was also examined by CD [10]. Analysis of the IFN-τ spectra predicts that the 

secondary structural elements derived from CD spectra indicate approximately 70% α-helix. The 

remainder of the molecule is either predicted to be random or a combination of β sheet and turn. Since it 

is known that algorithms that predict secondary structures from CD spectra are most accurate at 

identifying α helices, we are confident that IFN-τ is mainly α helical. The CD spectra for the synthetic 

peptides of IFN-τ were also obtained. The peptides IFN-τ(1–37), IFN-τ(62–92), IFN-τ(119–150), and 

IFN-τ(139–172) all show the presence of α helix, while IFN-τ(34–64) and IFN-τ(90–122) are mainly 

random. The presence of an α helix in the peptides supports the CD analysis of the intact protein and 

also roughly indicates the location of helical segments. 

The secondary structure of IFN-τ, including the location of the α helices and loop region, was then 

predicted using a neural network-based computer program called PHD that relies on sequence 

alignments of all proteins related to the target sequence [25,26]. When this prediction is correlated with 

the CD data, peptides that possess considerable α helicity are predicted to contain entire helical 

segments, and conversely, peptides with little helicity are predicted to be within loop regions. 

A model of the 3-D structure of IFN-τ was constructed using a distance geometry-based homology 

modeling method with mouse IFN-β acting as a template. The distance constraints were generated 

between residues within IFN-τ that are homologous to residues of IFN-β. Dihedral-angle restraints of α 

helices were generated from the secondary-structure prediction of IFN-τ. No constraints were applied to 

the 13-residue carboxy tail of IFN-τ, which is absent in IFN-β, since it is likely to be flexible in a 

manner similar to other proteins such as IFN-γ. Additional distance constraints were added from putative 

disul- 


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Figure 4 

Stereo view of IFN-τ model. Highlighted sequences are from 1–37, 62–92, 

and 139–172. 

Page 445 

fide bridges between residues 1 and 99 and residues 29 and 139. Several structures were generated using 

distance geometry routines, and the energy was minimized and averaged to yield a final model [16]. A 

similar model was built by Senda et al. (unpublished results) using a homology modeling method. This 

model was also built using the x-ray coordinates of IFN-β and shows a similar topology to the IFN-β 

three-dimensional structure (Figure 4). The most striking feature of both models is that those 

discontinuous regions, previously determined to be functionally important, are localized to one side of 

the molecule and found to be spatially contiguous (Figure 4). This observation is consistent with 

multiple binding sites on IFN-τ interacting simultaneously with the Type I IFN receptor and emphasizes 

the importance of structural modeling in the understanding and interpretation of functional data. 

III. Type II IFN 

A. Functional Sites on the IFN-γ Molecule 

The production of IFN-γ-neutralizing antibodies specific for an N-terminal peptide of human IFN-γ 

provided the first evidence that the N-terminus of IFN-γ contained an important functional site [27]. A 

similar approach was used to produce N-terminus-specific neutralizing antisera against murine IFN-γ 

[28]. Subsequent studies using IFN-γ synthetic peptides to map the epitope specificity of monoclonal 

antibodies to murine IFN-γ showed that N-terminal specific monoclonal antibodies neutralize IFN-γ 

antiviral activity [29]. In receptor- 


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competition studies, murine IFN-γ N-terminal peptide consisting of residues 1–39 [IFN-γ (1–39)] 

blocked both binding to receptor and antiviral activity of IFN-γ [30]. Overlapping peptides of other 

regions of the IFN-γ molecule failed to block binding and function of IFN-γ [31]. Thus the combination 

of peptide mapping of epitope specificities and receptor competition using peptides has identified the Nterminus 

as a structurally and functionally important region of the IFN-γ molecule. This region is 

highlighted in the sequence of human IFN-γ found in Table 3. 

Interestingly, site-specific antibodies to the C-terminus of murine IFN-γ, which were induced using the 

peptide consisting of residues 95–133 [IFN-γ (95–133)], also neutralized IFN-γ activity, however IFN-γ 

(95–133) failed to block binding of IFN-γ to receptor and IFN-γ activity simultaneously. Antibodies to 

internal peptides failed to block both antiviral activity and binding of IFN-γ to receptor. In studies with 

recombinant murine IFN-γ receptor, which consisted of the entire α chain except for the transmembrane 

domain, the C-terminal peptide did block binding of IFN-γ to receptor [32]. Thus we have the interesting 

paradox wherein the IFN-γ C-terminal peptide blocked binding of IFN-γ to the recombinant, soluble 

receptor and yet did not block binding to the cell-surface receptor. One interpretation of these findings 

has allowed us to formulate the “velcro-key” model of binding to receptor that involves both N- and Cterminal 

domains of IFN-γ (Figure 5). The N-terminus binds in the “lock and key” manner characterized 

by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct 

from that for the N-terminus, most likely through its polycationic region, which is conserved across 

species barriers. Binding of this type would exhibit high affinity and low specificity, similar to a piece of 

velcro. The C-terminal peptide of IFN-γ would therefore act as a poor competitor for cell-surface 

binding due to its low specificity alone. This interaction becomes specific in the context of the whole 

IFN-γ molecule and may increase the affinity of receptor binding. 

An alternative explanation that may also account for the inability of the C-terminal peptide to compete 

for cell-surface interactions is that its binding site is located not on the extracellular domain of the 

receptor, but rather on the intracellular domain. The primary differences between the cell-surface form 

of the IFN-γ receptor and (2) the accessibility of the recombinant receptor's cytoplasmic domain. A 

synthetic peptide corresponding to the membrane proximal region of the cytoplasmic domain of the 

murine IFN-γ receptor was able to bind IFN-γ and specifically compete with the binding of IFN-γ 

(95–133) to fixed/permeabilized cells [33]. 

Studies by others have reaffirmed the importance of both the N- and C-terminal regions of IFN-γ in 

function. Using recombinant DNA techniques, it has been shown that deletion of residues from the Nterminus 

of the molecule 


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Figure 5 

Velcro-key model of IFN-γ binding to its 

receptor. (From Reference 25. Copyright 

1992. The American Association of 

Immunologists.) 

Page 447 

results in decreased receptor binding [34]. Deletions or substitutions at the C- terminus have a direct 

effect on function of the molecule [35–38]. Epitope mapping of neutralizing monoclonal antibodies has 

also revealed an internal region of the molecule (from residues 84–94) as being functionally important 

[39]. This sequence bears strong homology to the nuclear localization sequence (NLS) of the SV40 large 

T antigen and has recently been demonstrated to be fully functional as an NLS for IFN-γ [40]. Thus, 

internal regions of the IFN-γ molecule are also likely to play an important functional role. 

B. IFN-γ Receptor α Chain Sites of Interaction with IFN-γ 

Both the human and the murine IFN-γ receptors consist of a ligand-binding subunit and a speciesspecific 

cofactor molecule. It is through interaction with this cell-surface receptor complex that IFN-γ 

exerts its biological effects. The IFN-γ molecule and its N-terminal peptide IFN-γ (1–39) bind 

specifically to the 


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cell-surface receptor and to a recombinant, soluble form of the ligand-binding chain of the receptor [25]. 

Synthetic peptides corresponding to the sequence of the extracellular domain of the ligand-binding 

subunit were used to define the region of the receptor to which the N-terminus of IFN-γ binds. Receptor 

peptide MIR (95–120) competed most strongly with IFN-γ binding to both cell-surface and recombinant, 

soluble receptor [41]. Additionally, antisera to this peptide and the adjacent overlapping peptide, MIR 

(118–143), inhibited the binding of IFN-γ to the recombinant, soluble receptor. Therefore, the receptor 

domain responsible for binding the N-terminus of IFN-γ is defined by the region encompassing residues 

95–120 of the ligand-binding subunit of the IFN-γ receptor and may extend further into the neighboring 

sequence. 

Antibodies to the C-terminal region of IFN-γ have been shown to be potent neutralizers of IFN-γ activity 

[29]. However, no cell-surface binding site for the C-terminus of IFN-γ could be localized using either 

antisera or synthetic peptides. Furthermore, as indicated above, the C-terminal IFN-γ peptide, IFN-γ 

(95–133), competed specifically with the intact IFN-γ molecule for binding to a recombinant, soluble 

form of the receptor, which consists of both the extracellular and the intracellular domains [42]. It was 

hypothesized that since the intracellular portion of the soluble receptor was accessible, in contrast to that 

of the cell-surface receptor, the C-terminus of IFN-γ might indeed be binding to this region. In studies 

using synthetic peptides corresponding to the cytoplasmic domain of the murine IFN-γ receptor, only 

peptide MIR (253–287) specifically bound both murine IFN-γ and its C-terminal peptide, MuIFN-γ 

(95–133) [33]. This peptide corresponds to the membrane proximal region of the receptor's cytoplasmic 

region. Antibodies to this receptor peptide inhibited the binding of the C-terminus of murine IFN-γ to 

the receptor in cells which had been fixed and permeabilized. Analogous binding studies with human 

IFN-γ and its C- terminal peptide, HuIFN-γ (95–134), yielded a similar result [43]. Surprisingly, the 

binding of the IFN-γ C-terminal peptides to their cytoplasmic binding sites is not species restricted, 

which is in contrast to the binding of the whole molecule at the cell surface. Both human and murine 

IFN-γ and their C-terminal peptides bound equally well to receptor peptides of either human or murine 

origin [43]. Thus, a receptor binding site for the C-terminus of the IFN-γ molecule has been localized to 

the membrane proximal region of the ligand-binding subunit's cytoplasmic domain (Figure 6). 

Previously, there have been several reports of human IFN-γ having activity on murine cells when 

administered cytoplasmically [44–46]. With the identification of a cytoplasmic binding site for IFN-γ, 

which is not species restricted, the question arose as to whether this might be the basis for these earlier 

observations. Thus, C-terminal IFN-γ peptides of both human and murine origin were used to stimulate 

murine macrophage lines P388D 1 WEHI-3. Macrophages were chosen particularly for their capacity to 

nonspecifically endocytose material, 


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Figure 6 

Proposed receptor activation pathway for IFN-γ. (From Reference 53. Copyright 1995. 

The American Association of Immunologists.) 

Page 449 

and we took advantage of this as a means of introducing the IFN-γ peptides into these cells. The IFN-γ Cterminal 

peptides induced a potent antiviral state in the murine macrophages and upregulated expression 

of MHC class II molecules, both in a dose-dependent fashion [43]. These effects were demonstrated to 

be sequence specific, as a scrambled version of the murine C-terminal peptide lacked activity. 

Furthermore, a truncated form of the murine C-terminal peptide, lacking the sequence of basic amino 

acids (RKRKR), was also without activity. The absence of activity of this truncated peptide was linked 

directly to a loss of its ability to bind to the receptor [43]. Therefore, interaction of IFN-γ, via its Cterminus, 

with its cytoplasmic binding site is important for function and requires the presence of a 

region of basic amino acids near the C-terminus of the molecule. 

C. Structural Biology of IFN-γ and the IFN-γ Receptor 

Structure-function studies of IFN-γ carried out using the synthetic peptide approach and site-specific 

antibodies indicated that both the N- and C-terminal regions of the protein were not only functionally 

important, but also accessible at the surface of the molecule. The x-ray crystal structure of human IFN-γ 

has been determined and reveals that in the IFN-γ homodimer both the N- and the 


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Figure 7 

Stereo view of IFN-γ [40]. Regions 1–39 of chain A and 95–119 of chain B of the dimer 

are highlighted. 

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C-terminus were indeed accessible [3]. Figure 7 illustrates the close proximity of the N- and C-termini 

of IFN-γ. The subunits of the homodimer are oriented head to tail, such that the N-terminal helix-loophelix 

(corresponding to residues 1–39) of one IFN-γ molecule interacts with the C-terminus of the 

second IFN-γ molecule. 

As mentioned above, synthetic peptides were also instrumental in identifying the region of the receptor 

to which the N-terminus of IFN-γ binds. Recently, the crystal structure of a complex between murine 

IFN-γ and the murine IFN-γ Rα subunit has been determined [5]. The synthetic peptides and 

corresponding antisera had predicted an interaction of murine IFN-γ residues (1–39) with receptor 

region (95–143). The crystal structure confirmed these observations, indicating an interaction of IFN-γ 

residues (1–42) with receptor residues (108–132). However, the crystal structure did not define an 

extracellular binding site for the C-terminus of IFN-γ. There has been some speculation that the basic 

amino acid residues of the C-terminus may interact with an acidic patch on the receptor's extracellular 

domain, which would support the previously mentioned “velcro-key” model, but that the crystallization 

conditions precluded this interaction [5]. It is quite possible that such an interaction may occur as a 

transitional state prior to the internalization of the C-terminal portion of IFN-γ and interaction with the 

cytoplasmic region of the receptor. An alternative explanation for the apparent lack of a binding site for 

the IFN-γ C-terminus on the receptor's extracellular face is that its primary site of interaction is 


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with the cytoplasmic portion of the receptor as described above. Thus, the orientation of the C-terminal 

portion of the IFN-γ molecules in the receptor complex should be such that they are situated near to the 

cell membrane. When one examines the structure of the receptor-ligand complex, it is easy to see that 

this is indeed the case. Studies are currently under way to determine the crystal structure of the complex 

between the cytoplasmic domain of the IFN-γ receptor and the IFN-γ molecule, in particular the Cterminus. 

With regards to the definition of sites of interaction between receptors and ligands, the 

synthetic peptide approach has repeatedly proven to be an accurate indicator of structurally important 

regions of the IFN-γ/IFN-γ R system. 

D. Signal Transduction by IFN-γ 

Within the past several years some of the immediate-early signal transduction events initiated in 

response to IFN-γ stimulation have been elucidated. Treatment of cells with IFN-γ leads to the rapid 

activation of two protein tyrosine kinases, JAK1 and JAK2 [47]. The JAK kinases are a newly emerging 

family of protein kinases important in signaling via cytokines and growth factors. These proteins are 

unrelated to the src family of tyrosine kinases and are characterized as being larger, having two putative 

phosphotransferase domains and containing no characteristic SH2 or SH3 domains [48–51]. Members of 

the Janus kinase family are found associated with the cytoplasmic domains of cytokine and growth 

factor receptors at or near to the membrane proximal region [51]. 

In the resting cell, JAK1 and JAK2 are found associated with the α and β/AF-1 chains of the IFN-γ 

receptor, respectively [52]. These kinases as well as the ligand-binding chain of the IFN-γ receptor are 

tyrosine phosphorylated in response to IFN-γ treatment [47,53,54]. This leads in turn to the tyrosine 

phosphorylation of a latent cytoplasmic transcription factor, known variously as p91, Stat 91, or Stat 1 α 

on tyrosine residue 701 [55]. It is interesting to note that the IFN-α signal-transduction pathway partially 

overlaps with that of IFN-γ. Stimulation of cells by IFN-α leads to the activation of JAK1 and another 

Janus family kinase, Tyk2 [56,57]. In turn, this cascade leads to phosphorylation of two latent 

cytoplasmic transcription factors, p84 (Stat 1β) and p113 (Stat 2β) in addition to the p91 (Stat 1α) 

activated by IFN-γ. 

The identification of tyrosine kinases that directly associate with the subunits of the IFN-γ receptor lead 

to the question of how the binding of IFN-γ might affect these proteins. Recently, the synthetic peptide 

method was used to identify two regions of the murine IFN-γ receptor's α chain as being important for 

interaction with the kinase JAK2 [58]. One of these regions lies in the distal portion of the cytoplasmic 

tail (residues 404–432), while the other (residues 283–309) is nearer to the membrane proximal region to 

which the C-terminal part of IFN-γ binds (residues 253–287). The fact that there are adjacent binding 

sites for JAK2 and IFN-γ implied a potential for interaction between the IFN-γ 


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Page 452 

ligand and the machinery of signal transduction, namely JAK2. It was found that both intact murine IFNγ 

and its C-terminal peptide (95–133) are capable of specifically mediating an increase in the degree of 

association between the recombinant, soluble IFN-γ receptor and JAK2. These findings were further 

supported as IFN-γ and IFN-γ (95–133) caused an increase in the amount of JAK2 coprecipitating with 

the receptor from intact murine macrophages [58]. This has been the first such demonstration of an 

extracellular cytokine ligand participating directly in interaction with cytoplasmic signaling elements. 

E. IFN-γ as a Candidate for Rational Drug Design 

The IFN-γ molecule is a potentially attractive model for the rational application of drug-design 

strategies. Reagents exist that are capable of either positively or negatively modulating the in vivo 

effects of IFN-γ. An initial target is quite simply at the level of receptor-ligand interaction. Synthetic 

peptide analogs of the N-terminal region have been successfully applied in vitro to inhibit interaction of 

intact IFN-γ with cell-surface receptors [30]. Interestingly, it has been observed that the N-terminal 

region of mouse IFN-γ has the ability to interact with the human receptor [59]. It was shown that the 

mouse peptide IFN-γ (1–39) had a 10-fold greater ability to block the binding of human IFN-γ to cellsurface 

receptors. This was shown to be correlated with a more stable structure in solution for the 

murine peptide and illustrates the importance of stable structure to receptor binding, which may be 

exploited when designing peptide mimetics. The solution structure of this peptide has also been 

determined and could provide the beginning steps for determining the structural requirements of an 

antagonist [7]. Future studies could focus on cocrystallization of the peptides with receptor or NMR 

studies of peptide domains of the receptor and IFN-γ. Recombinant, soluble forms of the extracellular 

domain of the ligand-binding subunit of the receptor have also been used in analogous fashion both in 

vitro to inhibit cell-surface binding and in vivo to interfere with disease progression [60]. Therefore 

prevention of potentially deleterious effects of IFN-γ may be achieved by preventing initial interactions 

with receptor molecules at the cell surface. 

A second candidate region lies within amino acid residues 84–94 of human IFN-γ and the corresponding 

region of its murine homologue. This portion of the molecule functions as a nuclear localization signal 

and, therefore, is also an attractive target for drug design. In cells treated with IFN-γ, the IFN-γ molecule 

traffics rapidly to the nucleus of the cell, usually within one to two minutes. When the IFN-γ molecule is 

crosslinked to its receptor, the resultant receptor-ligand complex migrates to the nucleus [40]. The 

implication is that this sequence may therefore be of use in artificially targeting proteins from the 

cytoplasm directly to the nucleus. This is potentially a very attractive method 


Document 

for directed subcellular targeting of synthetic transcriptional activators or repressors that might not 

otherwise be directed to the nucleus. 

Page 453 

Finally, the C-terminal region of IFN-γ offers another possibility. With respect to the induction of an 

antiviral state, or the upregulation of MHC class II molecule expression, synthetic peptides 

corresponding to the C-terminal 39 amino acids of either human or murine IFN-γ function as potent 

agonists. This activity is due to the interaction of these peptides with the cytoplasmic domain of the IFNγ 

receptor and the associated protein tyrosine kinases. Furthermore, in contrast to the stringent species 

specificity of intact IFN-γ, the action of the C-terminal peptides agonists is not limited by species 

constraints. This property therefore renders the C-terminal portion of the IFN-γ molecule an attractive 

model for the development of IFN-γ agonists and antagonists. The identification of the C-terminus of 

IFN-γ as that part of the molecule that contacts the cytoplasmic portion of the receptor implies that in 

developing IFN-γ agonists, the primary focus should be on this region of the protein. Corresponding 

antagonists may be developed based upon the portion of the receptor to which the IFN-γ C-terminus 

binds. 

IV. Conclusion 

The interest in IFNs as therapeutics has existed from their initial discovery in 1957. Since then scientists 

have been trying to understand the mechanism of their action and apply that knowledge to the treatment 

of many different diseases, meeting with some success. The effort now is to understand how IFNs work 

at the molecular level, with the goal being to design better, more specific therapeutics. Through 

structure/function studies, we now know where the functional sites lie on many of the IFNs. We also 

know the sites of interaction with their receptors and second messenger systems. From these studies, 

initial candidates for structure-based drug design have been identified. Although more work is needed to 

further characterize the IFNs and their receptor systems, the challenge now is to apply our existing 

knowledge and create second generation molecules that can modulate the many activities of these 

fascinating proteins. 

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30. Magazine HI, Carter JM, Russell JK, Torres BA, Dunn BM, Johnson HM. Use of synthetic peptides 

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18 

The Design of Anti-Influenza Virus Drugs from the X-ray Molecular 

Structure of Influenza Virus Neuraminidase 

Joseph N. Varghese 

Biomolecular Research Institute, Melbourne, Victoria, Australia 


Page 459 

Influenza has plagued humankind since the dawn of history and continues to affect a significant 

proportion of the population irrespective of age or previous infection history. These periodic epidemics 

that reinfect otherwise healthy people have devastated communities world wide. Some pandemics like 

the 1917–1919 “Spanish flu” were responsible for the death of tens of millions of people throughout the 

world. The origins, spread, and severity of influenza epidemics have been a puzzle that has only in the 

last two decades been adequately addressed. In early times it was thought that the disease was the evil 

influence (sic) of the stars, and other extraterrestial objects. At present it is generally accepted that the 

disease is of viral origin, spread by aerosols produced by infected animals, and the continual production 

of new strains of the virus results in reinfection of the disease (reviewed in Reference 1). 

There are three types of influenza virus classified on their serological cross-reactivity with viral matrix 

proteins and soluble nucleoprotein (A, B, and C). Only type A and B are known to cause severe human 

disease. Type B is only found in humans, while type A has a natural reservoir in birds and some 

mammals like pigs and horses [2]. Influenza, an orthomyxovirus, is a 100 nm lipid-enveloped virus 

(Figure 1) containing an eight-segment negative single-stranded genome [3]. Two of the segments code 

for the surfaces glycoproteins, hemagglutinin (which binds to terminal sialic acid), and neuraminidase 

(which 


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Figure 1 

A schematic diagram of an influenza virus particle that illustrates 

its constituent components and morphology. The surface antigens hemagglutinin 

and neuraminidase are attached to the lipid and matrix protein shell that 

encapsulates the eight negative-stranded RNA genes of the virus and 

associated nucleoprotein and polymerase. 

Page 460 

cleaves terminal sialic acid) and which appear as spikes protruding out of the viral envelope. The viral 

target in humans is the upper respiratory tract epithelial cells. Replication (see Figure 2) begins with 

penetration of the virion through the mucin layer covering the epithelial surface, followed by attachment 

to the viral receptor by the hemagglutinin. Penetration of the cell is achieved by endocytosis and the 

virion core is released after the fusion of the virion and vesicle membrane mediated by the 

hemagglutinin. Fusion is enabled by a conformational change in the hemagglutinin made possible by 

lowering the pH of the endosome by the M2 ion channel protein. Following replication, the progeny 

virions are released by budding off the cell membrane [4,5]. 


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Figure 2 

A simplified schematic of the replication cycle of an influenza virion in the host 

respiratory epithelia. Details of viral transport through mucin and pathways of 

viral spread on budding from the epithelial cell membrane during replication is 

not understood. Neuraminidase activity is important for release of budding 

progeny virions, desialyation of viral glycoproteins, and probably facilitates 

transport through sialic-acidrich mucin. 

Page 461 

Release of virions occur 8 hours post infection and the onset of infection is sudden, resulting in pyroxia, 

muscular and joint pain, and a dry cough [6]. Virus shedding continues for up to a week, when a rise in 

virus-specific antibody clears the virus from the host. The vulnerability of the host succumbing to 

viremea during this week of rising viral titer is mediated by interferon induction [7] 48 hours post 

infection, which attenuates viral replication until the cell-mediated immune response begins to clear the 

virus. The severity of the illness is thought to depend on the level of cross protection arising from 

antibodies raised from previous influenza infections [19]. The course of the illness can be debilitating, 

and no effective treatment is available at present to halt the progression of the disease. Death can result 

for susceptible populations (neonate and elderly) primarily as a result of secondary infections [8]. This 

chapter shall examine a structural basis for the continual emergence of new influenza strains, and the 

reasons current vaccines against influenza fail to protect against all 


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strains of influenza. The discovery of the active site of influenza neuraminidase and the exploitation of 

its structural conservation shall be discussed in terms of the design of potent neuraminidase inhibitors. 

The potential therapeutic use of these inhibitors as antiviral drugs against influenza virus infections shall 

be examined. 

A. Antigenic Variation 

The plethora of different strains of virus that are responsible for the continued reinfection of virus in 

humans is primarily related to mutations in the viral genes of two surface glycoproteins, hemagglutinin 

and neuraminidase [9]. The current paradigm for this genetic variation [10,11] is that these mutations 

arise primarily from incremental changes in the amino acid sequences of these glycoproteins by 

selection pressure of the immune system of the infected host. This mechanism termed “Antigenic Drift” 

accounts for most of the strain variation within a particular subtype of influenza. 

However, infrequently a mutation arises by genetic reassortment of viruses from different animal hosts 

(“Antigenic Shift”) whereby an entirely new gene for one of the surface glycoproteins is generated that 

is significantly different (~50%) in amino acid sequence from the parent virus. This is the mechanism by 

which new subtypes of influenza arise and are primarily responsible for the major pandemics that occur. 

Strains of influenza virus are classified by type (A, B, or C), geographic location, date of original 

isolation, and the subtype of the hemagglutinin and neuraminidase antigens. There exist 9 known 

subtypes (N1 to N9) of neuraminidase and 13 known subtypes (H1 to H13) of hemagglutinin for 

influenza A in all animal populations. Two neuraminidase (N1 and N2) and three hemagglutinin (H1, 

H2, and H3) subtypes of influenza A have occurred in strains that have infected humans since 1933 

when isolates were first characterized [12]. Prior to 1933 there is indirect evidence of antigenic shift 

occurring in human populations [13]. The N1 subtype was associated with virus isolated between 1933 

and 1957, after which time the N2 subtype appeared in the Asian influenza. No major change in the 

structure of neuraminidase has occurred since, although the hemagglutinin subtype has changed from H2 

to H3 in 1968 in the Hong Kong pandemic, and H1N1 reappeared in 1978 as the Russian pandemic. 

Influenza B, which infects only human hosts, has only one subtype, but like type A undergoes continual 

antigenic drift. 

B. Current Therapeutics 

Amantidine and Rimantidine are the only class of drugs that have been approved for therapy. At high 

concentration (>50 mg/mL) Amantidine is thought to buffer the pH of the endosome and prevent the 

conformational 


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change of the hemagglutinin necessary for fusion. Drug-resistant mutants arise where the hemagglutinin 

trimers are thought to be less stable than the wild type [14]. At low concentrations (

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could not again be agglutinated by either the eluted virus or fresh virus preparations. This activity is now 

attributed to neuraminidase, which is one of the two integral membrane glycoproteins of influenza virus 

(for reviews see References 24 and 25). 

A. Function 

Neuraminidase is an exoglycosidase that destroys the hemagglutinin receptor by cleaving the αketosidic 

linkage of terminal sialic acid [(N-actylneuraminic acid (Neu5Ac))] to an adjacent sugar 

[26,27]. Viral hemagglutinin binds specifically to Neu5Ac-containing receptors on the surface of 

susceptible cells [28]. Neuraminidase, which also removes terminal sialic acid from a range of 

glycoconjugates, plays an important, but not completely understood, role in the viral replication cycle. 

Without neuraminidase activity viruses [29] were thought to be immobilized by mucosal secretions in 

the upper respiratory tract. By removing terminal sialic acid from the sialic-acid-rich mucous layer 

[27,30] protecting target cells, neuraminidase could facilitate penetration of the virus to the cell surface. 

It has been shown that neuraminidase-deficient virus [31] can still replicate in vivo, albiet at a much 

reduced rate [32]. This shows that neuraminidase does not play an essential role in viral entry, 

replication, assembly or budding in mice, but has an important role in the spread of the infection by 

preventing aggregation at the cell surface and possible immobilization in the mucin by hemagglutinin. 

Once replication is initiated in the infected cell, the freshly synthesised viral glycoproteins have to be 

desialylated to prevent self-aggregation at the infected host cell surface by hemagglutinin binding to 

terminal sialic acid on these glycoproteins. Finally on elution of progeny virions from infected cells, 

neuraminidase activity is required to facilitate viral escape from the cell surface. 

Inactivation or inhibition of neuraminidase during budding has been observed to result in aggregation of 

virions on the cell surface [33–35]. Inhibition of this glycohydrolase could provide a means of 

controlling this disease by slowing the rate of viral attachment and subsequent release of progeny virions 

allowing the host immune system to eliminate the virus while the number of infected cells is low. 

B. Morphology 

There are between 50 to 100 neuraminidase spikes per virion [36] which is approximately 10% of the 

visible spikes projecting out of the surface of the virion [37]. These spikes can be removed from the 

virus by treatment with detergent [38]. Electron microscopic images of the neuraminidase spikes [39] 

reveal a mushroom-shaped molecule made up of a boxlike head of about 80 × 


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80 × 40 Å with a narrow centrally attached stalk (15 Å wide and 100 Å long), which terminates into a 

hydrophobic knob anchored in the viral envelope. The detergent-released spikes can be digested by 

pronase to release the neuraminidase “heads,” which retain full antigenic and enzyme activity [40,41]. 

The molecule was found to be a tetramer of molecular weight 240,000, reducing to 200,000 when 

treated with pronase [42]. A low resolution x-ray image of crystallized neuraminidase heads [43] 

established that the enzyme had circular 4-fold symmetry. 

III. Molecular Structure of Neuraminidase 

Crystals of pronase-released heads of the N2 human strains of A/Tokyo/3/67 [44] and A/RI/5+/57 were 

used for an x-ray structure determination. The x-ray 3-dimensional molecular structure of neuraminidase 

heads was determined [45] for these two N2 subtypes by a novel technique of molecular electron density 

averaging from two different crystal systems, using a combination of multiple isomorphous replacement 

and noncrystallographic symmetry averaging. The structure of A/Tokyo/3/67 N2 has been refined [46] 

to 2.2 Å as has the structures of two avian N9 subtypes [47–49]. Three influenza type B structures [50] 

have also been determined and found to have an identical fold with 60 residues (including 16 conserved 

cysteine residues) being invariant. Bacterial sialidases from salmonella [51] and cholera [52] have 

homologous structures to influenza neuraminidase, but few of the residues are structurally invariant. 

A. Structural Topology 

The protein fold consists of a symmetric arrangement of six four-stranded antiparallel β sheets arrange 

as blades of a propeller (Figure 3), the propeller axis being approximately parallel to but titled away 

from the circular 4-fold axis of the tetramer. This tilt angle varies between the known subtypes. This 

topology has now also been found in the seven β sheet propeller structure of bacterial methylamine 

dehydrogenase [53] and galactose oxidase [54], and the eight β sheet propeller structure of methanol 

dehydrogenase [55]. 

Each sheet of neuraminidase has a “W” topology (+1,+1,+1) [56] with four strands connected by reverse 

turns (Figure 3). The first strand of each sheet enters from the top, approximately parallel to and near the 

propeller axis; the fourth strand exits from the bottom, approximately perpendicular to the propeller axis. 

Top and bottom surfaces of the head refer to the faces of the tetramer away and towards the viral 

membrane respectively. Each sheet thus has a characteristic 90° right-hand twist between the inner- and 

outermost strand. The six sheets and their connections to each other are topologically identical. 


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Figure 3 

(a) A MOLSCRIPT [107] stereo diagram of the neuraminidase tetramer viewed 

from above down the symmetry axis. The different shaded arrows represent β 

strands comprising the six β sheets that form the “propeller” framework of 

each subunit. (b) A stereo diagram of a neuraminidase monomer viewed down 

the 4-fold axis, and α-sialic acid is shown bound in the active site 

of the enzyme, which lies in a pocket formed by the six β 

sheets near the pseudo six-fold axis of each subunit. 

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The first strand of each sheet is connected across the top of the submit to the fourth strand of the 

preceding sheet. The N-terminus lies across the bottom of the subunit, and builds the fourth strand of the 

sixth sheet before entering the first sheet. The C-terminus strand builds the third strand of the sixth 

sheet, enters the subunit interface from the bottom, and runs parallel with the outermost (fourth) strand 

of the sixth β sheet. 


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B. Protein Structure 

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The 6 β sheets of the subunit, although topologically identical, vary in size and detailed structure. 

However, the six sheets can be considered as maintaining an approximate 6-fold symmetry relationship 

to each other about an axis parallel to the mean direction of the first strand of each sheet and through the 

centroid of these directions. Four disulfide bridges are formed between adjacent sheets as well as two 

between the sheets, which distort the sheet structure. Similar pairing has now been found in one of the 

domains of CD4 [57]. Also short β bulges occur on the inner and outer strands of most of the sheets. The 

packing of the β sheets is stabilized by hydrophobic interactions at the sheet interfaces and hydrogen 

bonding at the periphery of the sheets. In addition the intersheet disulfide bonds and ion pairs stabilize 

the structure. 

In general the loops connectioning the β sheets are shorter and tighter on the bottom of the subunit 

compared to the outer loops, which tends to be more elaborate. The intersheet loop connecting the fourth 

and fifth loop is the most extensive in the structure and is stabilized primarily by a disulfide bridge 

between Cys318 (sequence numbering of A/Tokyo/3/67 will be used throughout) and Cys337, a 

conserved ion pair Asp330—Arg364, and a putative Ca 2+ ion binding site. This calcium binding site has 

approximate octahedral coordination with the main-chain oxygens of residues 293, 297, 345, and 348, a 

carboxylate oxygen of Asp324, and a water molecule in N2 and type B neuraminidases. In N9, the mainchain 

oxygen at 343 is replaced by a water molecule. Calcium has been shown to be necessary for 

neuraminidase activity [58] and this site is connected to the active site via conserved residues; however, 

the functional role of calcium in the structure is unknown, although it has been shown that calcium is 

essential for the thermostability of the molecule [59]. 

C. Carbohydrate Structure 

Carbohydrate at four N-linked glycosylation sites were observed in N2 neuraminidase at residues 86, 

146, 200, and 234 in the x-ray structure. Two N-acetylglucosamines were resolved at Asp86 and 

Asp234, both at the bottom surface of the monomer. The carbohydrate at Asp200 consists of eight sugar 

residues with linkages consistent with known mannose-rich simple N-linked carbohydrates [60]. This 

oligosaccharide emerges from the side of the monomer and covers a neighboring subunit (see Figure 4). 

The oligosaccharide site at Asn146 is the most conserved of all neuraminidase glucosylation sites, 

except that of the neurovirulent virus A/WSN/33 [61]. The absence of this glycosylation site in 

A/WSN/33 has been shown to confer neurovirulence in mice [62]. It is a complex sugar containing Nacetylgalactosamine 

[63] that is not found in any other of the known oligosaccharides of influenza virus 

glycoproteins and is the only glycopeptide antigenically related to chick embryo “host antigen” 


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Figure 4 

A CPK model of two tetramers of A/Tokyo/3/67 neuraminidase heads as seen 

in the crystal structure. The four carbohydrate spikes emanating from the top 

of the molecule (at Asn146) interdigitate and form an open “barrel” structure. 

They form an unusual crystal contact. The dark spheres represent carbohydrate 

and the light spheres represent protein. The carbohydrate at Asn200 starts 

from one subunit and covers a neighboring subunit on the same tetramer. The 

other carbohydrates lie on the 

underside of the tetramer. 

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[63,64]. The oligosaccharide appears as a spike emanating from the top of the monomer, forming a 

crystal contact with a neighboring tetramer in crystals of A/Tokyo/3/67 neuraminidase. The four 

carbohydrate spikes of a tetramer form an open “barrel” structure of eight carbohydrate chains with the 

neighboring tetramer, with no apparent intercarbohydrate contacts. This oligosaccharide may play an 

important but as yet unidentified role in neuraminidase structure or activity. 

D. Antigenic Variation in Neuraminidase Structures 

Comparison of all known sequences of approximately 390 residues of the neuraminidase globular head 

[24], indicates that only 54 (excluding 16 conserved cysteine residues) are invariant (Figure 5a). Apart 

from 21 residues involved 


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Figure 5 

(a) Stereo image of a CPK atomic model of an influenza virus neuraminidase 

tetrameric head viewed distal to the viral membrane. The darker-shaded atoms 

represent totally conserved residues that for the most part form the enzyme 

active-site pocket. The lighter shaded atoms represent strain-variable 

residues and carbohydrate. (b) A stereo image of the enzyme active-site 

pocket of a subunit of neuraminidase with the same shading scheme. 

Page 469 

in preserving the structural integrity of the molecule [46], the main clustering of these invariant residues 

is within the enzyme active site (Figure 5b), where 17 are in the active site and 16 are neighboring the 

active site. This is a cavity on the upper surface of the molecule into which sialic acid has been observed 

to bind [65,66,50]. Excluding the active-site pocket, strain variation occurs over the entire surface of the 

neuraminidase heads. The active site was found to be in 


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Figure 6 

Stereo image of a ball-and-stick model of sialic acid bound to the active site of 

Tern/N9 neuraminidase. The hydrogen-bond interactions with conserved residues 

are shown as dotted lines. Nitrogen atoms are shaded black, oxygen atoms 

are shaded dark gray, and carbon atom are shaded light gray. 

Page 470 

a pocket of totally conserved (over all animal subtypes) residues [65]. In this way the enzyme active-site 

pocket is surrounded by highly variable surface residues that prevent immune recognition of the active 

site by antibody molecules [25]. The x-ray diffraction studies of neuraminidase—antibody complexes 

have shown that the footprint of an antibody in the complex is larger than the exposed surface of the 

conserved region of the active site [67–69]. These structural results indicate that antibodies are unable to 

exert mutational pressure on the conserved active site because they cannot bind there without engaging 

strain-variable residues as part of the binding surface. However, as antibodies bind to strain-variable 

elements of the structure, the virus can overcome host immune pressure by point mutations of the 

residues that do not have a catalytic or structural role [70,48] but are able to disrupt the 

antigen—antibody binding interface. The rapid emergence of these escape mutants explains the failure 

in producing a universal vaccine for influenza. 

E. The Enzyme Active Site 

The structures of N-acetyl neuraminic acid (sialic acid Neu5Ac) and the 2-deoxy-2,3-dehydro-N-acetyl 

neuraminic acid (Neu5Ac2en) inhibitor complexed with N2 neuraminidase [66] revealed the nature of 

the interactions of the 


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molecules in the active-site pocket (Figure 6). Sialic acid binds in the active site in the α-anomer and, in 

a distorted half-chair conformation, through the same face as used in its interaction with hemagglutinin 

[71]. The carboxylate group of the sugar interacts with three guanidinium groups of argine residues 118, 

292, and 371 and has an equatorial conformation with respect to the sugar ring (this group, axial toward 

the floor in the undistorted structure, is probably held equatorial by interactions with these arginine 

residues). The NH group of the 5-N-acetyl side chain interacts with the floor of the active-site cavity via 

a bound water molecule. The oxygen of the 5-N-acetyl side chain is hydrogen bonded to the N ε of Arg 

152, while the methyl group lies in a hydrophobic pocket near Ile222 and Trp178. The last two hydroxyl 

groups of the 6-glycerol side chain are hydrogen bonded to carboxylate oxygens of Glu276 and the 4hydroxyl 

is directed to a carboxylate oxygen of Glu119. The glycosidic oxygen O2 interacts with a 

carboxylate oxygen of Asp151. Similar binding of sialic acid in the active site was observed in type B 

virus [50]. 

Comparison of active sites of N2, N9, and type B neuraminidase [72] show there are no significant 

differences between active-site orientations, except for some minor displacements of Arg224 and 

Glu276, where the major interactions with the 6-glycerol group of sialic acid occur. However there are 

differences in the water structure in the active sites of the different subtypes. The Gly405 residue (in N2 

and N9) is replaced by a tryptophan in type B, which displaces four water molecules that lie in a solvent 

pocket bounded by arginines at residues 371 and 118. The Val240 residue (in N2 and N9) is replaced by 

a methionine in type B, which displaces two water molecules that form a channel under Arg 224, 

decreasing the flexibility of the active site of type B in this region. These waters are not displaced in the 

sialic acid/neuraminidase complex in N9 and N2 and would alter the hydrogen-bonding pattern of the 

complexes when compared to type B. 

Comparison of the active sites of influenza neuraminidases and bacterial sialidases [51,52] indicates that 

there is considerable conservation of the catalytic site at the carboxylate-binding end. The residues 

Asp151, Arg118, Glu277, Arg292, Val or Ile349, Arg371, Tyr406, and Glu425 are conserved over all 

known viral and bacterial strains. The arginyl residues 118, 292, and 371 position the 2-carboxylate 

group and the Val(or Ile)349, Glu425, and Glu277 are important in positioning the triarginyl cluster. The 

residues Asp151 and Tyr406 are presumably important in bond cleavage, but the precise mechanism is 

still unclear. These eight residues (Figure 7) are thus most likely to be conserved in all neuraminidases. 

Differences between viral, bacterial, and mammalian neuraminidase structures may correspond with the 

different role these enzymes have in vivo. These differences are likely to be in the interactions of the 6glycerol, 

5-N-acetyl, and 4-OH groups of silaic acid. In the influenza virus, the turnover rate must be 

balanced against the requirement to maintain 


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Figure 7 

Stereo image of the active site of Tern/N9 showing the active-site residues 

surrounding 4-guanidino-Neu5Ac2en (black). Those residues that are conserved 

in both influenza and bacterial neuraminidase are shaded gray, and those that are 

conserved only in influenza virus neuraminidase are not shaded. 

sufficient sialic acid at the cell surface to enable attachment via the hemagglutinin. This balance may 

require some configuration of residues in the active site not directly responsible for catalysis but only 

involved in the binding and release of sialic acid. 

IV. Neuraminidase Inhibitor Design 

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Earlier screening programs [73] failed to identify potent inhibitors of viral neuraminidase. The first 

inhibitor synthesized [74] with a K i value in the micromolar range was Neu5Ac2en. This was based on 

the proposed transition state of the reaction, where the anomeric carbon (C2) bound to the ketosidic 

oxygen has a trigonal state. Several analogues of Neu5Ac2en were synthesized soon after, and the most 

potent of these, a trifluoracetyl derivative, had a K i of only 0.8 μM [75]. While this compound showed 

that in cell culture it retarded virus shedding [34,76], it failed as an effective antiviral agent in animals 

[77]. 

The x-ray structure of Neu5Ac2en/neuraminidase complexes have been determined for N2 [66], type B 

[78], and N9 [79]. The Neu5Ac2en molecule binds in the active site of neuraminidase with the 

carboxylate oxygen atoms placed in the same location as the carboxylate of sialic acid. The 5-N-acetyl, 4- 


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hydroxy, and 6 Neu5Ac2en -glycerol are positioned isosterically in the two molecules. 

Page 473 

An alternative approach to develop sialidase inhibitors has been made using synthetic thioglycoside 

analogs of gangliosides such as Neu5Aca(2-S-6)Glcb(1-1)Ceramide [80] were shown to inhibit different 

subtypes of human and animal influenza virus with K i values of up to 2.8 μM. These metabolically 

stable ganglioside analogs contain a thioglycosidic linkage to the terminal neuraminic acid that resists 

cleavage by the enzyme. Several flavonoid neuraminidase inhibitors have been isolated from plant 

extracts [81], one of which 5,7,4'-trihydroxy-8-methoxyflavone, was a more potent inhibitor than 

Neu5Ac2en. Recently, in vivo anti-influenza virus activity of a Kampo (Japanese herbal medicine) 

preparation has shown promising results in inhibiting influenza virus replication in mice [82], but the 

mode of action of these compounds is unclear. 

A. Enzyme Mechanism 

The similar positioning of the carboxylate oxygens and ring of Neu5Ac2en and sialic acid suggests that 

Neu5Ac2en is probably a transition state analog. As Neu5Ac2en has a higher affinity for neuraminidase 

than sialic acid, a mechanism of catalysis was proposed [83] that involves the distortion of the substrate 

by the formation of a oxycarbonium ion intermediate, which has a similar structure to Neu5Ac2en. 

However the structural results from sialic acid/neuraminidase complexes suggest that the tighter binding 

of Neu5Ac2en more likely comes from the relaxation of the conformational strain arising from the 

transition from chair to boat of the pyranose ring of sialic acid in the active site [66]. 

Evidence for a sialyl cation transition state by isotopic effects [84] support the existence of a 

oxycarbonium ion intermediate. However the structural basis for neuraminidase activity is still unclear. 

It has been suggested [66] that the tyrosyl oxygen of Tyr406, assisted by the sialic acid carboxylate 

itself, could stabilize the developing charge on the oxycarbonium ion intermediate. The reaction would 

be completed by the activation of a water molecule by a deprotonated Asp151 and its attack on the 

carbonium, resulting in the formation of the α-anomer of sialic acid [85]. However the pH-activity 

profile of neuraminidase [86,87,78] suggests a bell-shaped profile, which indicates normal activity from 

a pH range of about 4.5 to 9. This would indicate that the role of Asp151 as the acid group in the 

catalysis is unclear. A nonspecific proton donor has been proposed [78], probably a water molecule as 

the acid group, with a deprotonated Tyr406 stabilizing the oxycarbonium ion, and a proton transferred 

from water to the departing aglycon group. It has also been postulated that a proton is eliminated at C3 

leading to the transformation 


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of the oxycarbonium ion into Neu5Ac2en, which is produced irreversibly at low levels from sialic acid 

by the enzyme [78]. An S N1-type mechanism has been suggested [88] that is facilitated by an activated 

water molecule, which can be expelled upon inhibitor binding. The catalytic mechanism could possibly 

proceed without an acid group: the electrostatic potential of the enzyme could lower the barrier 

preventing the breaking of the ketosidic bond and the solvent could protonate the aglycon after release 

[89]. Clearly details of the enzyme mechanism have yet to be elucidated definitively. Structural 

considerations indicate that only Tyr406 (and possibly Glu277) and the triarginyl cluster are essential in 

the enzyme mechanism and that Asp151 is implicated. 

B. Inhibitor Design Principles 

All the nearest-neighbor interactions between sialic acid or Neu5Ac2en and the protein are with totally 

conserved amino acids. Thus an inhibitor designed to bind only to the conserved active-site residues of 

neuraminidase would inhibit neuraminidase activity across all strains of influenza. This would enable 

the development of an antiviral drug that would affect the spread of viral replication potentially in three 

ways, i.e., transport through the protective mucosal layer, desialyation of freshly synthesized viral 

glycoproteins, and elution of progeny virions from infected cells. 

The development of potent inhibitors was based on the structural information of the N2 neuraminidase 

conserved active site and its complex with sialic acid and Neu5Ac2en [66]. There are no reports of de 

novo molecules designed to fit into the cavity, and the most useful approach was to consider molecules 

that were structurally related to Neu5Ac2en. This involved the design of molecules that would bind 

isosterically to Neu5Ac2en but which were modified to increase the number of favorable interaction 

with the protein. The method of Goodford [90] enabled the calculation of favorable binding sites for a 

variety of chemical probes. The validity of this method was indicated by its ability to identify the 

positions of the carboxylate binding site of sialic acid as an energy minima for a carboxylate probe 

(Figure 8a), and the successful prediction of known bound-water sites in the active sites by a water 

probe. Utilizing this methodology, predictions of energetically favorable substitutions to Neu5Ac2en 

were examined [91]. A replacement of the hydroxyl at the 4-position of the pyranose ring of Neu5Ac2en 

by an amino group was identified by this procedure as an energetically favorable substitution. A 

protonated primary amine probe identified a favorable binding site of -16 kcal mol -1 at this location and 

in a pocket in the active site near two conserved glutamate residues Glu119 and Glu227 (Figure 8b). 

This suggested that the substitution of the 4-hydroxyl group by an amino group would increase the 

overall binding interactions by forming a salt link with Glu119. Furthermore the substitution of the 4hydroxyl 

group with a much bulkier guanidinyl group would lead to even tighter binding as a result 


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Figure 8 

Stereo image of the residues in the active site of Tern/N9 complexed with 

4-guanidino—Neu5Ac2en overlayed with GRID maps [90] (caged mesh contours) 

for (a) a carboxylate oxygen probe, contoured at -12 kcal/mol; (b) an amino 

nitrogen probe, contoured at -12 kcal/mol. 

of lateral interactions of the terminal nitrogens of the guanidinyl group and the carboxylate groups of 

Glu119 and Glu227. 


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This led to the design and syntheses of 4-amino-Neu5Ac2en and 4-guanidino-Neu4Ac2en [91] which 

bound to A/Tokyo/3/67 with a K i of 50 nM and 0.2 


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nM, respectively. These compounds were later shown by x-ray studies of 4-guanidino and 4-amino- 

Neu4Ac2en complexed with A/Tokyo/3/67 neuraminidase to bind close to that predicted by the design 

studies. However details of the interactions of the guanidinyl group of the 4-guanidino-Neu4Ac2en with 

the glutamic acid groups (Glu119 and Glu277) in the floor of the active site were slightly different. This 

was confirmed on a higher resolution x-ray study (Figure 9) of a 4-guanidino-Neu4Ac2en complexed 

with Tern N9 neuraminidase [72]. One of the primary guanidinyl nitrogens of 4-guanidino-Neu4Ac2en 

is hydrogen bonded to the main-chain oxygen at residue 178, a carboxylate oxygen of Glu227, and a 

water molecule. The other primary guanidinyl nitrogen interacts with the main-chain oxygen of residues 

178 and 151. The secondary guanidinyl nitrogen interacts with the carboxylate of Glu119 and Asp151. 

The interactions with Glu119 are electrostatic and van der Waals in character and lack hydrogenbonding 

geometry (postulated in the design study) as the carboxylate group of Glu119 stacks parallel to 

the guanidinyl group. Furthermore, theoretical energy-minimized structures of the complex using 

AMBER [92] converged to the x-ray structure only if the protein nonhydrogen atoms were kept rigid in 

the x-ray structure [72]. Otherwise this resulted in active site residues showing large distortions in their 

conformation. This is an example of the difficulty in correctly modeling even modest changes in the 

interactions of an inhibitor/active site complex. 

The 4-guanidino analog shows potent inhibition of neuraminidase activity in all known wild strains of 

influenza. Furthermore it is very specific to in- 

Figure 9 

Stereo image of the Tern/N9 4-guanidino-Neu5Ac2en complex showing the 

hydrogen-bond interactions (dotted lines) of the inhibitor with conserved residues 

in the active site of the enzyme. Nitrogen, oxygen, and carbon atoms are 

shaded black, dark gray, and light gray, respectively. 


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fluenza, as it shows weak inhibition to bacterial, para influenza, and mammalian neuraminidases 

[91,93]. This is possibly due to the specific interactions of the 4-guanidino group within the subpocket 

of the active site of influenza neuraminidase that is not conserved in other neuraminidases. In bacterial 

neuraminidases [51] this pocket is much smaller, and would prevent the binding of the 4-guanidino 

group in this region of the active site. This is consistent with the proposition that the interactions of 

sialic acid with the active site of neuraminidases are function specific at the C4, C5, and C6 position of 

sialic acid and that modification at these positions confer specificity to the target enzyme [72]. Other 

approaches [94] to structure-based design of inhibitors have to date produced only millimolar inhibition 

of neuraminidase activity. 

V. Antiviral Activity 

In vitro inhibition of viral replication in tissue culture was demonstrated earlier [34] for the trifluro 

derivative of Neu5Ac2en, but its antiviral activity in vivo was not demonstrated [77]. As a consequence 

of this, efforts were directed towards hemagglutinin, which was then considered a better target for 

antiinfluenza drugs. The interest in neuraminidase inhibitors as anti-influenza drugs has only been 

revived with the success of 4-guanidino-Neu5Ac2en and its analogs in attenuating viral titer in mice 

when administered directly into the lungs [91,95]. 

A. Inhibition In Vitro 

Von Itzstein and co-workers [91] have shown that the 4-amino-and 4-guanidino-Neu5Ac2en inhibit 

influenza strains A/Singapore/1/57 and B/Victoria/102/95 in MDCK cells with IC 50 values (the 

concentration required to inhibit plaque formation in MDCK cells by 50%) of 1.5 mM and 0.065 mM (4amino) 

and 0.014 mM and 0.005 mM (4-guanidino) respectively. These IC 50 values, in particular for the 

4-guanidino compound, are well below those found for amantadine, ribovarin, and Neu5Ac2en. 

Furthermore in comparison to Neu5Ac2en, the 4-guanidino-Neu5Ac2en inhibitor was 100-fold less 

active against human lysosomal sialidase and over 1000-fold more active against a wide range of 

clinical isolates of influenza A and B, including amantidine and rimantadine resistant variants [93]. 

The inhibition of virus replication in MDCK cells has been confirmed [96] and this knowledge 

prompted the extension of the inhibition studies to human respiratory epithelium cells in vitro [97], 

which indicated high antiviral activity for strains of A(HINI) and A(N3N2) isolates. They also found 

that delayed administration of the drug—after viral replication was well estab- 


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lished—was associated with inhibition of virus replication. However viral titer was higher (1.3 log 10 

compared to 4.0 log 10 at 10 mg/mL concentration of the drug) for the delayed administration compared 

with viral titer when the drug was present throughout the period of viral exposure. The clinical 

significance of this in the treatment of established infections has yet to be explored in detail, although 

preliminary clinical trails [98] have indicated some positive results. 

B. Administration and Inhibition In Vivo 

The earlier work of Palase and Schulman [77] indicated that the failure in inhibiting viral replication in 

mice after intranasal and subcutaneous treatment with Neu5aC2en would also occur for Neu5Ac2en 

analogs. This failure of Neu5Ac2en as an antiviral treatment in animal models can now be ascribed to 

the rapid excretion of the compound [99] thereby not delivering sufficient concentration of the inhibitor 

to the infected tissue. It had been shown [91] that the antiviral activity in mice is considerably more, 

when the 4-guanidino compound is administered intranasally than when the drug is injected 

intraperitoneally. This can be attributed to the localization of sufficiently high concentration of inhibitor 

in the lining of the nasal and respiratory epithelia where influenza virus replication is believed to occur. 

Animal trials with ferrets challenged with influenza virus have shown the 4-guanidino Neu5Ac2en 

compound is effective in studies [91] involving prophylactic administration of the drug. When the drug 

is administrated intranasally, 50 μg/kg, twice daily, one day before infection with the virus and the 

succeeding six days, it substantially reduces virus titer in nasal washing and abolishes fever that usually 

appears 3 days after infection. 

The drug is currently undergoing clinical trials, and the initial results from a double blind, randomized, 

placebo-controlled trial using this compound have been positive both for early treatment and 

prophylaxis of experimental inoculation of human volunteers [98] with influenza A/Texas/91. 

C. Drug Resistance 

Although the active site of influenza virus has been conserved in all known field strains of the virus, the 

possibility of drug resistance needs to be addressed. Experience with influenza and other viruses, in 

particular HIV, have shown [100] that drug-resistant mutants arise very rapidly, resulting in the 

effectiveness of antiviral drugs being short lived. One attempted solution to the problem is the use of 

several drugs during therapy [101], making it more difficult for the virus the develop resistance. 

In the case of influenza virus, there have to date been no reports of drug resistance from field strains. 

However it has recently been reported [102,103] 


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that 4-guanidino-Neu5Ac2en-resistant mutants can arise from multiple serial passages of virus in 

MDCK cells in the presence of the inhibitor. It has been demonstrated [104] that almost all of the 

mutations arise in the hemagglutinin receptor binding site and not on the neuraminidase. These altered 

HA variants, which have weaker binding to HA receptors, appear to arise as a result of increased 

inhibition of the neuraminidase by the drug. This is consistent with the earlier proposition that the rate of 

desialylation of receptor is critically related to the rate of attachment to receptor, for the virus infection 

and elution. The decreased activity of the neuraminidase by the drug selects HA mutants with decreased 

binding to receptors. 

However a drug-resistant neuraminidase mutation has been isolated [103,105] that results in a single 

active-site residue mutation—with apparently unaltered activity—of glutamic acid 119 to glycine. The 

crystal structure of this mutant and its complex (Figure 10) with 4-guanidino-Neu5Ac2en has been 

determined [105] and the structure suggests that the decrease in inhibitor binding arises from the loss of 

stabilizing interaction with the 4-guanidino group of the drug [72] and alterations in the solvent structure 

of the active site. This alteration arises from a water molecule that binds near the location of one of the 

carboxylate oxygens of the glutamic acid in the wild type molecule. The location of the 4-guanidino- 

Neu5Ac2en drug in the complex with the mutant enzyme is isosteric compared to the drug/wildtype 

complex [72]. The only differences are the interactions with residue 119. 

Figure 10 

Stereo image of the Glu119Gly Tern/N9 mutant complexed with 

4-guanidino-Neu5Ac2en. The inhibitor binds in a similar conformation as with 

the wild type enzyme. The atom labeled “Wat” represents the water 

molecule found in the mutant enzyme that assumes the role of the glutamic acid 

in the wild type enzyme. Nitrogen, oxygen, and carbon atoms are shaded 

black, dark gray and light gray, respectively. 


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It has now been over a decade since the structure of influenza virus neuraminidase was determined 

[45,65]. The development of the 4-substituted Neu5ac2en analog inhibitors dates from 1987 when the 

structure of sialic acid and Neu5Ac2en complexed with neuraminidase were determined to sufficient 

accuracy to permit modeling of potential inhibitors [66]. The development was a multidisciplinary 

collaboration of biochemists, crystallographers, molecular modelers, and synthetic chemists and 

culminated in the synthesis [106] and biological testing of the compounds [91]. Preliminary data on the 

efficacy of these drugs on humans indicate its effectiveness in both prophylaxis and treatment of the 

disease [98]. The 4-guanidino compound is in Phase 2 trials (October, 1995). While drug resistance of 

the virus has been observed in vitro, it will be interesting to see if variants arise in animal studies as they 

do for Amantidine. This is one of the first rationally designed antiviral drugs to be synthesized and 

portends well for this methodology to be used as an additional weapon in controling the many pathogens 

that have plagued humanity for so long. 


The author would like to acknowledge Peter Colman, my collaborator in neuraminidase crystallography, 

Mike Lawrence for the GRID maps shown here and reading this manuscript, Brian Smith for discussions 

on enzyme mechanisms, Jenny McKimm-Breschkin for discussions on drug resistance, Bert van 

Donkelaar for technical support, and Paul Davis for computing support. 

References 

1. Kilbourne ED. Influenza. New York: Plenum, 1987. 

2. Webster RG, Schafer JR, Suss J, Bean Jr WJ, Kawaoko Y. Evolution and ecology of influenza 

viruses. In: Hannoun C, et al. eds. Options for the Control of Influenza Virus II. Amsterdam: Excerpta 

Medica, 1993:177–185. 

3. Lamb RA. Genes and proteins of the influenza virus. In: Krug RM, ed. The Influenza Viruses, ed. 

New York: Plenum, 1989: 1–87. 

4. Murphy JS, Bang FB. Observations with the electron microscope on cells of chick chorio-allantoic 

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19 

Rhinoviral Capsid-Binding Inhibitors: Structural Basis for Understanding 

Rhinoviral Biology and for Drug Design 

Vincent L. Giranda 

Abbott Laboratories, Abbott Park, Illinois 

Guy D. Diana 

ViroPharma, Inc., Malvern, Pennsylvania 


A cure for the common cold has been sought after so long that it has become a clicheé: “We can 

_____(do something difficult) but we can't cure the common cold”. There are, unfortunately, a number 

of factors that conspire to make the cure for the common cold ephemeral. In spite of these difficulties, 

dramatic progress has been made in producing chemotherapies for human rhinoviruses (HRVs), which 

are the major cause of the common cold in humans [1]. Although most colds are generally both mild and 

self-limiting, they are responsible for both a large proportion of visits to physicians and lost work time 

[2]. Billions of dollars are spent in the United States alone on symptomatic relief from this disease. The 

ubiquitousness of this disease has led many people to seek a cure for many years (the discovery of the 

class of HRV inhibitors described here is over 20 years old). This chapter will describe the influence of 

structure-based approaches in the design of a class of antipicornaviral agents called capsid-binding 

inhibitors. 

Any effort to inhibit HRV replication, and thus cure many common colds, is made more difficult by 

three factors. First, there are at least 102 described serotypes of HRVs, and it seems likely that there are 

many more serotypes not yet described [3]. Rhinoviruses are responsible for about 40–60% of the colds 

in humans [1,4]. Therefore, a chemotherapeutic agent would need to be effective 


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against most of the HRV serotypes to be useful in approximately 50% of common colds. This 

percentage may be somewhat higher because some of the other causes of cold-like illnesses, particularly 

the enteroviruses (e.g., Coxsackie and echoviruses), are also inhibited by capsid-binding compounds 

[5–7]. In order to be efficacious, drugs must necessarily have a broad spectrum of activity. 

The second difficulty in producing HRV chemotherapy stems from the relatively innocuous nature of 

HRV infection. Compounds must be able to be very safely administered, with a minimum of drug-drug 

interactions, if therapy is to be acceptable. An analogy may be drawn between common headache 

remedies and common cold chemotherapy. Common headache cures such as nonsteroidal 

antiinflammatory agents and acetaminophen clearly can cause serious side effects (gastrointestinal 

bleeding or catastrophic liver failure) particularly if misused [8]. In spite of this possibility, serious 

complications from these agents are quite rare. One would suspect that an antirhinoviral agent would 

need to be at least as safe as headache remedies. 

The third major difficulty in developing cold cures arises from the fact that the HRVs are RNA viruses. 

When presented with any selective pressure, including chemotherapeutic or antibody challenge, RNA 

viruses mutate rapidly [9]. This ability to mutate is most clearly illustrated in influenza viruses (RNA 

viruses), where new strains continuously arise to circumvent immunity in a population. Influenza A 

viruses have been shown to mutate around the anti-influenza drug Amantadine, after a single passage 

through a susceptible human host. The mutated viruses shed from a host treated with Amantadine are 

now resistant to Amantadine. These mutated viruses appear to be as virulent as the parent strain of virus 

[10]. 

Any effort at antirhinoviral therapy must attend to these three issues: (1) serotypic diversity; (2) 

exceptional safety; and (3) viral resistance. Therefore, any structure-based approach cannot concentrate 

on potency alone, but must also attend to these three issues as well. The requirement for inhibiting 

multiple targets has been addressed in tangible ways using structure-based design and will be discussed 

here. Safety issues have been addressed in limited published data from clinical and preclinical studies, 

but structure-based design has not played any significant role in addressing these problems. One might 

argue that structure-based approaches have aided in the design of clinical backups. These backups are 

then brought forward after an initial drug fails for safety reasons. Drug-resistant mutations created in the 

laboratory have also been examined structurally and will be discussed. The importance of resistance 

developing in the clinical setting has not yet been answered. 

This chapter will first introduce the target, the HRVs, and describe their anatomy and life cycle. 

Emphasis will be placed on the viral capsid and its disassembly or uncoating. How structural 

information has helped in our under- 


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standing viral physiology will be highlighted. This will be followed by a description of capsid-binding 

antirhinoviral compounds. We will leave discussions of other HRV targets, e.g., proteases, to other 

authors more knowledgeable in these subjects. Drug structure—activity relationships will be discussed, 

followed by a discussion of drug resistance. Finally clinical trials and future prospects for capsidbinding 

inhibitors in any antirhinoviral armamentarium will be discussed. 

II. The Human Rhinovirus Anatomy 

The human rhinoviruses are picornaviruses (pico = small; rna = RNA), a family of small (300 Å in 

diameter), positive sense, single-stranded RNA viruses. Other generas in this family include the 

enteroviruses (e.g., polioviruses); the aphthoviruses (e.g., foot-and-mouth disease virus); the 

cardioviruses (e.g., mengovirus, EMC virus); and the heparnaviruses (e.g., hepatitis A virus). With the 

exception of the heparnaviruses, a crystallographic structure is known for at least one of the viruses in 

each genera [5,11–17]. These structures show remarkable similarities, which will be described. In spite 

of these similarities, caution should be exercised when trying to generalize data gathered in one genus to 

other members of the picornavirus family. 

The picornaviruses share an icosahedral structure (Figure 1). The icosahedral protein coat that 

encapsidates the viral RNA is made up of 60 symmetrically arranged protomers. Each of these 

protomers is comprised of four viral polypeptides, termed VP1 through VP4. This was the extent of our 

structural knowledge of the picornaviruses until the 1985 structure of HRV14 was published by Michael 

Rossmann and coworkers [16]. This was followed rapidly by structural determination of a variety of 

other picornaviruses. These structures provided the framework on which to base current paradigms for 

the picornaviral life cycles, particularly with regard to their assembly, attachment, and uncoating. 

The VP1, VP2, and VP3 all contain a core eight-stranded antiparallel β barrel (Figure 2). These 

polypeptides have surfaces on both the exterior and interior of the virion particle, facing both solvent 

and viral RNA. The fourth polypeptide, VP4, is considerably smaller than the others and does not 

contain the eight-stranded barrel motif. The VP4 resides entirely on the interior surface of the virion, in 

close association with the viral RNA. The N-terminus of VP4 is known to be myristoylated in both rhino- 

and polioviruses [18,19]. 

Three regions of the picornavirus structure deserve special attention because they appear to play crucial 

roles in the viral life cycle as well as bear on the function of the capsid-binding compounds. These 

regions are the canyon, the VP1 hydrophobic pocket, and the β cylinder. 


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Figure 1 

Schematic illustration of the icosahedral rhinovirus 14. 

(a) Shown is the icosahedron comprised of 60 copies 

each of VP1 (light gray), VP2 (black), and VP3 (gray). 

The shaded circles around each five-fold axis indicate 

the canyon positions. Also indicated is the approximate 

position of the VP1 hydrophobic pocket that lies 

underneath the surface of the virion. (b) An 

icosahedral pentamer is expanded with one viral protomer 

shown as a protein ribbon diagram. (c) This pentamer is 

seen in a cutaway view. Here VP1 is white, VP2 and VP4 

black, and VP3 gray. A capsid-binding compound is 

depicted as black spheres inside the VP1 ribbon diagram. 

The cross hatched regions on the (c) schematic (right) 

indicate areas that disorder when HRV14 crystals are 

exposed to acid. 


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A. The Canyon 

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The HRVs have been divided into the major and minor receptor groups based on two identified cellular 

receptors [3]. The major group, which is comprised of approximately 90 serotypes, binds to the 

intercellular adhesion molecule 1 (ICAM-1) [20]. The minor group, about 10 serotypes, binds to the low 

density lipoprotein receptor family [21]. 

The canyons are depressions approximately 15 to 20 Å deep that encircle each icosahedral five-fold axis 

(Figure 1). When first seen in HRV 14, these canyons were postulated to be the site at which a cellular 

receptor would bind. Subsequent electron-microscopic data revealed that ICAM-1 does indeed bind in 

the canyon as predicted, although in a somewhat different orientation than early models [22,23]. These 

canyons allow the receptor binding sites to escape immunological surveillance because the canyons are 

too narrow to allow an immunoglobulin to contact the canyon floor. Directly underneath the floor of the 

canyon lies a second important structure, the VP1 hydrophobic pocket. 

B. The VP1 Hydrophobic Pocket 

The VP1 hydrophobic pocket is the site where the capsid-binding compounds reside. This hydrophobic 

pocket in VP1 was not initially apparent in the HRV14 structure because it exists in a closed 

conformation in the native HRV14. The addition of a capsid-binding antiviral agent induces the pocket 

to open. This was first seen by Smith and coworkers when the first crystal structure of a capsid-binding 

drug, WIN 51711, was solved bound in HRV14 [24]. (WIN is the designation for a Sterling Winthrop 

compound.) This was a seminal event in the structure-based design of these capsid-binding compounds; 

before this structure was solved the exact site at which the compounds bound was unknown. 


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Figure 2 

Panels (a), (b), and (c) depict VP1, VP2, and VP3 β barrels, respectively. In all 

cases the view is such that the virion exterior would be on the right side of 

the page. A capsid-binding compound is depicted as gray spheres as it is 

found inside VP1. 


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Subsequent studies have shown this pocket is present in every known HRV and enterovirus structure. 

Page 493 

This pocket is inside the β barrel of VP1, directly underneath the canyon floor where ICAM-1 binds 

(Figure 1c). The proximity of the hydrophobic drug-binding site to the receptor-binding site explains the 

effects these compounds have on viral attachment in specific HRV serotypes (see below). 

C. The β Cylinder 

The β cylinder is a complicated structure that lies on the inside of the viral coat (Figure 1c). There is one 

β cylinder at each icosahedral five-fold axis. The β cylinder is formed by winding the five-fold related 

VP3 N-termini around the symmetry axis. In close association with this cylinder is the N-terminal 

regions of VP1 and VP4. 

The myristoyl moiety attached to VP4 is observed in the poliovirus structures. This moiety is in close 

association with the cylinder formed by VP3 [19]. In HRVs, density consistent with the myristoyl 

moiety is also seen near the β cylinder [25]. In the HRVs, structural disorder of the first 25–28 Nterminal 

residues of VP4 obscures the connection between VP4 and the myristoyl group. The addition of 

myristic acid to protein is seen in many viral and cellular 


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proteins. It is typically a signal moiety that directs the protein to which it is attached towards cellular 

membranes [26]. 

Page 494 

Above the β cylinder is an ion, thought to be Ca 2+, which lies right on the five-fold axis and coordinates 

to the five adjacent VP1 polypeptides [25,27]. The proximity of the β cylinder to structures shown later 

to become external during uncoating (portions of VP1, VP4) suggests that it may be important in 

uncoating. 

III. The Human Rhinovirus Life Cycle 

The HRVs must undergo a number of transitions to replicate (Figure 3). First, they must attach to the 

cell surface at a cellular receptor. They are then internalized into the cell via the endosomal 

compartment. Following internalization, they must eject their RNA out of the viral capsid, through a 

lipid bilayer, and into the cytosol in a manner that preserves the integrity of the RNA. Replication 

Figure 3 

A schematic diagram depicting some of the 

required steps in viral replication. 


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of the RNA and production of a polyprotein ensues. The polyprotein is then processed by viral proteases 

to form the viral polypeptides. The coat must then assemble, package RNA, and leave the cell. The 

capsid-binding compounds' effect on propagation have been shown to occur at the attachment, 

uncoating, and assembly steps (25,27–30). The relative effect on each of these steps appears to be 

variable and has not yet been extensively characterized. The most universal effect appears to be on the 

uncoating step. 

During uncoating several different rhinovirus subparticles are observed. These are thought to be 

intermediates in the uncoating process [32–33]. The fully infectious 149S particle becomes an 125S Aparticle, 

which has lost VP4 but still maintains viral RNA. This A-particle is no longer infectious. The Aparticle 

then releases RNA to become an 80S empty shell. 

The formation of these types of particles can be induced by association of the virion with the cell 

receptor or acidification [32,34]. In poliovirus, attachment to cells has been shown to lead to a particle 

that has lost VP4 and has externalized the N-terminal region of VP1, which normally resides on the 

virion interior. 

There has been considerable debate about how the HRVs accomplish the transfer of RNA from inside 

the virion into the cell cytosol. This step is crucial for productive uncoating. An important question 

concerns the requirement for acidification of the endosome for HRVs to release their RNA. Evidence 

that appeared to conflict was found in a number of studies using either entero- or rhinoviruses (35–39). 

This question was later addressed by experiments that specifically separated entero- and rhinovirus 

behavior [40]. These experiments showed that HRVs, unlike poliovirus, require a pH-lowering step for 

productive infection. This pH lowering is likely to occur in the endosomal compartment. It should be 

noted that HRV and enteroviruses have been classified historically based on their resistance to acid: 

HRVs are acid-labile, while enteroviruses are stable in acid. Consequently, differences in behavior 

between the rhino- and enteroviruses in an acidic environment within the cell are not surprising. 

To replicate the changes in HRV that might be induced by acidification in the endosome, HRV14 was 

acidified in the crystalline state and examined via x-ray diffraction. When compared to the native 

HRV14, the acidified HRV14 capsid becomes disordered in three regions: the Ca 2+ ion on the five-fold 

axis; a region of the β cylinder and the adjacent portion of VP4; and the GH loop [41]. The GH loop is 

the region of structure that connects β-strands G and H and lies directly between the hydrophobic pocket 

and the receptor binding site (Figure 4). It forms the roof of the VP1 hydrophobic pocket and the floor of 

the canyon at the receptor binding site. 

Mutants that are resistant to acid in vitro were isolated [41,42]. These mutants cluster about the GH loop 

(Table 1, Figure 4), and typically would be thought of as mutants that stabilize protein structure (e.g., 

larger or more 


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Table 1 VP1 Acid- and Drug-Resistant Mutations 

Phenotype Mutations in VP1 

Acid resistant H1078L, H1078Y, N1100K, N1100T, D1101E, N1145S, 

W1163R, V1188A, V1191A, T1216I, M1221L, M1224L, 

A1225V 

Drug resistant (compensation) N1100S, N1105S, V11531, N1219S, S1223G 

Drug Resistant (exclusion) V1188L, V1188M, C1199F, C1199Y, C1199W, C1199R 

branched amino acids) [43–46]. The proximity of these mutations to the GH loop coupled with the 

loop's movement on acidification suggest that the GH-loop movement plays an important role in 

uncoating. 

It has also been suggested that binding of ICAM-1 to the virions may also play a role in uncoating. In 

support of this hypothesis, it has also been observed 


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Figure 4 

A ribbon diagram of HRV14 VP1 bound to WIN 61605 (small gray spheres 

with black bonds). The VP1 region that disorders under acid conditions is 

depicted by black color on the ribbon diagram. Residues that can be 

mutated to be acid stable, drug resistant (compensation type) or to either 

phenotype are shown as black, white, and gray ball-and-stick models, 

respectively. The majority of mutations are near the site of drug binding 

as well as the site of acid-induced disorder in VP1. 


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that in some HRVs (particularly HRV14 and to a lesser extent in HRV3, but not HRV16) the addition of 

ICAM-1 itself, in the absence of acid, can induce structural changes that mimic uncoating in the capsid 

[34]. However, these changes are unlikely to lead to productive uncoating because they occur in the 

extracellular space. Therefore, the binding of ICAM alone, at the cell surface, is unlikely to be sufficient 

to cause productive uncoating of HRVs. 

It seems that the hydrophobic pocket in HRVs is maintained to allow an induced transition around the 

GH loop that is required for productive uncoating. This transition could be induced by acidification in 

the endosome, receptor binding, or a combination of the two. Filling this pocket in VP1 with a drug or 

naturally occurring factor would inhibit this transition, thus inhibit uncoating. As expected, binding of 

compounds in the VP1 pocket has also been shown to inhibit intracellular uncoating as well as either 

acid-or heat-induced uncoating of the virus [28,29,47]. 

An attractive hypothesis suggests that the GH loop transition precedes or allows the externalization of 

VP4, which would be required for the formation of the uncoating intermediate particles. Remember, 

VP4 contains the myristoyl moiety, which can signal VP4 to associate with a membrane. The VP4 

would drag the N-terminal region of VP1 (to which it is closely associated) to the exterior of the virion. 

This would result in a particle with the N-terminus of VP1 exposed, as has been observed in poliovirus 

[48–50]. The sequence of this exposed region of VP1 suggests that it can form an amphipathic helix. 

The VP1 helices could then insert into the membrane and form a pore, which could allow the passage of 

RNA through the lipid bilayer into the cytosol. This is reminiscent of the pore formation by colicin [51]. 

The observation that both the Ca 2+ ion plus the β cylinder and VP4 become disordered under acidic 

conditions are consistent with this hypothesis. These are regions that would need to disorder to allow the 

externalization of VP4 and the N-terminus of VP1. 

IV. Capsid-Binding Compounds 

Capsid-binding compounds were discovered long before the emergence of the HRV crystal structure 

[52]. They were initially discovered on screening of compounds that had been produced by an insect 

pheromone project at Sterling Winthrop. Examples of compounds known or presumed to bind in this 

pocket are shown in Figure 5 [52–69]. The prototypical WIN drug contains an oxazoline ring attached to 

a phenoxy group, which is in turn linked by an aliphatic chain to an isoxazole ring. The three rings will 

be termed A, B, and C here (Figure 6). Compounds of this type were the first to be shown to inhibit the 

viral uncoating and also to stabilize the virion to heat-induced denaturation [29]. 


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Figure 5 

Some compounds which have been shown to inhibit picornavirus 

replication. These are thought to bind in the VP1 hydrophobic 

pocket. References are indicated on the figure. 

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The structure of the first compound to be solved in complex with HRV 14 was found to be bound in an 

extended conformation within the VP1 hydrophobic pocket [24]. The compound is almost entirely 

buried within the capsid of the virion. Since in the native HRV14 the pocket exists in a closed 

configuration, binding required large motions in VP1 to accommodate the drug. These 


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Figure 6 

Some WIN compounds depicting different rings 

and linkers. Note WIN 52452 has no C ring. 

motions, of up to approximately 4.5 Å, are most pronounced in the region of the GH loop. 

In contrast, HRV1A and HRV16, a minor and major receptor group virus respectively, have their 

pockets in open conformations even in the absence of drug [13,15]. For these serotypes, drug binding 

typically shifts positions of capsid atoms a maximum of 1 to 2 Å, smaller than those shifts seen in 

HRV14. Drugs bind in these pockets in a fashion similar to that seen in HRV14, extended and almost 

entirely buried by VP1. 

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The structures of five HRVs have been solved to date: HRVs 1A, 3, 14, 16, and 50. In all of these 

HRVs, as well as the polio- and coxsackie viruses, VP1 hydrophobic pockets have been observed 

[5,12,13,15,17,24,70,71] (HRV3, Zhao, R. et al., personal correspondence; HRV50, Giranda V. L. et al., 

unpub- 


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Figure 7 

HRV14 VP1 hydrophobic pocket schematic with WIN 61605 

illustrating some of the terminology commonly used to describe 

this pocket. Notice the GH loop separates the pocket from the 

canyon floor. 

Page 500 

lished data). These pockets all share similar features and have been described as foot shaped, with a 

hydrophobic toe region, a heel region capable of hydrogen bonding, and a pore region near the ankle of 

the foot (Figure 7). 

Many of the picornavirus structures have been shown to have electron density in their VP1 pockets even 

in the absence of any added drug. These densities have been modeled as fatty acids or similar 

compounds [12,15,56,70–72]. The occurrence of these pocket factors have led some to hypothesize that 

these factors perform a similar function as do capsid-binding inhibitors, that is, to stabilize the virions 

[15,24,41,73,74]. 

Teleologically one could argue that the virion would pick up a fatty acid in its VP1 pocket before its 

egress from the cell, which would then stabilize the virion in transit to new hosts. The HRVs are known 

to be stable for long periods of time on surfaces and the dominant mode of transmission is thought to be 

hand-to-hand contact [75,76]. When a new host cell is reached, the stabilization factor might exit the 

pocket. This would allow the necessary conformational transition (probably at the GH loop) to occur and 

allow productive uncoating. 

A. Multiple Targets 

The use of these structures in a traditional structure-based drug design approach has been limited by the 

large number of unknown target serotype structures. There have been useful studies that have grouped 

picornaviruses based on their susceptibility to various antiviral agents (see below) [7,77]. However, in 

order to design a truly broad-spectrum single drug, the structural elements common among many 

serotypes must be considered. 


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Because of the large number of serotypes, a successful inhibition strategy needs to consider whether it is 

better to have a drug that is exceedingly potent against a small subset of HRV serotypes (e.g. 30%) or a 

drug that is somewhat less potent but effective against most serotypes (e.g. 90%). Because of these 

considerations, different parameters are required to describe viral inhibition. 

Two important values that have been used extensively to describe potency for antipicornaviral 

compounds include the mean inhibitory concentration (MIC) and the MIC 80. The MIC is the 

concentration that inhibits the viral progeny production by 50% in a cell-based plaque assay. The mean 

MIC is the average MIC over the number of serotypes against which the compounds have been tested. 

The MIC 80 is the MIC at which at least 80% of the serotypes tested will be inhibited by at least 50%. 

Another way to think about the MIC 80 is that it is the MIC value for the serotype that is at the 80th 

percentile rank for viruses inhibited. For example, if ten viruses were tested, the MIC 80 would be the 

MIC concentration of the drug that inhibits the 8th most sensitive virus [15]. 

B. Potency and Binding Energetics 

It would be reasonable to assume that the activity of a drug (MIC) against a specific serotype would be 

related to its binding energy. This is an important consideration because algorithms that are used to 

predict potency rely on estimations of binding energy. The only experiment completed to directly study 

this correlation suggests a rough correlation in a small number of samples. This study however was 

limited to a small number of compounds in a single chemically similar series [78]. It is unclear whether 

this relationship will hold over diverse chemical entities. 

One could imagine a series of compounds that binds, but is ineffective at stabilizing the virion to any 

extent, thus ineffective in inhibiting viral replication. This appears to be what was observed when 

fragments of WIN compounds that only contained the A and B rings were examined. These fragments 

were less able to stabilize the virus to heat-induced denaturation than intact compounds. Although the 

binding constant for these compounds has not been determined, the diminished thermostabilization 

occurred at concentrations of compound sufficient to allow the compounds to be seen bound in the VP1 

pocket via x-ray crystallography [79]. This observation suggests that the drug binding and inhibition 

have been decoupled to some degree. 

Another question concerning correlating binding affinity to viral inhibition is the number of sites per 

virion required to be occupied before the virion can no longer uncoat: all 60, or a few? Further, is there 

any positive or negative cooperativity in the interaction? These questions have not yet been answered. 


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C. Structure-Activity Relationships 

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Structural features, as stated above, have been found that are common to the known HRV structures. 

These features place constraints on compounds that have been demonstrated in a large number of 

structure—activity relationships. Shape considerations, hydrophobicity requirements, hydrogen-bonding 

requirements and compound flexibility will be discussed. 

Pocket-Shape Considerations 

Compound Length. The results of x-ray studies on several HRV serotypes have shown that the 

hydrophobic pockets vary in size, but all pockets impose structural constraints on compounds. The 

pockets, which almost completely enclose the compounds, require that both drug length and width must 

be limited. The results of structure—activity relationships for one series of WIN compounds have shown 

that the optimum length of the aliphatic linking region between the phenoxy (B ring) and isoxazole (C 

ring) ring is between 3 and 6 carbons (Table 2). This optimum length will vary in other series based on 

the extent of substitution on the A and C rings (see below) [54,80–82]. This effect on linker length is 

obvious both for individual serotypes like HRV14 and for the MIC 80, which measures many serotypes. 

This effect of pocket length can also be seen when substituents are added to either the A or C ring which 

would lengthen the compounds. Additions of alkyl chains of four or more carbons tend to decrease 

potency, whereas shorter chains may increase potency (Table 3) [54,83]. 

In studies on compound length, HRV14 appears to be more sensitive to longer compounds than are 

many other rhinoviruses. This is consistent with the observation that the hydrophobic pocket of HRV14 

is longer than that of HRVs 


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1A, 16, and 50. Cluster analysis examining the sensitivity of 100 different picornaviruses against 15 

different inhibitors showed that the sensitivities of different viruses depends on the shape of the pocket, 

with shorter pockets preferring shorter compounds [7]. This analysis is consistent with the structural 

observations, i.e., the viruses with long pockets (e.g., HRV14 and polioviruses) cluster together. A 

cluster distinct from that which contains HRV14, and prefers shorter compounds, is the group that 

contains the shorter pocket viruses: HRVs 1A, 16, and 50 (Figure 8). 

It is interesting that these drug sensitivity groups do not correspond to the receptor binding groups of the 

viruses. For example, both HRV14 and 50 are major receptor group viruses, but the pocket of HRV50 is 

clearly shaped more like the pocket of the minor receptor group virus 1A. 

The argument that the two different drug sensitivity groups may constitute distinct classes of HRVs has 

been bolstered by genetic examination, which has shown that clusters with similar amino acid sequences 

correlate well with the drug sensitivity [84]. As with the pocket shapes, these genetic clusters do not 

correlate with the two receptor groups into which the HRVs are placed. 

Attempts have been made to break through the length barrier imposed by the pocket structure. One 

could conceive of a drug that could pass through the pore at the heel of the pocket and connect the VP1 

hydrophobic pocket to the canyon floor. Such a drug may have activity that could inhibit uncoating via 

stabilization of VP1 as well as inhibit attachment by directly blocking the receptor site. Attempts have 

been made to synthesize just such compounds, with extended “tails” attached to the C-ring. While these 

compounds bind, their tail does not extend through the pocket pore, but rather coils up within the pocket 

[85]. 


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Figure 8 

Solvent accessible surfaces (dot surfaces) of (a) HRV1A, (b) 14, 

(c) 16, (d) 50 filled with WINs 56291, 61605, 56291, 61209 

respectively (ball and stick). In all cases the cutaway view of the 

pocket has the viewer looking from inside the virion. The pocket 

of HRV14 is narrower and longer than the others and this 

is reflected in the drug structure—activity relationships (Tables 1–3). 


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Page 506 

Compound Width. Bulk considerations have also been examined for the phenoxy or B ring [80]. In 

HRV14, which has a long narrow pocket, compounds with no substituents on the phenoxy ring were the 

most potent. However most other serotypes were more sensitive to compounds with substituents on the 

phenoxy ring, either a dimethyl or a dichloro. The serotypes that preferred disubstituted compounds 

included HRV1A and 50, both known to have wider, shorter pockets (Table 4). This is also in agreement 

with the drug-clustering analysis discussed above [7]. 

Induced VP1 Pocket Changes. The shape constrains discussed above are by nature somewhat 

qualitative. The precision with which one could predict the binding energy of a compound based on its 

shape is limited by the ability of the pocket to conform to its occupant. Conformational changes within 

the pocket are most pronounced between native and drug-bound HRV14, but present to a lesser extent in 

both HRV1A and 16. The extent of conformational changes produced by different drugs on the same 

virus may differ. This observation is illustrated in a study of a compound SCH 38057 in HRV14. This 

compound has a substantially different structure than the WIN compounds, and when bound to HRV14, 

induces changes in HRV14 that are also quite different from changes resulting from the binding of a 

WIN compound [62]. 

The ability of compounds to effect conformational changes in the capsid when bound and the variability 

of these changes both between drug classes and viral serotypes have made a priori predictions of 

potency based on shape considerations very difficult. Comparative Molecular Field Analysis (CoMFA) 

studies has been useful, when examining similar compounds in a single serotype (HRV14), in 

demonstrating spatial constraints [85], but generalizing these constraints over many serotypes and drug 

classes is much more difficult. 


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Hydrophobicity Requirements 

Drug binding is enhanced by hydrophobicity in that portion of the drug that binds to the pocket toe. 

Quantitative structure-activity relationship (QSAR) analysis of these compounds have consistently 

shown that the most predictive parameter of antiviral activity is a measure of hydrophobicity, the 

octanol:water partition coefficient (logP) [80,82,85]. These studies have also consistently shown that 

there is no apparent correlation between electrostatic potential or dipole moment and potency. 

Page 507 

This evidence suggests that the drug—pocket interactions in the toe of the pocket are low-intensity 

hydrophobic interactions. Supporting this hypothesis is the finding that many structurally diverse 

molecules can be accommodated in this region, and even structurally similar molecules can bind in 

distinctly different conformations. Closely related structures have been shown to shift along their long 

axis by as much as 1.6 Å with respect to their phenoxy substituents [55]. There is even more variability 

with respect to the isoxazole placement (Table 5, Figure 9). No single hydrogen-bonding or electrostatic 

interaction appears to predominate within the pocket toe. 


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Figure 9 

A solvent-accessible surface of HRV14 (from the 61605 bound 

structure, dot surface) overlaid with WIN 61605 (ball and stick), 

51711, 56826, and 56291 (a). The atoms closest to the toe pocket 

wall (top) for each compound are in approximately the same 

position, while the isoxazole ring (C ring) positions vary significantly. 

The atom closest to the toe pocket wall is quite different in (b), 

which compares WIN 61605 (ball and stick) to R61837 (tubes) and 

SCH 38057 (lines). 


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The correlation of potency with logP might suggest that A rings with the lowest hydrogen-bonding 

potential would be the most potent. This has not been shown to be true. In fact, rings with heterocyclic 

nitrogen atoms are preferred to furan and thiophene rings, which would be expected to have less 

hydrogen-bonding potential [86,87]. This observation even extends to tetrazole rings, which have often 

been used in pharmaceutical design to replace hydrophilic groups such as carboxylates or esters. 

Explanations for the variability in potency due to A-ring changes have not been satisfactory. These 

heterocycles lack any consistent pattern of hydrogen bonds with the pocket residues (Table 6) [88]. Like 

QSAR analysis, structural analysis has not been able to show any relationship between hydrogenbonding 

groups in the A ring, or dipole moment, and potency [80,85]. 

Extensive structural characterization of many different A-ring heterocycles has not yet been done. 

Difficulties predicting relative potency of these compounds a priori stem from the lack of understanding 

of solvation/desolvation effects as well as difficulties in characterizing the low-intensity hydrophobic 

interactions. Consequently, it seems likely that new structure—activity relationships about the A-ring 

heterocycle will continue to be determined based on empirical findings. 

Hydrogen-Bonding Requirements 

Capsid-binding compounds with a hydrogen-bond accepting atom in the region of the drug that binds to 

the VP1 pocket heel appear to demonstrate greater potency than do their non-hydrogen-bonding 

counterparts (Table 7) [55,89]. Studies using HRV14 where Asn1219, a hydrogen-bond donor in the 

pore region, has been mutated to Ala have shown that the compounds bind as well in the mutated virus 

as in the native virus [27]. This might suggest that the hydrogen bonding to Asn1219 at the pocket pore 

is unimportant. Recent structures of compounds in HRV14 have shown that other hydrogen-bond donors 

can function in place of Asn1219, even if Asn1219 is still present. These groups in HRV14 are the 

hydroxyl of Ser1107 and the backbone nitrogen of Leu1106. Other hydrogen-bonding groups are present 

in other rhinoviruses that coordinate to tightly bound waters, which can also act as hydrogen-bond 

donors [55,56]. This provides a flexible hydrogen-bonding network that can then 


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accommodate a variety of different hydrogen-bonding groups in the C ring of the drug (Figure 10). 

Page 510 

This hydrogen-bonding network has also been observed in all classes of capsid-binding compounds that 

have had their structures determined in HRVs [58,62]. Reports of compounds that bind to the capsid and 

inhibit picornavirus uncoating, but would seem to have little hydrogen-bonding potential (e.g., 

dichloroflavan) have not been structurally examined [60]. The effect of adding a hydrogen-bonding 

group to these compounds near the pocket pore is unknown, but one might expect that the potency of the 

compound would improve. Remember that WIN compounds without a hydrogen-bonding group at the 

pore region still have antiviral activity, albeit this activity is much weaker than that of an analogous drug 

with the hydrogen-bonding group present. 


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Figure 10 

Possible hydrogen bonds in the pore region of HRV14 bound to WIN 

61605 (ball and stick). The waters (spheres W1 and W2) could 

potentially form hydrogen bonds to WIN 61605 as well as the side 

chains of Asn1219 and Ser1107 as well as the backbone of Leu1106 

(residues highlighted as tubes). The viewer is looking 

from the virion exterior. 

Page 512 

The fluidity of the hydrogen-bond donation network at the pore could allow a large number of 

structurally distinct molecules to bind in this location. If the function of the pocket is to bind fatty acids 

or other structurally diverse pocket factors and thus stabilize the virion. The flexibility of the hydrogenbonding 

network at the pore seems ideally suited for such a purpose. 

Drug Flexibility 

The WIN compounds all contain a flexible linking region that allows them to conform to differently 

shaped interiors of VP1 hydrophobic pockets. This flexibility is seen in many compounds that share the 

WIN-drug mechanism of action. However, some compounds do not contain a region as obviously 

flexible as an aliphatic linker (e.g., those with unsaturated rings as linkers, Figure 5). 

Flexibility in the aliphatic linking region of the WIN compounds has been explicitly studied and the 

results suggest that such a property is important for 


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broad spectrum activity (Table 8) [56,90]. While certain less-flexible compounds may have increased 

potency for a particular serotype of HRV, these compounds have lower potency versus other serotypes. 

It is not known if the effect of flexibility is an equilibrium or kinetic effect. The flexibility might allow 

the compounds to expand or contract to fill available space in the VP1 hydrophobic pocket. 

Alternatively, the flexibility may allow the compounds to achieve a conformation required to enter or 

leave the pocket, but this conformation would not be seen in the crystallographic experiment. If this is 

true, modeling of the equilibrium structure of compounds in the pocket will not be accurate predictors of 

compound potency. 

Structure—Activity Relationship Summary 

The combination of classical structure—activity relationships combine with the structures of several 

compounds in a number of HRV serotypes have led to a paradigm for designing potent compounds. The 

effects of pocket shape, requirements for hydrophobicity in the toe of the pocket, as well as the potential 

for hydrogen bonding in the heel region appear to be strong determinants of antiviral potency. The 

requirement for a broad spectrum has so far limited a more detailed paradigm, such as one that might 

exist for designing inhibitors to a specific serine protease. In spite of this vagueness of antirhinoviral 

structure—activity relationships, they have been useful in guiding new compound synthe- 


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sis. Large numbers of compounds, which might have been synthesized in the absence of any structural 

knowledge, have been eliminated from the list of inhibitors to be created, because these compounds 

would not have fit well into the VP1 pocket due to its finite size and hydrophobic nature. 

The goal of being able to predict with greater accuracy the potency and spectrum of compounds before 

they are synthesized awaits three developments: the structures of more HRVs and compounds, the 

ability to more accurately model hydrophobic interactions, and probably the most difficult, the ability to 

predict changes in the HRVs that occur due to drug binding. 

V. Viral Resistance 

As would be expected for any virus, particularly an RNA virus, resistance to capsid-binding compounds 

has been observed in the laboratory. The effect such mutations have on the life cycle of the virus is 

important. Mutations that occur in regions where changes are poorly tolerated, because that region 

serves a vital function in the viral life cycle, are likely to lead to less virulent viruses. The almost 

universal inhibition of rhino- and enteroviruses by capsid-binding compounds, coupled with the 

observation that all of the viruses with determined structures have a VP1 hydrophobic pocket, suggests 

that these pockets serve an important and similar function. It seems unlikely that so many viruses would 

maintain such a pocket if it were not a selective advantage. Drug-resistance mutants have been classified 

into two groups, exclusion mutants and compensation mutants. 

A. Exclusion Mutants 

The exclusion mutants' behavior has been readily and adequately determined by biochemical and 

crystallographic means [91,92]. The mutations occur within the hydrophobic pocket of VP1 and 

thermostabilization studies have shown that these mutations preclude the binding of drug in the VP1 

pocket. One mutation site in HRV14 has been located at position 1188, which is on the side of the 

pocket closest to the viral interior, away from the canyon. Mutations at this site that convey resistance 

are Val rarrow.gif Leu or Val rarrow.gif Met. In both cases the mutation is to a larger side chain, 

which would be expected to fill the pocket. The crystal structure of the Val rarrow.gif Leu mutation 

confirms this hypothesis and demonstrates that the Leu side chain occupies space that would normally 

be occupied by an antiviral drug. 

The second site found in HRV14 is at Cys1199. Mutations to Phe, Tyr, Trp, and Arg all confer 

resistance at this site. Again, all of these mutations are to larger side chains. The hypothesis that these 

mutations function by excluding 


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drug from the pocket is confirmed by crystallographic analysis showing that the Phe side chain in the 

Cys rarrow.gif Phe mutation occupies the site which would be occupied by a drug if it were bound. 

B. Compensation Mutants 

Page 515 

More intriguing and more difficult to understand are the compensation mutants. These viruses bind 

compounds within their hydrophobic pockets, but are still able to replicate and are therefore able to 

compensate for the bound drug (Table 1, Figure 4). These mutations, like the mutations that convey acidresistance 

phenotypes, cluster about the hydrophobic pocket of VP1 [27,28,92]. 

Two hypothesis have been presented in order to explain the behavior of the compensation mutants. The 

first suggests that there is a link between the binding of receptor by virion and its ability to bind 

compounds in the VP1 pocket. The second hypothesis suggests that the effect of a mutation on protein 

stability leads to the drug-resistant phenotype. These two hypotheses are not mutually exclusive. 

Linked-Binding Hypothesis 

The compensation mutants can be subdivided into those that occur on the canyon floor and those that are 

inside the VP1 hydrophobic pocket. The linked-binding hypothesis suggests that the subset of 

compensation mutations that occur on the canyon floor increase the binding affinity of receptor. An 

increase in binding for one of these mutants, Val1153 rarrow.gif Ile, has been observed [28]. This 

increased binding would then in turn cause a decrease in the affinity of the capsid for the drug. This 

would result in the drug being less effective in inhibiting uncoating. 

The second subset of compensation mutants are those that occur inside the hydrophobic pocket but do 

not interact with the receptor binding site. These mutations might directly decrease drug-binding affinity 

in the VP1 pocket due to decreased hydrophobic interactions caused by the smaller amino acid side 

chains [27]. In both subsets of mutations, the binding affinity of a drug is reduced either directly or 

through the effects of receptor binding. This decreased affinity for drug would be displayed as a drugresistant 

phenotype. 

Stabilization—Destabilization Hypothesis 

An alternative explanation for the behavior of the drug compensation mutants suggests that these 

mutants allow for increased conformational flexibility in the capsid. This increased conformational 

flexibility would compensate for the decrease in flexibility, which is manifested by decreased acid or 

thermal lability, induced by the binding of drug. The increase in flexibility would allow a 


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conformational transition to occur, presumably near the GH loop, that would be required for viral 

uncoating. The conformational transition would lead to productive uncoating, even in the presence of 

bound drug. 

Page 516 

This hypothesis suggests that drug-resistant mutations would be of the type that destabilize protein 

structures. Such mutations are typically from larger to smaller side chains or from highly branched to 

less branched side chains [43–45]. Four of five observed compensation mutants are of this type (Table 

1). The remaining mutant, Val1153 rarrow.gif Ile, while a priori might not be predicted to destabilize 

the capsid, has been shown experimentally to decrease viral thermostability when compared with native 

HRV14 [28]. 

The advantage of the stabilization-destabilization hypothesis is that it allows for a single mechanism of 

resistance for both compensation mutants occurring in the hydrophobic pocket as well as those that are 

outside the pocket near the receptor binding site. Both of these sites are close to the GH loop of VP1 that 

becomes disordered under acidic conditions. This stabilization—destabilization hypothesis also explains 

the behavior of acid-resistant mutants, which are predominately of the type which should stabilize a 

protein to conformational changes. Wild-type viruses have a preferred stability profile, which allows 

uncoating under the proper conditions. Mutations or addition of drug can perturb this profile, resulting in 

decreased replication. A second perturbation might then compensate for the first, restoring virulence 

(Figure 11). This behavior has been observed in HRV1A, in which mutants have been isolated that 

require presence of drug to replicate. These mutations are more acid-labile 

Figure 11 

The effects of various rhinovirus manipulations. The solid line depicts 

the native rhinovirus uncoating profile. Mutations and drugs can 

effect this profile to make the virus more or less stable to 

pH- or temperature-induced changes. 


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Page 517 

than the wild-type virus. At the optimal drug concentration for growth, the pH-stability of these 

mutations is equivalent to that of native HRV1A in the absence of drug. Increasing drug concentration 

beyond that which is optimal for growth further increases the acid-stability of these mutant viruses so 

that they are more acid-stable than native virus without drug (Daniel Pevear, personal communication). 

C. Clinical Resistance and Virulence 

The clinical importance of these mutations has not been clearly demonstrated. In at least one case a drugresistant 

mutant was able to grow in vitro in single-cycle growth experiments as well as wild-type 

HRV14 [28]. Frequently, however, drug-resistant or acid-stable mutants will not grow as well in cell 

culture as native virus [93]. 

The final analysis of the clinical importance of resistant mutants awaits the results of clinical trails. In 

the one study published to date, HRV2 mutants of the compensation type (resistant to the capsid-binding 

compound chalcone) were tested in healthy human volunteers [65]. The drug-resistant mutants caused 

significantly fewer colds than the normal virus. Mutants that require the presence of drug to grow did 

not cause any apparent disease. In this single case it appears that the mutant viruses were not as virulent 

as the parent strain. 

VI. Animal and Clinical Studies 

The idea that an inhibitor that binds to a nonenzymatic, nonreceptor site of a virion could inhibit viral 

replication in vivo would a priori be considered to be an unlikely scenario by most in the field of 

structure-based design. If this idea were proposed knowing only the structure of the native virus 

(especially HRV14, which has a closed-pocket conformation) skepticism would abound. This type of 

project arose not from a structure-based approach, but from the tried-and-true screening approach. The 

compounds were first shown to be effective in inhibiting viral replication in vitro [52]. This was 

followed by an experiment showing that these compounds could inhibit at least one enterovirus in a 

mouse model [94]. 

In this study, suckling mice were inoculated with an echovirus and if untreated they developed a fatal 

paralytic illness. When treated either prophylactically or within a few days of the viral inoculation, 

virtually all of the mice were protected. This is dramatic proof of the concept that these compounds can 

inhibit picornaviral infection in vivo. 

There are important differences between mice and men and between enteroviral paralytic illness and 

rhinoviral upper respiratory tract infections. 


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Page 518 

The differences between mice and men should be obvious to the casual reader. Enterovirus-induced 

paralytic illnesses are systemic infections, and the virus must at some point move through the body and 

be subject to circulating drug. Upper respiratory infections resulting from rhino-or enteroviruses tend to 

be localized to the pharynx, which would require that drug titers remain high in this region of the body. 

In spite of these difficulties, there have been some dramatic successes in clinical trials of antirhinovirals, 

although clearly there are still obstacles to overcome. Two different classes of antipicornavirus 

compounds have been shown to be successful at inhibiting at least some types of infection when 

administered prophylactically. The WIN compound 54954, given orally, has been shown to inhibit 

Coxsackie virus A21 in humans [6]. The Jaansen compound R77975 has been shown to inhibit HRV9 

when administered intranasally 6 times daily [95,96], but R77975 has not been shown to inhibit 

infection when used therapeutically (after symptoms occur). Further clinical trials with WIN 54954 were 

suspended due to the developments of side effects. More recently VP 63843 has been shown to have 

dramatically improved effect when compared with WIN 54954 in the prophylactic treatment of 

Coxsackie virus A21 infection in humans and it will be tested therapeutically. 

The inability of R77975 to show a therapeutic effect is more likely due to poor pharmacodynamics 

rather than a fundamental inability for this compound to inhibit an established infection. The 

pharmacodynamic problem was witnessed in the R77975 study, which showed that administration 

intranasally 3 times a day did not yield prophylactic protection. Yet if administered 6 times daily the 

prophylaxis occurs. Therefore if more potent and metabolically stable compounds are found, more 

positive clinical results would be expected. 

VII. Future Directions 

The structures of the picornaviruses (native, with receptor bound, in the presence of acid, with a myriad 

of compounds bound, and of acid- and drug-resistant mutants) have yielded valuable information about 

possible molecular mechanisms for their uncoating. These same studies have suggested the mechanism 

by which these uncoating inhibitors work. A by-product of this research is the hypothesis that these 

compounds may mimic naturally occurring factors that occupy the VP1 pocket. The hunt for these 

natural compounds and their significance is underway. 

This understanding of the mechanism of action, as well as the structure-activity studies, have also 

yielded valuable information for future development of antipicornaviral drugs. The determination of 

compound's size, shape, and other physical requirements for activity will also be of assistance. 


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Page 519 

From the animal and human experiments it is clear that the cure for some common colds is within reach. 

These therapies are also quite likely to be efficacious in enteroviral diseases, for example in cardiogenic 

coxsackie virus infection [97]. 


We would like to acknowledge that there have probably been hundreds of people who have contributed 

to work reviewed here. We would like especially to thank the many people at ViroPharma, Sterling 

Winthrop, Purdue University, and the University of Wisconsin at Madison with whom we have worked 

over the years. We would also like to thank Dirksen Bussiere and Yvonne Martin for reading and editing 

this manuscript prior to submission. 

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20 

The Integration of Structure-Based Design and Directed Combinatorial 

Chemistry for New Pharmaceutical Discovery 

Roger Bone and F. Raymond Salemme 

3-Dimensional Pharmaceuticals, Inc., Exton, Pennsylvania 

I. New Challenges For Drug Discovery 

Page 525 

Rapid advances in cell and molecular biology, together with comprehensive genome sequencing efforts, 

are providing detailed correlations between specific pathological conditions and discrete molecular 

targets. The same tools of recombinant DNA technology that identify key gene targets also provide the 

means for target biosynthesis in quantities sufficient for both the high-throughput screening of 

compound libraries for leads and the structure-based refinement of leads using x-ray crystallography and 

NMR spectroscopy. 

The rapid expansion in genomics data makes it inevitable that targets will be identified whose functions 

are so poorly understood that the most rapid and efficient way to establish their involvement in disease 

will be through the development of prototype drugs. New approaches to drug discovery that are able to 

integrate many different types of information are needed to seize this opportunity and drive an optimally 

efficient discovery process. 

In what follows, we describe a practical integration of structure-based design and combinatorial 

chemistry aimed at enhancing the effectiveness of both approaches. Three-dimensional structures 

provide the information required to most efficiently direct the design and optimization of new lead 

compounds. Combinatorial chemistry technologies, which are based on high-throughput automated 

methods of chemical synthesis, produce new classes of lead compounds and permit the rapid generation 

of structure—activity relationships 


Document 

Figure 1 

An integrated technology for drug discovery combines the 

precision of structure-based design with the parallelism of 

combinatorial synthesis. Chemi-informatics systems track 

and integrate all data emerging from the discovery cycle. 

Page 526 

(SAR). Chemi-informatics plays a key role in this integration by assuring that properties important in 

drug development are both factored into compound design and cumulatively tracked throughout the 

discovery process (Figure 1). The product of this approach is a permanently useful set of drug-design 

parameters. The integration of these technologies promises to produce an increase in the efficiency of 

drug discovery and may ultimately offer a means for reducing the aggregate failure rate of compounds 

selected for development. One paradigm for achieving the integration of these technologies is described 

below. 

II. Structure-Based Design 

Structure-based drug design has become a highly developed technology that is in active use in most 

major pharmaceutical companies. Structure-based design is an iterative process in which lead 

compounds identified by screening, de novo design, or mechanism-based features are systematically 

elaborated to improve potency and specificity [1,2]. The process involves successive rounds of structure 

determination of lead—target complexes, design of lead modifications using molecular modeling tools, 

synthesis of new drug leads, and measurement of the chemical and biological properties of the modified 

leads using screens for target 


Document 

Page 527 

function. Iterative refinement and optimization of drug leads is an effective strategy for generating 

potent preclinical candidates. Structure-based design can also be used to design new chemical classes of 

compounds that present similar substituents to the target using a template or scaffold which is 

chemically distinct from previously characterized leads [3,4]. 

Structure determination typically relies on x-ray crystallography or high-field nuclear magnetic 

resonance to directly visualize the 3-dimensional structure of a molecular target and the structures of 

complexes of the target with drug leads. Alternately, many targets fall into identifiable classes that 

frequently enable the development of homology models of the 3-dimensional target structure or a 

mechanism-based strategy for drug-lead generation. Ongoing genome sequencing efforts have led to the 

identification of hundreds of potential therapeutic targets, many of which represent possible sources of 

crossover pharmacology. Homology modeling is a key feature of an integrated drug discovery effort 

because it allows this genomics information to be utilized early in the development of target ligands or 

in the engineering of ligand specificity. 

Although structure-based design is an effective technology, current limitations center on the inability to 

quantitatively predict how specific modifications of the lead will actually affect ligand binding affinity 

[5,6]. This reflects the complexity of the drug-binding process and our inability to accurately predict the 

conformational response of macromolecular structures to ligand binding. In addition, we have only 

limited ability to accurately calculate molecular energy parameters or to accurately estimate the effects 

of factors such as polarizability, solvation, and entropy that may have an important influence on drugbinding 

energetics. Although computational methods will continue to improve, most design work (and 

algorithms) still relies heavily on heuristic rules (Figure 2) that have been developed through experience 

and that guide the structural and medicinal chemists in the systematic modification of lead compounds 

[7]. As a practical consequence, many cycles of serial lead modification are required in order to produce 

molecules of suitable potency and specificity to be considered preclinical drug candidates. 

Structural information can increase the efficiency with which pharmacokinetic or toxicological liabilities 

in lead compounds are eliminated by suggesting where compounds can be modified so as to alter drug 

properties without affecting target potency. Structural data can also be used to direct de novo design of 

alternate and distinct chemical classes of lead compounds, each of which might be expected to have a 

different pharmacological profile [3,4]. New chemical compound classes can also be designed from 

existing lead compounds by recombining substituents and core regions (scaffolds) from existing lead 

compounds. Chemically distinct lead series can then be optimized in parallel so that when a preclinical 

candidate is found to have inadequate drug properties, a backup is immediately available for preclinical 

evaluation. As 


Document 

Figure 2 

Some heuristic rules frequently used in structure-based drug 

design. The positions of “bound” water molecules are key 

indicators for lead modification sites. 

outlined below, both the scaffold modification and structural recombination strategies are key 

components of an integrated drug discovery technology that combines structure-based design and 

combinatorial chemistry. 

III. Combinatorial Chemistry 

Page 528 

Combinatorial chemical technology enables the parallel synthesis of organic compounds through the 

systematic addition of defined chemical components using highly reliable chemical reactions and robotic 

instrumentation [8–11]. Large libraries of compounds result from the combination of all possible 

reactions that can be done at one site with all the possible reactions that can be done at a second, third, 

or greater number of sites. Combinatorial chemical methods can potentially generate tens to hundreds of 

millions of new chemical compounds as mixtures, attached to a solid support, or as individual 

compounds. The first combinatorial libraries with millions of members were oligopeptide and 

oligonucleotide libraries for which reliable and versatile chemical reactions already existed [10,12,13]. 

However, these libraries were not generally useful as a source of leads for small-molecule 

pharmaceuticals due to the relatively high molecular weight of the resulting leads and other limitations 

of oligonucleotides and oligopeptides as drugs. More recently, “drug-like” libraries have been produced 

that offer much greater utility as a source of drug leads (Figure 3). However, practical limitations of the 

versatility and reliability of the chemical reactions used to make these libraries have resulted in 

somewhat smaller sets of compounds [14–18]. 


Document 

Figure 3 

Generation of combinatorial libraries. 

Traditional combinatorial libraries based on 

polypeptides or polynucleotides were built up 

by oligomer condensation and achieved a 

high level of diversity through conformational 

flexibility. Small-molecule libraries of drug-like 

molecules can be built up using a variety of 

strategies that focus on maximizing chemical 

diversity while (generally) minimizing 

conformational flexibility. 

Page 529 

From the drug discovery perspective, large combinatorial libraries have the same utility as conventional 

pharmaceutical or natural-product compound libraries, i.e., as a source of leads for new drugs. The 

design of large combinatorial libraries is driven by the requirement that individual reactions be highly 

reliable and versatile, while producing libraries with the highest possible degree of chemical diversity. 

Individual steps must be optimized, the compatibility of building blocks must be examined thoroughly 

and the synthesis must be automated. As a consequence, a significant investment in time and resources 

must be made before a library can actually be produced. Designed and used in this way, large 

combinatorial libraries provide a new source of screening leads. However, libraries with maximum 

diversity, which may be used for “blind” lead discovery, do not address the principle rate-limiting step 

in drug discovery, the elaboration of a suitable SAR around the lead compound after an active lead has 

been discovered. 


Document 

While initial combinatorial chemical strategies have focused on the exhaustive synthesis of all members 

of a given library, it is inevitable that advances in automated chemistry and equipment will soon make it 

possible to synthesize a vastly greater number of compounds than it will be practical to 


Document 

Page 530 

screen. For example, for a simple library created using amine and acid condensations onto a given amino 

acid scaffold, over a million compounds can be produced from commercially available reagents. If this 

simple library is expanded to incorporate the hundreds of commercially available or easily synthesized 

amino acids scaffolds, the number of compounds that can possibly be made increases to greater than 

10 8. Although biological, chemical, and physical assays can be automated so that hundreds of thousands 

to millions of compounds can be screened annually, the process is expensive and the reliability of the 

screening process decreases when measurements are made on mixtures of multiple compounds. Various 

estimates of the number of drug-like molecules with molecular weight (MW)

Document 

Figure 4 

The information flow in a drug discovery process that 

connects elements of structure-based design and 

combinatorial chemistry for 

drug discovery. 


Page 531

Document 

ciency of drug discovery (Figure 4). The process begins with the knowledge-based design of a library 

template or scaffold and involves the synthesis of small subsets of library members. As with structurebased 

design approaches, the process is an iterative one in which SAR data and structural data guide not 

only the iterative selection of library members for testing, but also the design of later generation library 

scaffolds. The integrated process is described in detail in the next few paragraphs. 


Document 

Figure 5 

Generation of a virtual combinatorial library by finding 

substituents of a custom scaffold that can be 

accommodated in the binding site of a molecular target 

or meet other 3D structural criteria. Once the virtual 

library is synthesized in the computer, individual 

members can be selected using structural or additional 

criteria and synthesized using automated equipment. 

Page 532 

The first step in the process is the generation of a chemical template or scaffold that can be derivatized 

at multiple sites using reliable chemical reactions to produce a large combinatorial library (Figure 5). 

This custom chemical template is designed based on the structure of the target using the same heuristic 

set of rules used for traditional structure-based drug design. Useful 3-dimensional pharmacophore 

models are best derived from crystallographic or nuclear magnetic resonance structures of the target, but 

can also be derived from homology models based on the structures of related targets or 3-dimensional 

quantitative structure-activity relationships (3D QSAR) derived from a previously discovered series of 

active compounds [20]. In addition, the mechanism of action of the target or any other information that 

exists regarding the target or the target class can be used in the design to maximize the chances of 

finding hits [21,22]. The combinatorial libraries are designed so that a few thousand to millions of 

discrete molecules can be produced by reaction of the custom-designed template with appropriate 

proprietary and commercially available chemical building blocks. 

The next step in the process involves the implementation of the automated synthesis and generation of 

the library. Significant lead time is anticipated before a library can be produced because even the most 

reliable chemical reactions require optimization if they are to be carried out by a robot, particularly if 


Document 

Page 533 

the reactions are to be implemented with the template attached to a solid support. When the synthesis is 

optimized and fully automated, thousands to millions of compounds are accessible. 

One of the key features of this process is that all of the compounds that can potentially be made by 

elaboration of a custom scaffold are first “made” in virtual form in a computer. Each of the chemical 

reactions required to create the library is encoded in a program that then systematically combines the 

template and the building blocks to create a 2-dimensional representation of each member of the library. 

Next, 3-dimensional representations are created and molecular-property descriptors are calculated for 

each member of the library. Molecular property descriptors encompass molecular connectivity, dipole 

moments, calculated partition coefficients, and many other calculable molecular properties. The virtual 

library of compounds can then be computationally screened and the library members ranked according 

to their ability to interact with the target receptor or 3-dimensional pharmacophore model [23–25]. The 

compounds can also be ranked by their inability to interact with any number of alternative targets whose 

inhibition is undesirable, or their ability to meet any range of desired chemical or physical properties 

that may be important in drug pharmacology. Alternately or additionally, compounds can be ranked 

according to their ability to span or sample the physical-chemical property space to produce the most 

diverse set of compounds for initial screening [26,27]. 

Small sublibraries of the large virtual library that best satisfy the selection criteria are chemically 

synthesized using automated methods and then the biological and/or chemical properties of each 

compound are measured using automated assays. The SAR data that emerge from the assays are stored 

in a central database and used in the selection process to drive additional rounds of sublibrary selection, 

synthesis, and assay. Multiple mathematical models are developed to correlate the computed structure 

and properties of each synthesized library member with the biological, chemical, or physical properties 

that are measured during each cycle of testing [28]. A key feature of this approach is that compounds 

can be selected not only on the basis of which are predicted to perform the best in the target assay but 

also on the basis of their ability to perform the best in the target assay but also on the basis of their 

ability to distinguish between or validate the SAR models that are generated. The observed and 

predicted properties of a given sublibrary are compared so that the set of assumptions upon which 

property refinement is based is constantly updated. In principle, this process can become completely 

automated so that leads are discovered and refined with very little manual intervention [28]. 

To achieve the greatest improvements in drug discovery efficiency, empirical data of various kinds must 

be collected throughout the iterative refinement process. It is desirable to obtain more accurate 

dissociation constants rather than IC 50 or single-point percent-inhibition values. In addition, the 3dimensional 

structures of interesting target—inhibitor complexes are determined 


Document 

Figure 6 

The combination of structure-based design and combinatorial 

chemistry can facilitate the generation of recombined 

compounds to rapidly produce potent compounds with 

maximum chemical and structural diversity. 

to provide information regarding how substituents are interacting with the target and when binding 

modes change. This information may be essential if SAR models are to remain predictive over large 

numbers of compounds. 

Page 534 

The integrated drug discovery process utilizes as much information as is available to find and optimize 

initial lead compounds. Because the automated synthesis of large libraries of compounds requires 

reliable and versatile chemical reactions, initial libraries are designed to discover new chemical lead 

classes or to develop SAR models. Both library and substituent designs evolve as hits are encountered 

and the structures of target—inhibitor complexes are determined. Single modifications on both the 

template and substituents may be made during the process based on the structures of target—lead 

complexes. When sufficient SAR data has accumulated and the structures of target complexes with key 

templates and substituents have been determined, potent compounds with the desired “drug like” 

compositions are designed and synthesized using a structure-based recombination strategy (Figure 6). 

The integration of these recent advances in drug discovery offers the possibility to substantially decrease 

the time required to find initial leads and develop them into prototype drugs. Similarly, the interval 

required to produce preclinical development compounds and backup candidates is also expected to 

shrink. Perhaps most interesting is the potential of this integrated technology, which is ultimately based 

on an abstract information model of the biological process, to dramatically increase the reliability of 

successful drug development 


Document 

Page 535 

by factoring in and refining important drug properties concurrently with the optimization of drug affinity 

and specificity for a specific molecular receptor. This objective is achieved by integrating assays and 

computational models that relate to important drug development issues (e.g., oral absorption, optimal 

pharmacokinetics, minimal toxicity, etc.) directly into the iterative design process. 

V. Chemi-Informatics 

The development of methods that can optimize the parallel refinement of drug potency and 

pharmacological properties is a key objective in enhancing the efficiency and productivity of drug 

discovery. To address this problem and to handle the dramatic increase in experimental data generated 

using robotic synthesis and assay methodologies, advanced informatics systems are required to collect 

and exploit data relating to the properties of the chemicals being produced. These systems will amplify 

the synergy between structure-based design and combinatorial chemistry and provide a means for 

reducing the aggregate failure rate of development candidates. 

The poor success rate for preclinical development candidates (about 1 in 20) results from our limited 

ability to predict such drug properties as intestinal absorption, excretion, metabolism, toxicity, efficacy, 

and side effects. Rendering the prediction more difficult is the certainty that these drug properties are 

composite properties that result from the operation of many biological processes. Recently, progress has 

been made towards understanding the underlying molecular basis for some of the individual components 

that contribute to the observed drug properties. For instance, the absorption of compounds into Caco-2 

cells in culture may be predictive of intestinal absorption [29–31]. Retention times of compounds during 

artificial membrane chromatography has also been correlated with oral absorption [32]. Recently, 

molecular transporters have been identified, cloned, and characterized that are responsible for the 

absorption of dipeptides and the excretion of organic cations [33,34]. The enzymatic basis for the 

metabolism of xenobiotics (cytochrome P450, glutathione transferase, etc.) has been known for some 

time, at least in part. In fact, many of the individual components of the composite biological processes 

can be developed into high-throughput assays, providing the opportunity to collect SAR data and 

develop SAR models. 

These observations, together with the integrated structure-based design and combinatorial chemical 

technology described here, define a comprehensive new strategy for drug discovery that has the 

potential to reduce the aggregate failure rate for development candidates (Figure 4). The creation of 

virtual libraries of compounds that are readily accessible through the use of automated 


Document 

Page 536 

synthesis methods allows for the first time an extensive and systematic investigation of structure-activity 

relationships related to drug properties. Small sublibraries can be selected, synthesized, and screened in 

high-throughput assays with the goal of developing predictive SAR models for individual components 

of the biological processes responsible for pharmacokinetic and toxicological properties of drug 

candidates. Eventually, it will be possible to assemble the predictive SAR models for various component 

processes to produce predictive models for bioavailability and toxicology. At this point it will be 

possible to use these models to guide the selection of sublibraries directed against therapeutic targets so 

that library members with the most “drug like” properties and the fewest liabilities are chosen. Steering 

away from compounds with undesired properties will focus the selection process on molecules with an 

improved probability of successful development. This strategy permits the simultaneous refinement of 

multiple chemical properties in addition to target efficacy and will shorten the drug-discovery process. 

Developing a system capable of collecting multivariate SAR data and exploiting the data to produce 

predictive SAR models is a major systems integration task. However, recent advances in computers, 

operating systems, and computational chemical tools now enables the implementation of a system that 

can track compounds, store chemical property data in a comprehensive relational database, and operate 

on virtual libraries in an iterative fashion to develop SAR models and refine chemical properties [28]. 

Tools for the production of virtual libraries have been developed by several groups and large virtual 

libraries can be produced within a few days using high-power computer workstations. A variety of tools 

also exist for selecting compounds based on criteria such as the ability to fit a target receptor [23], 

similarity or diversity [26,27], or any number of other properties that might be important for interaction 

with the target receptor. Statistical programs also exist that are up to the task of developing SAR models 

[20]. To obtain SAR models that have utility in the drug-discovery process it will be necessary to collect 

data on the properties of large numbers of compounds so that the models are predictive across diverse 

chemical classes of lead molecules. This requires implementation of a comprehensive relational 

database to collect and correlate SAR data. 

The challenge is to integrate these tools into a system that can collect and operate on vast arrays of 

chemical-property data in an intelligent fashion to direct and refine the properties of robotically 

synthesizable compounds. One approach that has been used successfully is to create a hierarchical clientserver 

system with a powerful central selection algorithm (Figure 4) that chooses compounds from 

virtual libraries based on their computed properties, supervises data analysis, and integrates results from 

experimental measurements [28,35]. This system, which has been termed DirectedDiversity in the 

author's laborato- 


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Page 537 

ries, holds the promise of a major reduction in the time required to produce new preclinical compounds 

with enhanced development potential. 


The preceding outlines a new drug-discovery paradigm that integrates structure-based design, directed 

strategies for combinatorial chemical synthesis, and a comprehensive chemi-informatics system for 

accumulating and analyzing information regarding chemical properties. Three-dimensional structures 

provide the information required to most efficiently direct the design and optimization of new lead 

compounds. High-throughput automated methods of chemical synthesis produce new classes of lead 

compounds and provide for the rapid generation of structure—activity data. Chemical informatics 

systems track chemical compounds, store chemical property data, develop predictive SAR models, and 

provide a means for intelligently directing the drug-discovery process. By applying this approach not 

only to the therapeutic target, but also to molecules involved in absorption, clearance, metabolism, or 

toxicology it will be possible to develop predictive models for bioavailability and toxicology. Ultimately 

this approach will greatly increase the cost effectiveness and efficiency of drug discovery by reducing 

the aggregate failure rate for development candidates. 

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35. Agrafiotis 

DK, Bone R, 

Jaeger EP, 

Rhind AW, 

Salemme FR, 

Soll RM. 

Directed 

Diversity®: a 

new paradigm 

for drug 

discovery. 

1996; in 

preparation.




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21 

Structure-Based Combinatorial Ligand Design 

Amedeo Caflisch 

University of Zürich, Zürich, Switzerland 

Claus Ehrhardt 

Novartis Pharma Inc., *Basel, Switzerland 


Page 541 

Structure-based ligand design is fascinating and challenging. Whenever it is possible to determine the 

three-dimensional structure of a pharmacologically relevant enzyme or receptor, computational 

approaches can be used to design novel high-affinity ligands. These methods can complement the broad 

screening efforts, which represent traditional lead discovery. 

In this chapter we focus on our approach to computer-aided ligand design. It is based on the docking of a 

diverse set of molecular fragments into the active site of a macromolecular target and on the use of a 

combinatorial strategy to connect them to form candidate ligands. The methodology is illustrated by an 

application to human thrombin, a trypsin-like serine protease fulfilling a central role in both hemostasis 

and thrombosis. The selective inhibition of thrombin is expected to prevent thrombotic diseases. 

Ligand-design programs are being developed at an ever increasing rate and some are related to various 

aspects of our ligand design approach. The LEGO software tool is based on the combination of multiple 

fragment docking, automatic connection by small linker units (one to four atom chains), and searching 

of three-dimensional databases for complementary molecules [1,2]. It has been implemented within the 

MOLOC molecular modeling system [3], which allows the visualization of the functionality maps and 

interactive model building of the growing ligands. Another related approach is that embodied in the 

program LUDI [4–8]. It makes use of statistical data from small-molecule 

* Formerly Sandoz Pharma Ltd. 


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Page 542 

crystal structures to determine binding sites of molecular fragments, i.e., discrete positions on the 

binding site surface suitable to form hydrogen bonds and/or to fill hydrophobic sites of the receptor. 

Alternatively, it uses simple rules or the output of the program GRID [9–12] to generate the interaction 

sites. Finally, the fragments fitted in the interaction sites are connected by linker groups. Other fragmentbased 

programs are GROUPBUILD [13]; GROW [14], HOOK [15], NEWLEAD [16], SPROUT [17], 

and TORSION [18]. These and other strategies for computer-aided structure-based ligand design have 

been reviewed by several contributors [13,19,20]. 

II. Docking Molecular Fragments 

A. Multiple Copy Simultaneous Search 

The present approach for ligand design is based on the combinatorial selection of molecular fragments 

optimally docked on the protein binding site to form a population of diverse candidate ligands. The 

multiple copy simultaneous search (MCSS) procedure combines the advantages of random distribution 

and simultaneous minimization of a set of replicas of a chemical fragment to obtain maps of 

energetically favorable positions and orientations (local energy minima) [21,22]. These maps, which 

contain all possible low-energy minima of a fragment-protein complex, are called functionality maps. A 

plethora of structural and thermodynamic data on inhibitor-enzyme complexes [23–26] suggest that the 

burial of nonpolar groups of the ligand in hydrophobic pockets of the protein is important for binding 

affinity and that intermolecular electrostatic interactions determine selectivity. For this reason, and 

because most of the known enzymes' binding sites have both hydrophilic and hydrophobic character, 

very diverse functional groups are used in MCSS. Representative examples include charged (e.g., 

acetate, benzamidine, methylammonium, methylguanidinium, pyrrolidine); polar (e.g., methanol, 2propanone, 

N-methylacetamide); aromatic (e.g., benzene, pyrrole, imidazole, phenol); and aliphatic 

(e.g., propane, isobutane, cyclopentane, cyclohexane) groups. Although most of these fragments are 

rigid, MCSS can also generate the functionality maps of flexible medium-size fragments, e.g., the amino 

acid side chains. Additional functional groups and more complex heterocyclic systems are currently 

being introduced to increase the diversity of the resulting ligands and to better characterize the 

specificity of the binding pockets (A. Caflisch and C. Ehrhardt, unpublished results). 

As shown by a flowchart in Figure 1, the method is fully automated, although certain critical parameters 

(e.g., number of replicas, radius of the sphere for random distribution, CHARMM parameters for the 

minimization) can be adjusted by the user to optimize it for specific applications. Several thousand 

replicas of a given group are randomly distributed inside a sphere 


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Figure 1 

Schematic representation of the MCSS procedure. Conditional 

statements are enclosed by diamonds. 

Page 543 

whose radius is chosen large enough to cover the entire region of interest. This can be a known binding 

site or the entire protein, if one wants to explore alternative binding pockets. The initial random 

distribution also can be performed inside a parallelepiped if the region of interest is elongated in one or 

two directions. A minimal distance can be given as input to avoid bad contacts between functional group 

atoms and protein atoms for the initial distribution. 

Subsets of between 500 and 3000 randomly distributed replicas of the same group are simultaneously 

minimized in the force field of the protein. The 


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Page 544 

interactions between the group replicas are omitted. The polar-hydrogen approximation (PARAM19) of 

the CHARMM force field is used [27]. In the application of the MCSS method to the sialic acid binding 

site of the influenza coat protein hemagglutinin [21], HIV-1 aspartic proteinase [22], and thrombin 

[20,28] the protein was kept fixed; hence, the forces on each replica consist of its internal forces and 

those due to the protein, which has unique conformation and, therefore, generates a unique field. The 

positions are compared every 1000 steps to eliminate replicas converging toward a common minimum. 

Further details concerning the methodology are given in References 21 and 22, while a critical 

assessment has been presented in Reference 20. 

B. Simple Approximations of Solvation Effects 

In previous applications of MCSS [21,22] the effects of the solvent were neglected, i.e., all proteinfragment 

interactions were calculated with a vacuum potential [27]. This choice was based on the 

principle that fast methods are necessary to perform effective searches of the binding site and that good 

candidate ligands subsequently can be ranked in terms of their binding free energy [20,28]. A possible 

difficulty with this approach is that minimized positions may be missed or misplaced due to the lack of a 

solvation correction during the MCSS minimization. 

Electrostatic Shielding 

In MCSS studies of thrombin [20,28], it was observed that minima of charged groups tend to cluster in 

the vicinity of charged side chains on the thrombin surface and in the S1 (basic groups) or S1' pocket 

(acidic groups). It is then necessary to estimate the electrostatic desolvation of both protein and fragment 

to obtain a realistic ranking of the minima [28]. As a simple test of the importance of electrostatic 

shielding, a distance-dependent dielectric function [29] was introduced instead of the unit dielectric 

constant in the vacuum potential. The overall shape of the acetate map did not change, but three more 

minima were found close to the Lys60F side chain in S1' [20]. It is difficult to find a physical meaning 

in favor of the distance-dependent dielectric function. Nevertheless, it is a simple and useful 

approximation, since it yields a smoother and more realistic potential surface than the vacuum 

Coulombic interaction. 

Hydrophobic Effect 

Aliphatic and aromatic fragments do not bear any partial charges in the polar-hydrogen approximation. 

Hence, MCSS determines their optimal position in the 


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Page 545 

protein binding site exclusively by van der Waals interactions. Minima of nonpolar fragments may be 

found in hydrophilic pockets because of the lack of an energy penalty for protein desolvation. A 

representative example of a cluster of propane minima in a mainly hydrophilic region of the HIV-1 

aspartic proteinase binding site is shown in Reference 20. In a simple attempt to approximate 

desolvation of polar regions of the protein, the attractive contribution of the van der Waals interaction 

energy was switched off between atoms of nonpolar MCSS fragments and all protein polar hydrogen, 

nitrogen, oxygen, and sp 2 carbon atoms. In addition, the van der Waals radius of nitrogen and oxygen 

atoms was increased from the PARAM19 default value of 1.6 Å to 2.2 Å and the van der Waals radius 

of the aliphatic carbons was reduced by 0.1 Å to avoid the too large van der Waals distance between 

carbons often produced by PARAM19. The modified force field yields thrombin functionality maps of 

propane, cyclopentane, cyclohexane, and benzene in agreement with structural data of known inhibitors 

(see Section II. C). In addition, these nonpolar groups are prevented from occupying hydrophilic 

pockets. 

As a further test of the modified force field, the MCSS procedure was used to generate the propane 

functionality map on the surface of the A peptide chain of the leucine zipper, i.e., residues 249–281 from 

the yeast transcriptional activator protein GCN4. Leucine zippers are composed of amphipathic α 

helices containing heptad repeats (abcdefg) in which hydrophobic residues are frequent at a and d. As 

the x-ray structure indicates [30,31], the two amphipathic α helices are held together by hydrophobic 

interactions between residues in the a and d positions (Figure 2). The B peptide chain was removed and 

MCSS was run separately with the original and the modified force field starting from the same random 

distribution of 5000 propane replicas around helix A. The sixty most favorable minima obtained with the 

modified force field are distributed in twelve clusters, seven of which match the Val and Leu side chains 

of the B helix involved in the interhelical interactions (Figure 2). Of the remaining five clusters, labeled 

A to E in Figure 2, B has minima in contact with Val and Ala side chains, D with Leu and Tyr side 

chains, while A, C, and E with both a Val or Leu side chain and the alkyl part of Lys side chain. The 

sixty most favorable minima obtained with the original force field form sixteen clusters (not shown). 

Only four of these match Val and Leu side chains of the B helix, while eight clusters desolvate one (or 

more) polar group on the hydrophilic (exposed) surface of the A helix. 

The functionality maps of polar groups generated with a distance-dependent dielectric and those of 

nonpolar groups obtained with the modified force field are closer to those obtained by using a 

continuum dielectric model [32–35] to postprocess the MCSS minima and estimate solvation effects 

[28]. Hence, simple modifications of the force field may result in more accurate projections of the 

binding free energy. Since these modifications are used during the 


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Figure 2 

Stereo view of the sixty propane minima (thick lines) obtained with the modified force 

field (see text) on the surface of the A peptide chain (medium lines) of the GCN4 leucine 

zipper (PDB code 2ZTA). Although the B peptide chain was removed during the MCSS 

procedure, its backbone and hydrophobic side chains are also drawn (thin lines) to show 

how the propane minima match the aliphatic groups of chain B. Hydrophobic residues 

are labeled at their C α atom.Five clusters of propane minima that do not match the 

hydrophobic side chain of the B helix involved in the interhelical interactions are 

labeled from A (top right) to E (bottom center) and discussed in the text. 


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minimization phase, a more realistic distribution of functional group minima is generated. 

C. Thrombin Functionality Maps 

Page 547 

Human thrombin is one of the best characterized enzymes from a structural point of view (Figure 3). It 

binds a series of diverse inhibitors without major rearrangements in its conformation, as shown by a 

number of x-ray crystallography studies [26,36–39]. Its S3 and S2 precleavage subpockets have 

hydrophobic character, whereas at the bottom of the S1 or recognition pocket the carboxy group of 

Asp189 is a salt bridge partner for basic side chains (Figure 3). D-phenylalanyl-L-prolyl-L-arginine 

chloromethane, PPACK, (Figure 4a), and Nα-((2-naphthylsulfonyl)glycyl)-DL-pamidinophenylalanylpiperidine, 

NAPAP, (Figure 4b) are the archetypal active-site inhibitors of 

thrombin. The crystal structure of the thrombin-NAPAP complex is shown in Figure 3. The PPACK and 

NAPAP inhibitors bind to the thrombin active site by occupying the S3 and S2 pockets with their 

hydrophobic moieties and by positioning their basic group (guanidinium of PPACK, benzamidine of 

NAPAP) into S1 to form a salt bridge with Asp189. 

In continuation of a project aiming at the structure-based design of low molecular weight, active-site 

directed inhibitors of human thrombin [40], MCSS was used to generate a series of functionality maps 

of the thrombin S3 to S2' pockets [20,28]. A detailed description of the thrombin functionality maps and 

the continuum approximation used to postprocess the MCSS minima is given in Reference [28]. From 

the analysis of the results for the nonpolar groups it is evident that hydrophobic moieties prefer to bind 

to the S3 and S2 pockets (Figure 5). The solvent exposed face of the Trp60D indole is another favorable 

site, though the intermolecular van der Waals interactions are much smaller. Binding to the S2' region is 

favored by interactions with the Leu40 side chain but implies a desolvation penalty because of the burial 

of part of the Arg73 guanidinium and/or the Gln151 side chain. The latter might be an artifact of the 

rigid protein structure used in the minimization, since the side chains of Arg73 and Gln151 are flexible 

enough to displace their polar groups towards a more exposed region. Binding to the neighboring Leu41 

side chain in S1' is highly unfavorable because of the concomitant desolvation of Lys60F. 

For polar groups with zero net charge there are several hydrophylic groups on the thrombin main chain 

that might be involved in strong hydrogen 


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Figure 3 

(a) Stereo view of the thrombin molecule in its complex with NAPAP. The side chains of 

thrombin involved in the binding of NAPAP and the disulfide bridges are shown in 

stick-and-ball representation with black sticks. NAPAP is shown in stick-and-ball 

representation with white sticks. (b) Zoom image of the active site region. Figures made 

with the program MOLSCRIPT [42]. 

Page 548 

bonds. These are 214CO, 216NH, 216CO, and 219NH in S1; 193NH and 195NH in the oxyanion hole; 

41CO in S1'; 40CO in S2'; and 147NH and 148NH on the autolysis loop, whose exposure is dependent 

on crystallization conditions and inhibitor type. 

Two main conclusions can be drawn from the analysis of the minimized positions of the charged 

functional groups. First, the minima with the lowest binding free energy have optimal hydrogen bonds 

with the Asp189 side chain in the S1 pocket. Representative examples are the lowest energy minimum 

of benzamidine (Figure 5) and the lowest energy minima of methylguanidinium and methylammonium 

(Figure 4 of Reference 28). Since the Asp189 side chain 


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Figure 4 

Chemical structure of PPACK (a) and NAPAP (b). 

Page 549 

is more buried than the side chain of Lys60F, the minima of positively charged groups interacting with 

the former have a more favorable binding free energy than those of the negatively charged groups close 

to the latter. This is due to reduced shielding of the charge—charge interaction and the smaller 

desolvation of the carboxylate oxygens of Asp189 compared to the amino group in Lys60F [28]. 

Second, polar groups on the protein surface are not ideal partners for a charged functional group because 

the high desolvation penalty for these groups might not be completely compensated for by the favorable 

electrostatic interaction energy. This finding is analogous to the results for the polar functional groups, 

i.e., their binding to a partially exposed charged side chain of the protein may result in an unfavorable 

total binding free energy. 


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Figure 5 

Stereo view of the three lowest energy minima of benzene obtained with the 

modified force field and the lowest energy minimum of benzamidine (thick lines for 

heavy atoms and thin lines for polar hydrogens) in the thrombin active site (thin lines). 

The inhibitor PPACK is also shown (medium lines), though it was removed during the 

MCSS procedure. Some C α atoms of thrombin are labeled. 

III. Connecting Molecular Fragments 

A. Computational Combinatorial Ligand Design 

Overview 

Page 550 

The recently developed program for computational combinatorial ligand design (CCLD) requires as 

input atomic coordinates and partial charges of the protein atoms, as well as the coordinates of the 

MCSS minima and the individual contributions to the free energy of binding [28]. An additional file 

contains a number of control parameters and, for each functional group used for MCSS, a list of atoms 

which can be used for connection (linkage atoms). The following procedures are performed during a 

regular execution of CCLD (Figure 6): The MCSS minima are first sorted according to their 

approximated binding free energies [28]; then a list of bonding fragment pairs and a list of overlapping 

fragment pairs are generated (see below). This is followed by the combinatorial generation of putative 

ligands (see below). 


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Figure 6 

Schematic representation of the CCLD program. Variable assignments are symbolized 

by :=. Conditional statements are enclosed by diamonds. [From Reference 28.] 


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Lists of Bonding Fragment Pairs and Overlapping Fragment Pairs 

Page 552 

The user has to specify for each functional group type which atoms are to be used for connection to 

other fragments. For each linkage atom CCLD generates a set of possible linkage points, i.e., points that 

will be used to determine the position and orientation of the link. All possible pairs of minimized 

positions are then analyzed and added to the list of bonding fragment pairs if they can be linked; 

otherwise, if two fragments have bad contacts they are added to the list of overlapping fragment pairs. A 

pair of bonding fragments may be connected by a linker unit, by a single covalent bond (1-bond), or by 

fusing two overlapping atoms belonging to different fragments (0-bond). The linker units are small since 

their function is to optimally connect two fragments without adding considerably to the molecular 

weight. The following linker elements have been implemented so far: Keto and methylene (2-bond), 

amide and ethylene (3-bond). The user is free to choose minimal and maximal values for the distance (d) 

between linkage atoms for each connection type. In the application to thrombin the following values in 

Ångstroms were used: d

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B. Candidate Ligands of Thrombin 

Page 553 

To test if CCLD is able to reproduce the known thrombin inhibitors, the functional groups of PPACK 

and NAPAP were given as input molecular fragments in the orientation derived from the crystal 

structures of the complexes. The CCLD program generated a set of candidate ligands that not only 

contained the PPACK and NAPAP structures but also a number of interesting hybrid molecules 

consisting of fragments from both inhibitors. A representative example is shown in Figure 7. This 

putative ligand consists of the C-terminal part of NAPAP, whose piperidine ring is connected at the 3position 

to the PPACK D-Phe by an amide linker. The latter has its carbonyl oxygen involved in a 

hydrogen bond with the Gly216 NH. 

In another run, the MCSS minima of benzamidine (Figure 5), benzene (Figure 5), cyclopentane, and 

cyclohexane [28] were used as starting molecular fragments. In a few seconds of CPU time of an SGI 

Indigo2 (R4400 processor), CCLD generated a series of molecules showing the same interaction 

patterns as those of known thrombin inhibitors, i.e., hydrophobic moieties in S3 and S2, hydrogen bonds 

with the polar groups of Gly216, and benzamidine in S1. One of these putative ligands is shown in 

Figures 8 and 9. It is involved in the same interactions as in the NAPAP-thrombin complex except for 

the hydrogen bond with the CO of Gly216. Its cyclohexane ring in S3 is connected to the 

Figure 7 

PPACK-NAPAP hybrid ligand (thick lines) generated by CCLD starting 

from the functional groups of PPACK and NAPAP (thin lines). The amide linker 

connecting the piperidine in 3-position to the D-Phe was created by CCLD. 


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Figure 8 

Minimized structure of a putative ligand suggested by CCLD (thick lines). 

The CCLD run used MCSS minima of benzamidine, benzene, cyclopentane, and 

cyclohexane. The putative ligand was minimized in the thrombin active site, whose residues 

within 8 Å of any atom of the ligand were allowed to move. The remaining residues of 

thrombin were kept rigid. The NAPAP structure is also shown (thin lines) as a basis of 

comparison. 


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Figure 9 

Stereo view of the minimized complex between the putative ligand shown in Figure 8 

and thrombin (thin lines). Intermolecular hydrogen bonds are drawn by dashed lines. 


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Page 555 

N-acylpyrrolidine ring in S2 by a single methylene linker. This is a novel design and the candidate 

ligand appears to be more rigid than NAPAP, since it has a smaller number of rotatable bonds. Hence, 

the penalty paid for the loss in entropy upon binding should be smaller for this CCLD hit than for 

NAPAP. 

In a study with a more diverse set of starting molecular fragments, ligands containing both a “core” 

similar to known inhibitors and additional intermolecular hydrogen bonds and/or van der Waals 

interactions were generated [28]. 

IV. Conclusion 

A combinatorial approach for the computer-aided design of putative ligands of proteins or receptors of 

known three-dimensional structure has been presented. Diversity of these candidate ligands is provided 

by first docking a set of diverse molecular fragments. For aliphatic functional groups a modified force 

field (switching off the attractive part of the van der Waals interaction with polar atoms of the protein) 

was introduced to better approximate solvation effects, thereby avoiding the docking of apolar fragments 

into hydrophilic cavities of the macromolecular target. The second part of the present ligand design 

approach consists of a combinatorial strategy for the connection of optimally docked molecular 

fragments by small linkage elements having optimal interactions with the target molecule. An 

application was presented in which candidate inhibitors of thrombin were found. 

It is important to note that the most difficult part of ligand design is the prediction of binding affinity. A 

method to estimate relative binding constants has recently been applied to a series of similar inhibitors 

of HIV-1 aspartic proteinase [41]. Our own efforts are concentrated on the development of an approach 

that will predict relative binding affinities and will be general enough to be used for any enzyme or 

receptor of known structure (A. Caflisch, D. Arosio, and C. Ehrhardt, work in progress). 

One of the purposes of this chapter was to show that structure-based ligand design is a fascinating and 

progressing research field. It is fascinating not only for its ultimate goal, i.e., the discovery of ethical 

drugs, but also because it is based on, and thereby increases, our understanding of molecular interactions 

and recognition phenomena on an atomic level. Another reason is its multidisciplinary character, which 

requires skills in different branches of science, from theoretical physics and chemistry to computer 

science and statistics. That structure-based computer-aided ligand design is a progressing field is evident 

in many chapters of this book. This is mainly a consequence of the methodological developments and 

the ever-increasing performance of computers. 


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We thank J. Apostolakis and Professor A. Plückthun for helpful discussions. The calculations were 

performed on an SGI Indigo2 and an eight-processor SGI Challenge (R4400 processors). The 

CHARMM program within the version 4.0 of the QUANTA software package (Biosym-MSI Inc) was 

used for some of the minimization performed in this work. The CCLD program is available from A. 

Caflisch. 

This work was supported by the Swiss National Science Foundation (Schweizerischer Nationalfonds 

grant nr. 3100-043423.95) and by Novartis Pharma Inc. 

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22 

Peptidomimetic and Nonpeptide Drug Discovery: Impact of Structure- 

Based Drug Design 

Tomi K. Sawyer 

Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 


Page 559 

Peptidomimetic and nonpeptide drug discovery has evolved to become an extraordinarily intriguing area 

of interdisciplinary research. It challenges synthetic, computational, and biophysical chemists, 

biochemists, pharmacologists, and drug delivery scientists to collaboratively discover lead compounds 

that exhibit sufficient potency, selectivity, metabolic stability, and in vivo pharmacological efficacy to 

warrant further development as drug candidates. Over the past two decades a highly focused effort in 

both industry and academia has advanced the rational transformation of “first generation” peptide lead 

compounds to significantly modified analogs having minimal peptide-like chemical structure [1–18]. 

Such work is typically illustrated by systematic backbone and/or side chain modifications, 

transformation into macrocycles, structure-conformation analysis (x-ray, NMR, CD), and computerassisted 

molecular modeling. These extensive structure-activity and structure-conformation studies have 

enabled the creation of prototype peptidomimetic “second-generation” analogs from initial peptide 

leads. Over recent years the emergence of 3D structural information on target proteins has significantly 

impacted peptidomimetic drug discovery strategies. Particularly noteworthy has been the advancement 

of the de novo design of chemically novel compounds that possess very limited peptide-like 

substructure, but include structure-based functional group modifications which may make new 

intermolecular interactions with the target protein (versus a “first generation” peptide lead compound). 

Furthermore, the integration of such structure-based drug design efforts with high-throughput 


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Page 560 

screening and synthetic chemical library generation is markedly reshaping peptide, peptidomimetic, and 

nonpeptide drug discovery research. In this chapter a few examples of peptidomimetic and nonpeptide 

drug discovery are detailed to highlight the scope of such work relative to a few specific targets (e.g., 

receptors, proteases, and signal transduction proteins) in which structure-based drug design has 

contributed in significant ways. 

A. Peptides: Molecular Diversity and φ-Ψ-χ Space 

Peptides exhibit extraordinary molecular diversity by virtue of their varying primary structures (Figure 

1). For many peptide hormones, neurotransmitters, and releasing factors the substructure of amino acids 

that contribute to molecular recognition (binding) and biological activity (signal transduction) at their 

target receptors have been determined [2]. Such work has led to the generation of pharmacophore 

models of either agonist or antagonist analogs and, in some cases, the design of peptidomimetics. Yet, 

for most peptide growth factors, cytokines, and large-sized (>50 amino acids) peptide hormones, the 

identification of the amino acid substructure which accounts for molecular recognition and signal 

transduction has been a difficult task, and proposals for pharmacophore models remain significant 

challenges. In this regard the term “pharmacophore” is defined as the collection of relevant groups 

(substructure) of a ligand which are arranged in three-dimensional space in a manner complementary to 

the target protein and are responsible for the biological property of the ligand as a result of binding of 

the ligand to its target protein [8b]. 

The three-dimensional structural properties of peptides (Figure 2) are defined in terms of torsion angles 

(Ψ, φ, ω, χ) between the backbone amine nitrogen (Nα), backbone carbonyl carbon (C'), backbone 

methine carbon (Cα), and side chain hydrocarbon functionalization (eg., Cβ, Cγ, Cδ, Cε of Lys) derived 

from the amino acid sequence. A Ramachandran plot (Ψ versus φ) may define the preferred 

combinations of torsion angles for ordered secondary structures (conformations), such as α helix, β turn, 

γ turn, or β sheet. With respect to the amide bond torsion angle (ω) the trans geometry is more 

energetically favored for most typical dipeptide substructures, however, when the C-terminal partner is 

Pro or other N-alkylated (including cyclic) amino acids the cis geometry is possible and may further 

stabilize β-turn or γ-turn conformations. Molecular flexibility is directly related to covalent and/or 

noncovalent bonding interactions within a particular peptide. Even modest chemical modifications by 

Nα-methyl, Cα-methyl or Cβ-methyl can have significant consequences on the resultant conformation 

[6; also, see Phe analogs in Figure 2]. 

The Nα-Cα-C' scaffold may be further transformed by introduction of olefin substitution (e.g., Cα- 

Cβ rarrow.gif C=C or dehydroamino acid [19]) or insertion (e.g., Cα-C' rarrow.gif Cα-C=C-C' or 

vinylogous amino acid [20]). Also the Cβ carbon may be substituted to advance the design of so-called 

“chimeric” amino acids 


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Figure 1 

Examples of native peptide hormones, neurotransmitters, and releasing factors 

Page 561 

[9]. Finally, with respect to N-substituted amides it is also noteworthy to mention the intriguing 

approach [21] of replacing the traditional peptide scaffold by achiral N-substituted glycine building 

blocks. Overall, such Nα-Cα-C scaffold or Cα-Cβ side chain modifications provide significant 

opportunities for expanding peptide-based molecular diversity (i.e., so-called “peptoid” libraries) as well 

as to extend our 3D structural knowledge of traditional φ-Ψ-χ space. 


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Figure 2 

Three-dimensional structural properties of peptides: backbone and side chain 


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B. Peptidomimetic Drugs: Concepts, Strategies, and Technologies 

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Historically, the major focus of peptidomimetic design has evolved from receptor-targeted drug 

discovery research and has not been directly impacted by an experimentally-determined 3D structure of 

the target protein. Nevertheless, a hierarchial approach of peptide rarrow.gif peptidomimetic molecular 

design and chemical modification has evolved over the past two decades, based on systematic 

transformation of a peptide ligand and iterative analysis of the structure-activity and structureconformation 

relationships of “second generation” analogs (Figure 3). Such work has typically 

integrated biophysical techniques (x-ray crystallography and/or NMR spectroscopy) and computerassisted 

molecular modeling with biological testing to advance peptidomimetic drug design. 

A plethora of sophisticated synthetic chemistry approaches have entered into the arena of peptide-based 

molecular design, including well-established applications of unusual amino acids and dipeptide 

surrogates, among other types of chemical modifications. Such backbone or side chain modifications 

may afford stability of the parent peptide to peptidases and have provided conceptual impetus for yet 

more sophisticated molecular design and peptidomimetic chemistry studies [1-18]. For example, a few 

of the known amide bond replacements (Figure 4) include: aminomethylene or Ψ [CH 2NH], 1; 

ketomethylene or Ψ[COCH 2], 2; ethylene or Ψ[CH 2CH 2], 3; olefin or Ψ[CH=CH], 4; ether or 

Figure 3 

Hierarchial approach in peptide rarrow.gif peptidomimetic structure-based drug design 


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Ψ[CH 2O], 5; thioether or Ψ[CH 2S], 6; tetrazole or Ψ[CN 4], 7; thiazole or Ψ[thz], 8; retroamide or 

Ψ[NHCO], 9; thioamide or Ψ[CSNH], 10; and ester or Ψ[CO 2], 11. These amide bond surrogates 

provide insight into the conformational and H-bonding properties that may be requisite for peptide 

molecular recognition and/or biological activity at receptor targets. Furthermore, such backbone 

replacements can impart metabolic stability towards peptidase cleavage relative to the parent peptide. 

The discovery of yet other nonhydrolyzable amide bond isosteres has particularly impacted the design of 

protease inhibitors, and these include: hydroxymethylene or Ψ[CH(OH)], 12; hydroxyethylene or 

Ψ[CH(OH)CH 2] and Ψ[CH 2CH(OH)], 13 and 14, respectively; dihydroxyethylene or 

(Ψ[CH(OH)CH(OH)], 15, hydroxyethylamine or Ψ[CH(OH)CH 2N], 16, dihydroxyethylene 17 and C 2symmetric 

hydroxymethylene 18. In the specific case of aspartyl protease inhibitor design (see below) 

such backbone modifications have been extremely effective, as they may represent transition state 

mimics or bioisosteres of the hypothetical tetrahedral intermediate (e.g., Ψ[C(OH) 2NH] for this class of 

proteolytic enzymes. 

Both peptide backbone and side chain modifications may provide prototypic leads for the design of 

secondary structure mimicry [11, 22–31] as typically suggested by the fact that substitution of D-amino 

acids, Nα-Me-amino acids, Cα-Me-amino acids, and/or dehydroamino acids within a peptide lead may 

induce or stabilize regiospecific β-turn, γ-turn, β-sheet, or α-helix conformations. To date, a variety of 

secondary structure mimetics have been designed and incorporated in peptides or peptidomimetics 

(Figure 5). The β-turn has been of particular interest to the area of receptor-targeted peptidomimetic 

drug discovery. This secondary structural motif exists within a tetrapeptide sequence in which the first 

and fourth Cα atoms are < 7 Å separated, and they are further characterized as to occur in a nonhelical 

region of the peptide sequence and to possess a ten-membered intramolecular H-bond between the i and 

i+4 amino acid residues. On of the initial approaches of significance to the design of β-turn mimetics 

was the monocyclic dipeptide-based template 19 [22] which employs side chain to backbone constraint 

at the i+1 and i+2 sites. Over the past decade a variety of other monocyclic or bicyclic templates have 

been developed as β-turn mimetics, and specific examples include 20 [23], 21 [24], 22 [25], 23 [26] and 

24 [27]. Most recently, the monocyclic β-turn mimetic 25 has been described [28] and illustrates the 

potential opportunity to design scaffolds that may incorporate each of the side chains (i, i+1, i+2 and i+3 

positions), as well as five of the eight NH or C=O functionalities, within the parent tetrapeptide 

sequence. tetrapeptide sequence modeled in type I–IV β-turn conformations. Similarly, the 

benzodiazepine template 26 has shown [29, 30] utility as a β-turn mimetic scaffold which also may be 

multisubstituted to simulate side chain functionalization, particularly at the i and i+3 positions of the 

corresponding tetrapeptide sequence modeled in type I–VI β-turn conformations. A recently reported 

[31] 


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Figure 4 

Backbone amide bond surrogates: Ψ[CONH] replacements 


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Figure 5 

Secondary structure modifications using β-turn α-turn, and β-sheet scaffolds. 

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γ-turn mimetic, 27, illustrates an innovative approach to incorporate a retroamide surrogate between the 

i and i+1 amino acid residues with an ethylene bridge between the N' (i.e., nitrogen replacing the 

carbonyl C') and N atoms of the i and i+2 positions, and this template allows the possibility for all three 

side chains of the parent tripeptide sequence. Finally, the design of a β-sheet mimetic, 28, provides an 

attractive template to constrain the backbone of a peptide to that simulating an extended conformation 

[32]. The β-sheet is of particular interest to the area of protease-targeted peptidomimetic drug discovery. 


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12640-0567a.gif 

Figure 6 

Contemporary drug discovery: integration of structure-based drug design, 

synthetic chemical libraries, and high-throughput mass screening technologies 

Finally, the convergence of structure-based drug design (biophysical and computational chemistry), 

synthetic chemical libraries, and high-throughput screening technologies have established a new 

paradigm for drug discovery (Figure 6). This powerful alliance of scientific disciplines is accelerating 

the identification of lead compounds and/or the optimization of drug candidates. The impact to 

academic, biotechnological and pharmaceutical research will be immense. 

C. Receptor, Protease, and Signal Transduction-Protein Targets 

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Structure-based drug design and peptidomimetic drug discovery has emerged as a powerful approach in 

many areas of pharmaceutical research, including receptor agonists and antagonists, protease inhibitors, 

and, more recently, in the rapidly developing area of signal transduction, in which the protein targets 

include catalytic and noncatalytic domains of a particular intracellular macro 


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molecule. A noncomprehensive listing of such receptor, protease, and signal-transduction protein targets 

is shown in Table 1. 

With respect to receptor-targeted peptidomimetic drug discovery, the most noteworthy success has been 

attained for G-protein-coupled receptor agonists and antagonists as well as, more recently, cell-adhesion 

integrin receptor antagonists (see below). However, it is important to state that the impact of highthroughput 

screening in the discovery of nonpeptide ligands (typically antagonists) at G-protein-coupled 

receptors has yielded extraordinary success. Although screening-based nonpeptide drug discovery will 

not be extensively reviewed here, the possibility of common pharmacophores between peptide and 

nonpeptide ligands may exist (limited cases) in relation to receptor binding. Nevertheless, in most cases 

receptor mutagenesis studies suggest the existence of different binding pockets for peptide and 

nonpeptide ligands, regardless of whether they both are functionally similar as related to agonism or 

antagonism [33]. With respect to both protease-and signal-transduction protein-targeted peptidomimetic 

drug discovery, the emergence of 3D structural information to provide high resolution molecular “maps” 

of the catalytic (or noncatalytic) domain has provided incredible opportunities for structure-based drug 

design. 

II. Peptide Ligand Lead Discovery and Structure-Based Drug Design 

As stated previously, peptidomimetic drug discovery was first advanced by molecular design concepts 

and chemical modification strategies focused on 


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Figure 7 

Convergent pathways in peptide, peptidomimetic, and nonpeptide drug 

discovery 

Page 570 

using receptor-targeted peptide ligands or “second generation” agonist or antagonist analogs to develop 

pharmacophore models (see Figure 3). However, this represents only a part of the sophisticated 

convergent pathways that exist currently to advance both peptidomimetic and nonpeptide drug discovery 

(Figure 7). In this scenario both native and foreign (biological or chemical origin) peptides may provide 

the opportunity for ligand structure-based drug design. From pharmacophore models of key peptide 

leads the iterative transformation to peptidomimetic “second generation” analogs may proceed through 

either peptide scaffold-or nonpeptide template-based approaches. Examples of such work are detailed 

below. 

Independent of the hierarchial approach to peptide ligand structure-based design of peptidomimetics is 

the work that encompasses screening-based nonpeptide lead discovery. Such nonpeptides may be of 

chemical or biological origin (see below), and historically have been identified by targeted or random 

screening approaches. Although they are not designed to be peptidomimetics in the chemical structure 

sense, many appear to be biological mimics (e.g., receptor agonists or antagonists, protease inhibitors, 

signal-transduction protein inhibitors, or antagonists) of a known native or foreign peptide. And, in few 


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cases, a pharmacophore model of a nonpeptide lead (or series) may show similarity to that of a 

pharmacophore model of a peptide ligand. Examples of such work are detailed below. 

A. Peptidomimetics: Receptor Agonists and Antagonists 

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Specific examples that illustrate peptide scaffold-and nonpeptide template-directed drug design 

strategies are shown in Figure 8 and include μ-opioid endorphin (END) agonist, 29 [34]; thyrotropinreleasing 

hormone (TRH) agonist, 30 [13]; fibrinogen (GPIIa/IIIb) antagonists, 31 [35] and 32 [36]; 

CCK A antagonist, 33 [37]; CCK B/gastrin antagonist, 34 [38]; endothelin antagonist, 35 [39]; growth 

hormone secretagogue (GHRP), 36 [40]; somatostatin agonist (partial), 37 [41], substance-P (NK 1) 

antagonists, 38 [42]; neurokinin-A (NK 2) antagonist, 39 [43]; and neurokinin-B (NK 3) antagonist, 40 

[44]. For the most part, the above compounds have been advanced as the result of extensive structureactivity 

studies and, typically, more focused structural studies (NMR) on a conformationally 

constrained, linear or cyclic peptide lead or series. Such structure-conformation activity studies have led 

to the development of pharmacophore models to guide iterative structure-based design strategies. 

One example is that of integrin receptor gpIIb/IIIa antagonists that are structurally derived from the 

tripeptide sequence Arg-Gly-Asp, which is common to gpIIb/IIIa protein ligands such as fibrinogen, 

vitronectin, fibronectin, von Willebrand factor, osteopontin, thrombospondin, and the collagens [45]. As 

shown in Figure 9, transformations of the linear peptide ligand Arg Gly-Asp-Phe by both peptide 

scaffold (at the Arg-Gly backbone) modification and substitution of the Arg side chain by a benzamidine 

moiety provided the peptidomimetic lead 31 that is active in vivo as an antiplatelet agent [35]. On the 

other hand peptidomimetics such as 32 illustrate nonpeptide template-based design strategies derived 

from iterative transformations of a cyclic peptide lead in which a 

γ-turn about the Asp residue was implicated in a pharmacophore model for the bioactive conformation 

[36]. Specifically, the benzodiazepinone substructure of 32 may effectively replace this predicted γ-turn 

conformation about the Asp, and the N-Me-Arg replacement with piperidine moiety was also compatible 

to high-affinity receptor binding. 

A second example is that involving the use of a glucopyranoside nonpeptide template by Hirschmann 

and coworkers [41, 46] for systematic functionalization to create novel peptidomimetics for the 

somatostain (SRIF) and substance-P (NK 1) receptors. As illustrated in Figure 10, the cyclic hexapeptide 

SRIF agonist provided a macrocyclic lead structure that was transformed to a glucopyranoside template 

designed to substitute for a postulated β turn about the Tyr-D-Trp-Lys-Thr substructure of the parent 

peptide ligand. The prototype 


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Figure 8 

Receptor-targeted peptidomimetics exemplifying ligand structure based drug design 


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Figure 9 

Peptide scaffold- and nonpeptide template-based design strategies: 

gpIIb/IIIa antagonists 

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peptidomimetic 37 was found to be a moderately potent SRIF-like agonist (partial) in cellular assays 

[41]. This discovery extends previous studies on TRH (see peptidomimetic 30, Figure 8) which utilized 

a cyclohexane ring system as a nonpeptide template to functionalize with the pyroglutamic acod and His 

side chains as well as the C-terminal carboxamide group of the parent peptide ligand [13]. However, in 

the comparative analysis of analogs of the SRIF-mimetic 37 it was also found that N-acetylation of the 

Lys side chain moiety yielded a potent antagonist of substance-P (NK 1 receptor). This indicated that 

slightly different functionalization of the nonpeptidic glucopyranoside template was quite compatible 

with NK 1 receptor molecular recognition. Intuitively, a “reversed design” strategy to convert the latter 

glucopyranoside-based NK 1 ligand to a cyclic peptide was next investigated (Figure 10), and 

successfully led to the discovery of a novel cyclic peptide ligand also exhibiting potent NK 1 receptor 

binding and antagonism [46]. 

The above examples of peptide scaffold- or nonpeptide template-based peptidomimetic agonists or 

antagonists illustrate various strategies to elaborate bioactive conformation and/or pharmacophore 

models of peptide ligands at their receptors. In many cases, receptor subtype selectivity has also been 

achieved by systematic structural modifications of prototypic leads of peptidomimetics. Thus, although 

the 3D structures of G-protein-coupled receptors (GPCRs) remain as elusive (except for models 

constructed from homology-based low- 


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Figure 10 

Peptide scaffold- and nonpeptide template-based design strategies: somatostatin 

agonist and substance-P (NK 1) antagonists 

resolution 3D structures of bacteriorhodopsin or rhodopsin, see below) the development of 

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pharmacophore models using the hierarchial approach in peptide rarrow.gif peptidomimetic structurebased 

drug design (see above, Figure 3) remains quite promising to creative science. Indeed, targetedreceptor 

screening of synthetic chemical libraries of highly modified peptide molecules (e.g., Nsubstituted 

Gly “peptoids” [21] is rapidly expanding our database of structurally diverse ligands. Such 

structurally unique lead compounds will provide the opportunity for further pharmacophore modeling 

strategies to be used in the discovery of novel agonists or antagonists at GPCR and other receptor types. 

B. Peptidomimetics: Protease Inhibitors 

Specific examples of peptidomimetics which illustrate peptide scaffold- and nonpeptide templatedirected 

drug-design strategies as applied to protease in- 


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Figure 11 

Protease-targeted peptidomimetics derived by ligand structure-based drug 

design 


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hibitors (Figure 11) include renin inhibitors, 41 [47] and 42 [48]; HIV protease inhibitors 43 [49] and 44 

[50]; angiotensin-converting enzyme inhibitors 45 [51] and 46 [52]; collagenase inhibitor, 47 [53]; 

gelatinase inhibitor, 48 [54], stromelysin inhibitor, 49 [55]; elastase inhibitor, 50 [56]; thrombin 

inhibitors, 51 [57] and 52 [58]; and interleukin-converting enzyme inhibitor, 53 [59]. In general, the 

design of protease inhibitors has focused on both the natural substrate structure and the mechanism of 

substrate cleavage to provide “first-generation” inhibitors. Also, these initial leads are typically peptide 

scaffold-based to provide the possibility for β-sheet conformation, which may permit “extensive” Hbonding 

between the backbone amide groups of the inhibitor and complementary H-bond donor or 

acceptor groups of the enzyme active site. Furthermore, the traditional approach to designing protease 

inhibitors includes the substitution of nonhydrolyzable amide surrogates (see below Figure 4) at the P1- P1' cleavage site. Specificity to a particular protease may sometimes be extrapolated directly from the 

primary structure of the substrate (e.g., human renin substrate specificity is conferred from the 

angiotensinogen N-terminal octapeptide sequence ~His6-Pro-Phe-His-Leu Val-Ile-His13~ in which 

refers to the cleavage site). In many cases substitution of the scissile amide (substrate) by a “transition 

state” bioisosteres or an electrophilic ketomethylene moiety have provided tight-binding “first 

generation” pseudopeptide inhibitor leads. 

One example of protease inhibitor design that illustrates the peptide scaffold-based approach is that for 

HIV protease inhibitors. Albeit over the past several years HIV protease inhibitor research has become a 

highly advanced example of iterative structure-based drug design (see below), the first discoveries of 

pseudopeptide and peptidomimetic inhibitors of this aspartyl protease were not made with knowledge of 

the 3D structure of the target enzyme. Specifically, the natural product pseudopeptide pepstatin (Figure 

12), a typical inhibitor of the aspartyl protease family of enzymes, was determined to be weakly potent 

against HIV-1 protease [60]. Relative to pepstatin, the central P 1-P 1' statine (i.e., Sta or 

LeuΨ[CH(OH)]Gly) moiety was further evaluated within the context of an “optimized” N- and Cterminal 

amino acid sequence using a chemical-library strategy [61]. As shown in Figure 12, a resultant 

pseudo-tetrapeptide Ac-Trp-Val-Sta-D-Leu-NH 2 was found to be a relatively potent HIV protease 

inhibitor. In another approach, a designed renin inhibitor (55; Figure 12) was determined to be a highly 

potent HIV protease inhibitor [62]. Further optimization studies led to the discovery [49] of the first 

bonafide peptidomimetic inhibitor of HIV protease (Tba-ChaΨ[CH(OH)CH 2] Val-Ile-Amp; 43) of HIV 

protease. This compound (43) provided the first evidence of cellular anti-HIV activity and, therefore, 

supported proof-of-concept studies related to the therapeutic significance of targeting HIV-1 protease. 

Replacement of the peptide scaffold by the pyrrolidinone-type β-sheet mimetic 28 in a 


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Figure 12 

Peptide scaffold-based design strategies: HIV protease inhibitors 


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chemically-related P 1-P 1' PheΨ[CH(OH)CH 2] Phe-modified lead has been reported [63] to yield 

effective peptidomimetic inhibitors of the HIV-1 protease (56; Figure 12). The pyrrolidinone-type lead 

has shown enhanced cellular permeability relative to its peptide backbone-type counterparts. In a third 

approach guided by HIV substrate-based design, the cleavage site dipeptide Phe-Pro was substituted by 

the “transition state” bioisostere to provide the highly potent and selective HIV protease inhibitor 57, a 

P 1-P 1' PheΨ[CH(OH)CH 2N] Pro-modified heptapeptide [64]. As compared to this pseudopeptide, a 

pioneering effort focused on peptide ligand structure-based design provided a second series of highly 

potent, selective, and cellularly-active HIV protease inhibitors [50] as represented by the recently FDAapproved 

anti-HIV drug 44 (Saquinavir). The design of still more HIV protease inhibitors having novel 

chemical structures (e.g., C 2-symmetric scaffolding, P 1-P 1' “transition state” bioisostere cyclization, and 

achiral nonpeptide template replacement) has progressed at an extraordinary pace (for reviews see 

Reference 65), and in a majority of cases such work has been strongly impacted by knowledge of the 3D 

structure of the target enzyme and/or inhibitor complexes of it (see below). 

A second example of protease inhibitor design that properly illustrates the peptide scaffold-based 

approach is that of thrombin inhibitors. Work with these compounds led to the identification of highly 

potent, selective, and in vivo-effective lead compounds. A member of the serine protease family, 

thrombin cleaves a number of substrates (e.g., fibrinogen) and activates its platelet receptor (a G-proteincoupled 

receptor) by proteolysis of the extracellular N-terminal domain which results in self-activation 

(for a review see Reference 66). Initial lead inhibitors of thrombin were substrate-based, including the 

fibrinogen P 3-P 1 Phe-Pro-Arg sequence [67] and simple Arg derivatives such as Tos-Arg-OMe [68]. 

Also, the natural product cyclothreonide-A, a macrocyclic peptide containing a Pro-Arg ketoamide 

sequence, provided an inhibitory peptide ligand lead [69]. As shown collectively in Figure 13, 

compounds 52 and 58–60 provided the opportunity to try different strategies to advance the design of 

thrombin inhibitors. Particularly noteworthy from these early peptidomimetic lead discovery studies was 

the design effort [70] that led to the highly potent thrombin inhibitor 52 (Agatroban), a sulfonamidemodified 

Arg derivative, which incorporated an unusual cyclic amino acid substituent C-terminal to the 

P 1 moiety as opposed to reactive electrophilic groups (e.g., ketone, aldehyde, or boronic acid). 

Interestingly, replacement of the Arg side chain moiety within a structurally similar analog 60 by a 

amidinobenzyl group was shown [71] to be optimal when the stereochemistry at the P 1 α-carbon has a Dconfiguration 

suggesting that the mode of binding may be different for 52 versus 60. In this regard, xray 

crystallographic analysis of thrombin-inhibitor complexes (see below) have provided insight in the 

interpretation of the structure-activity relationships of the aforementioned lead compounds. 


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Figure 13 

Peptide scaffold-based design strategies: thrombin and tripeptidyl peptidase II 

inhibitors 


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The recent discovery of peptidomimetic inhibitors of the serine protease TTP-II (tripeptidyl peptidase-II) 

further illustrates the peptide scaffold-based design approach [72]. Specifically, relative to a known TTP- 

II cleavage site on the endogenous neuropeptide CCK-8 (i.e., Asp 1-Tyr[SO 3H]-Met arrowd.gif Gly-Trp- 

Met-Asp-Phe 8-NH 2) the design of a highly potent inhibitor 61 (Figure 13) was successfully achieved by 

iterative structure-based optimization of the P3-P1 sequence. Noteworthy of the relatively simple 

structure of the TTP-II inhibitor 61 was that it contains no functional group C-terminal to the P 1 αcarbon. 

Such absence of an electrophilic moiety, “transition state” bioisostere, or other type of 

nonhydrolyzable amide substituent is rather unique relative to most examples of substrate-based 

protease inhibitors. 

C. Peptidomimetics: Signal-Transduction Protein Inhibitors and Antagonists 

Beyond receptors and proteases exists the rapidly emerging area of signal-transduction protein-targeted 

drug discovery research. To date, a multitude of catalytic and noncatalytic proteins have been identified 

which are critical components of intracellular signal-transduction pathways. These signal-transduction 

proteins provide the molecular basis for communication from extracellular “effectors” (e.g., hormones, 

neurotransmitters, growth factors, and cytokines) to stimulate cells in specific and regulated manner. 

Signal-transduction pathways often involve protein-protein interactions, including examples of enzymesubstrate 

(e.g., kinases, phosphatases, transferases, and isomerases) as well nonenzymatic complex 

formation (e.g., “adapter” proteins, exchange factors, and transcription factors). As compared to receptor- 

or protease-targeted peptidomimetic drug discovery, there are significantly fewer examples reported in 

the field of signal-transduction research. Thus, in several cases peptide ligands (as “prototype 

peptidomimetic leads”) will be described to illustrate opportunities for structure-based drug design. 

Specific examples which illustrate peptide scaffold- and nonpeptide template-directed drug-design 

strategies are shown in Figure 14 and include: Ras farnesyl transferase inhibitors, 62 [73], 63 [74], 64 

[75], and 65 [76], Src SH2 domain antagonists, 66 [77], 67 [78], and 68 [79]; and the protein tyrosine 

phosphatase PTP1B inhibitor 69 [80]. 

An example of the signal-transduction protein-targeted inhibitor design which illustrates both peptide 

scaffold- and nonpeptide template-based approaches is that for the Ras farnesyl transferase inhibitor 

discovery. Such compounds show potential as new therapeutic agents for Ras-related carcinogenesis 

[81]. Substrate sequences for farnesyl transferase have the consensus ~Cys-AA 1-AA 2-Met motif (AA 

refers to Val or Ile). Both substrate-based 


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Figure 14 

Signal-transduction protein-targeted peptidomimetics derived by structure-based drug design 


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inhibitors [73–75,82,83] and, more recently, a novel non-Cys containing peptide inhibitors [76,84] have 

led to potent and cellularly active compounds. As illustrated in Figure 15, the “collected” substratebased 

inhibitor 70 was designed to covalently attach farnesyl to a peptide via a phosphinic acid linker 

replacement for [82], and this compound has been determined to be both potent against the target 

enzyme and cellularly effective. Relative to peptide substrate structure-based design efforts, 

peptidomimetics incorporating Ψ[CH 2NH]-substitutions (e.g., 62, [73]) or a benzodiazepinone 

replacement of the central dipeptide moiety (e.g., 63, [74]) have yielded high affinity inhibitors. Another 

series of very potent Ras farnesyl transferase inhibitors have been designed in which the central 

dipeptide has been substituted by various isomeric and/or homologated derivatives of amino benzoic 

acid (e.g., 64 [75]), including a particularly effective analog biphenyl derivative 71 [83]. The above 

studies indicated that both conformationally flexible or constrained peptide scaffolds as well as 

nonpeptide template replacements can be used to “link” the Cys and Met substructures. It is also 

important to point out that although compounds such as 62–64 have “free” sulfhydryl groups (Cys) there 

is no evidence that they become farnesylated, and therefore the binding mode and effect on catalytic 

function of the target enzyme are unique relative to their peptide substrate counterparts. Recently, a 

novel peptide inhibitor series, as exemplified by Cbz-His-Try(O-benzyl)-Ser(O-benzyl)-Trp-D-Ala-NH 2, 

has been discovered [76]. These inhibitors do not contain a Cys residue and structure-based design 

efforts have successfully led to a series of peptidomimetics (e.g., 65) having only one chiral center. 

Interestingly, this novel inhibitor series has been determined to competitively inhibit farnesyl 

pyrophosphate binding rather than the binding of peptide substrate to the target enzyme. Another Hissubstituted 

peptidomimetic inhibitor of Ras farnesyl transferase has been recently reported [84] as 

exemplified by 73, which was designed relative to a peptide substrate-based parent analog (72, Figure 

15). Although the 3D structure of Ras farnesyl transferase is not known, biochemical studies suggest 

that a divalent metal ion (e.g., Zn 2+) may coordinate with the above inhibitor sulfhydryl or imidazole 

groups at their corresponding binding sites on the target enzyme. 

Another example of the signal-transduction protein-targeted drug design that illustrates peptide scaffoldbased 

approaches is that for Src SH2 domain antagonist discovery. Such compounds show promise as 

new therapeutic agents for Src-related carcinogenesis, osteoporosis, and immune diseases [85]. The Src 

SH2 domain is a prototype example of a superfamily of intracellular signal-transduction proteins that 

possess structurally homologous SH2 domains that specifically recognize cognate phosphoproteins in a 

sequence-dependent manner relative to a critical phosphotyrosine (pTyr) residue (i.e., ~pTyr-AA 1-AA 2- 

AA 3-AA 4~ Furthermore, recent x-ray crystallographic studies of a several SH2 protein targets (e.g., 

phosphopeptide complexes) is greatly impacting the 


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Figure 15 

Peptide scaffold- and nonpeptide template-based design strategies: farnesyl transferase 

inhibitors 


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opportunity for iterative structure-based drug design in this field or research (see below [86]). Relative 

to Src SH2 domain antagonist lead discovery, peptide library studies [87] have shown the ~pTyr-Glu- 

Glu-Ile~ as a preferred consensus sequence. Peptide scaffold-based approaches to replace the internal 

dipeptide, Glu-Glu, by both flexible and rigid linkers have been explored [88] but were unsuccessful in 

yielding potent analogs. As shown in Figure 16, prototype peptidomimetics 66 [77] and 67 [78] illustrate 

a successful approach in which stereoinversion at the second residue (P+2 relative to the pTyr) to the Dconfiguration 

and side chain substitution to hydrophobic functionalities (e.g., cyclohexyl and naphthyl) 

which provides accessibility to the known hydrophobic binding pocket for the P+3 Ile side chain. 

Indeed, such compounds showed binding affinities essentially identical to that of the N- and Cterminally 

extended phosphopeptides containing the pTyr-Glu-Glu-Ile sequence. Substitution of the 

pTyr residue of 66 by the difluoromethyl-phosphonate modified analog F 2Pmp provides a more 

metabolically stable derivative 74 (Figure 16) and a prototype lead to advance the design of cellularly 

active second-generation compounds. More recently, structure-based drug design studies of compound 

65 have led to the discovery of potent and Src SH2-selective peptidomimetic lead compounds [89], and 

this is further detailed below as related x-ray crystallographic structures of Src SH2-phosphopeptide 

complexes. 

III. Nonpeptide Ligand Lead Discovery and Structure-Based Drug Design 

As previously illustrated in Figure 7, the convergent pathways to design drugs which act as mimics 

(agonists), antagonists, or inhibitors of native peptide (protein) ligands at their target receptors, 

proteases, signal-transduction proteins, and so forth, include “foreign” nonpeptides. The origin of such 

nonpeptides may be either biological (e.g., natural product) or chemical (synthetic compound collection 

or libraries) that have been subject to biochemical screening to identify leads for further molecular 

design and structure-activity studies. Over recent years the success of screening-derived nonpeptide lead 

discovery and iterative transformation to drug candidates has been quite impressive, and many aspects 

of this area of research have been reviewed [12, 15]. Nevertheless, it is intriguing to explore the 

potential relationship between peptide ligands (including peptidomimetic derivatives) and such 

screening-derived nonpeptide ligands as related to pharmacophore modeling and structure-based drugdesign 

studies. 


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Figure 16 

Peptide scaffold-based design strategies: Src SH2 domain antagonists 


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A. Molecular Diversity and Screening-Based Identification of Nonpeptide Ligands 

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Precedence for the success of nonpeptide drug discovery can be traced to the identification of morphine 

(75; Figure 17) as nonpeptide natural product agonist at μ-opioid peptide receptors [90]. To date, the 

robust momentum of nonpeptide drug discovery continues to be accelerated by sophisticated 

biochemical screening technologies. The scope of molecular diversity as well as therapeutic targets for 

such screening-based nonpeptide ligand lead compounds includes the following examples (Figure 17): 

substance P (NK 1 antagonist, 76 [91]; angiotensin AT 1 antagonist, 77 [92]; growth hormone-releasing 

peptide (GHRP) agonist, 78 [93]; cholecystokinin CCK A antagonist, 79 [94]; CCK B/gastrin antagonist, 

80 [95]; CCK A agonist, 81 [96]; endothelin antagonist, 82 [97]; gonadotropin-releasing hormone 

(GnRH) antagonist, 83 [98]; vasopressin V 1 antagonist, 84 [99]; gastrin-releasing peptide antagonist, 85 

[100]; glucagon antagonist, 86 [101], neurotensin antagonist, 87 [102]; angiotensin AT 1 agonist, 88 

[103]; oxytocin antagonist, 89 [104]; and HIV protease inhibitor, 90 [105]. A significant number of 

screening-derived nonpeptide leads have been identified for G-protein-coupled receptors (GPCRs), and 

in a majority of cases these compounds have been determined to be competitive antagonists. Albeit the 

3D structures of this receptor superfamily have not been directly determined, homology model-building 

and site-directed mutagenesis studies are impacting structure-activity analysis of agonist and antagonist 

ligands (peptide, peptidomimetic, and nonpeptide) for several GPCR targets (see below). 

B. Nonpeptides: Exploring Pharmacophore Relationships to Peptide Ligands 

Relative to a number of GPCR targets, there exists significant opportunity to compare chemical 

structures and 3D pharmacophore models of both peptide and nonpeptide ligands. Such comparative 

analyses can explore the possibility of similar 3D substructural elements that may account for their 

molecular recognition at the binding site(s) of the receptor. The fact that a vast number of screeningderived 

nonpeptide leads are multifunctionalized 5- to 7-membered ring heterocycles (e.g., alkaloid and 

benzodiazepine) and contain conformationally rigid substructural elements (e.g., biphenyl, spiro-bicyclic 

rings, and N-substituted amide or amine linkages) suggests the likelihood that such compounds are 

binding with highly favorable entropic driving forces as compared to the more conformationally flexible 

peptide ligands. In this regard, efforts to “rigidify” peptide-based scaffolding or replace it by nonpeptide 

templates has been the underlying theme of peptidomimetic design strategies, and concepts for the latter 

approach date back to the proposed used of cycloaliphatic ring systems that might be 

multifunctionalized to create “topographi- 


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Figure 17 

Nonpeptide drug discovery: examples from screening-based approaches 


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cally-designed” peptidomimetics (or, as also defined, “peptoids” [106]). Nevertheless, screening-based 

nonpeptide drug discovery has advanced a treasure of structure-function information to provide insight 

into both structure-based design and molecular recognition [12,15,107]. In a few (limited) cases, there 

exists a likely possibility of similar pharmacophoric features or substructural elements between 

nonpeptides and their peptide-ligand counterparts (Figure 18). 

Historically, drug discovery research on opioid GPCR receptor targets (e.g., μ, δ, κ) has provided insight 

to explore the pharmacophores of both agonist and antagonists derived from endogenous peptides (e.g., 

endorphin, endorphin, and dynorphin) versus nonpeptides (e.g., the μ-receptor selective agonist 

morphine and its N-allyl-substituted antagonist derivative naloxone). Relative to the N-terminal Tyr 

moiety (side chain and α-amino functionalities) of the endogenous opioid peptides [108], the N-methyltyramine 

substructure of morphine represents a likely common pharmacophore for agonist ligand 

binding to the μ-receptor (Figure 18). In the case of angiotensin II receptor antagonist drug discovery, it 

has been proposed [109] that a common pharmacophore may exist relative to the C-terminal His-Pro- 

Phe-OH sequence of angiotensin II and nonpeptide 91 (Figure 18). In fact, these studies provided design 

insight leading to the discovery of the drug candidate 77 (Lorsartan). A third example in which 

correlation between peptide and nonpeptide pharmacophore models becomes apparent is that of 

neuropeptide-Y (NPY) versus the benextramine-based derivative 92 [110] or arpromidine-based 

derivative 93 [111] as illustrated in Figure 18. In both cases, the C-terminal Arg-Gln-Arg-Tyr-NH 2 

sequence of NPY was modeled relative to the nonpeptide structures such that the guanido functionalities 

were superimposed upon the corresponding basic (i.e., guanido or imidazole) substructural elements of 

either 92 or 93. 

In some cases, the availability of x-ray crystallographic information of both the peptide and nonpeptide 

ligands may provide insight into the pharmacophore modeling studies. An example of this exists for 

oxytocin antagonist structure-based drug design studies [112]. As shown in Figure 19, pharmacophore 

models of both a cyclic hexapeptide oxytocin antagonists and conformationally-constrained, 

tolylpiperazine camphorsulfonamide nonpeptide antagonist (89) suggest the likelihood of common 

substructural elements that were key for molecular recognition at the oxytocin receptor, and led to the 

design of a highly potent derivative 94. Nevertheless, such comparative pharmacophore “mapping” 

studies are very simplistic since the 3D structures of the target receptors are not known. Furthermore, 

site-directed mutagenesis studies 


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Figure 18 

Comparative substructural elements of peptide and nonpeptide ligands: μ-opioid receptor 

agonists, angiotensin, and NPY receptor antagonists 


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Figure 19 

Comparative substructural elements of peptide and nonpeptide ligands: examples 

of oxytocin receptor antagonists and HIV protease inhibitors 


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often suggest that peptide and nonpeptide ligands have different modes of binding to their receptors (see 

below). It is also important to point out that the discovery of nonpeptide agonists will likely provide 

important structural information to advance our understanding of ligand binding and activation of 

receptors as well as insight for pharmacophore modeling. In this regard, nonpeptide agonists 78 (growth 

hormone-releasing peptide receptor) and 81 (cholecystokinin receptor CCK A subtype) are noteworthy 

exceptions to the rule that nonpeptide screening-based leads are antagonists (see above, Figure 17). 

Finally, beyond receptor targets the advantages of high-throughput screening of chemical files, natural 

products, and synthetic libraries are increasing for proteases as well as other enzyme and noncatalytic 

targets. An recent example of such efforts is the nonpeptide HIV protease inhibitor 90 [105], which was 

originally identified from screening a chemical file. Through iterative structure-based drug design 

studies, including x-ray crystallographic analysis of both ligand and inhibitor-enzyme complexes, the 

pyrone template has led to the discovery of highly potent, selective, and cellularly active lead 

compounds (see below). In retrospect, the original concept of superimposing the key substructural 

elements of the nonpeptide ligand 90 to a known peptidomimetic inhibitor of HIV protease is illustrated 

in Figure 19. A more detailed account of this effort and successful elaboration of the nonpeptide lead 

structure is described below. 

IV. Protein Target 3D Structural Models and Structure-Based Drug Design 

A significant impact in both peptidomimetic and nonpeptide drug discovery has emerged over recent 

years as the result of the determination of the 3D structures of protein targets by x-ray crystallography or 

NMR spectroscopy [113–119]. In addition, computational methodologies such as QSAR, 3D 

QSAR/CoMFA, homology-modeling, ligand docking, molecular dynamics, and mechanics, and solventaccessible 

surface visualization have greatly impacted such research. Furthermore, programs such as 

GRID, GROW, GrowMol, LEGEND, BUILDER, LUDI, FOUNDATION-SPLICE, and CONCERTS 

have enabled 2D and 3D database searching and de novo design [115,120,121]. Overall, the iterative 

cycle of structure-based drug design (Figure 20) has evolved to be an “engine of invention” for several 

examples of peptidomimetic and nonpeptide drug discovery. 


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A. Receptor Targets 

Figure 20 

Iterative cycle(s) for structure-based drug design 

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Amongst the known superfamilies of cell membrane anchored receptors, significant research has been 

focused on GPCR targets (Table 1). The GPCR superfamily of receptors consist of seven 

transmembrane-spanning (TM) helices. Sequence homology among them varies from 25–70% (for 

reviews see References 33, 122–132). The initial development of 3D structural models of GPCR targets 

has been developed from homology-building methodologies based on a low-resolution structure of 

bacteriorhodopsin [133]. Representative examples of recent studies that provide insight to 

pharmacophore modeling and structure-based drug design of peptide, peptidomimetic, and nonpeptide 

ligands are discussed below. 

3 G-Protein-Coupled Receptors 

Recent studies have explored several GPCR targets with respect to ligand-receptor binding interactions 

using site-directed mutagenesis to explore agonist 


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versus antagonist ligands, and such work provides insight to pharmacophore modeling. Specific 

examples of such work as focused on peptide, peptidomimetic, and/or nonpeptide ligands, and working 

3D structural models of their GPCR targets include angiotensin II AT 1 and AT 2 subtypes [134], 

neurokinin NK 1 and NK 2 subtypes [135], cholecystokinin/gastrin CCK A and CCK B subtypes [136], 

opioid μ-, δ-, and κ-subtypes [137], vasopressin V 1A subtype [138], bradykinin B 2 subtype [139], 

neurotensin [140] and α-melanotropin MC 1 subtype [141]. From such work it has been inferred that 

different binding-site interactions may exist for peptide versus nonpeptide ligands as based on their 

differential sensitivities to site-directed mutants of the native GPCR. The recent development of 3D 

structural models of the neurotensin [140] and α-melanotropin MC 1 subtype [141] GPCRs provide 

interesting case studies. Both examples provide the correlation of significant structure-activity databases 

and experimentally determined (NMR) structures of key peptide analogs with predicted molecular 

contacts at their respective target receptors. As illustrated in Figure 21, the proposed peptide agonist 

binding interactions for neurotensin and α-melanotropin analogs at their human GPCR targets may be 

used to further guide the molecular design and synthesis of “second-generation” peptidomimetic 

derivatives. 

In the first example, the neurotensin C-terminal octapeptide was subject to conformational searching 

(~Arg-Pro-Tyr~ sequences from the Brookhaven Protein Databank), manual docking to the homologybuilt 

neurotensin GPCR receptor model, and constrained molecular dynamics simulation to provide a 3D 

structure of the ligand-receptor complex [140]. A compact structure of the peptide in its complexed 

conformation was consistent with a Type-1 β-turn as previously determined by structural and structureactivity 

studies. Key molecular contacts predicted from this neurotensin GPCR model include 

hydrophobic interactions with the C-terminal Ile and Leu side chains, π-cation interactions with each 

Arg residue side chain, and a “cluster” of aromatic-aromatic interactions with the Tyr side chain. No 

electrostatic interactions were predicted, and the primary contact residues on the neurotensin GPCR 

model were those comprising the third extracellular loop. 

In the second example, the α-melanotropin (MC1) GPCR model was constructed [141] by homologybuilding 

methods relative to both bacteriorhodopsin and rhodopsin fingerprint maps, and the MSH 

superagonist peptides [Nle 4, D-Phe 7]-MSH and Ac-cyclo[Nle 4, Asp 5, D-Phe 7, Lys 10]-MSH 4–10-NH 2 were 

modeled in conformations derived from previous experimental studies (i.e., a Type-II β-turn at the 

common tetrapeptide sequence ~His-D-Phe-Arg-Trp~). Of the alternative binding modes that were 

described for the above the two MSH peptide ligands, one predicts the possibility of a network of 

aromatic-aromatic and hydrophobic interactions between the MC1 receptor and the D-Phe and Trp side 

chains of the MSH ligand (Figure 21). In addition, this MC1 receptor model predicts multiple 

electrostatic and π-cation interactions between 


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Figure 21 

GPCR 3D structural models for neurotensin and α-melanotropin agonists 


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the MC1 receptor and the Arg side chain of the peptide ligand. The primary contact residues of this 

particular MC1 receptor model were all transmembrane domain derived and lie within 4–7.5 angstrom 

(centroid to centroid). In conclusion, the development and refinement of 3D structural models of GPCR 

targets, iterative site-directed mutagenesis studies, and systematic testing of key agonists and/or 

antagonists will undoubtedly make a significant impact in the structure-based drug design of 

peptidomimetic and nonpeptide ligands at these receptors. Such work may be expected to synergize well 

with ligand-based pharmacophore modeling strategies which have become quite sophisticated in recent 

years as the results of advanced computational chemistry methodologies. 

B. Protease Targets 

Protease-inhibitor drug discovery illustrates significant success in both mechanistic and 3D structurebased 

drug discovery for each of the representative classes (i.e., aspartyl, serinyl, metallo, and cysteinyl) 

as exemplified in Table 2 (for a review see Reference 142a). In retrospect, pioneering achievements in 

the design of peptidomimetic inhibitors of angiotensin-converting enzyme (for reviews see References 

142b,142c) to have led to 45 (Captopril [51]) and 46 (Enalapril [52]). Such work has provided great 

impetus to the area of proteasetargeted drug discovery. Over the past two decades a pervasive effort 

integrating substrate-based inhibitor design, x-ray crystallography or NMR spectroscopy of inhibitorprotease 

complexes, high-throughput mass screening, and combinatorial chemical technologies has 

evolved to further advance this area of research. 

Aspartyl Proteases 

The aspartyl proteases include pepsin, renin, cathepsin-D, chymosin, and gastricsin as well as microbial 

enzymes (e.g., penicillopepsin, rhizopuspepsin, and endothiapepsin) and retroviral proteases (e.g., HIV- 

1 protease). The first high-resolution x-ray crystallographic structures of this protease family were 

determined for penicillopepsin [143], rhizopuspepsin [144], endothiapepsin [145], pepsinogen [146], 

and pepsin [147]. Based on homology-building strategies, 3D structural models of renin were 

subsequently constructed [148] to first guide the structure-based design of peptidomimetic inhibitors (for 

a review see Reference 149). Furthermore, in several cases the x-ray crystallographic structures of renin 

inhibitors were determined [150] as ligand-enzyme complexes with rhizopuspepsin, endothiapepsin, or 

pepsin. Eventually, the x-ray crystallographic structures of renin (apo/complexes) were achieved to 

provide high-resolution molecular maps of the target enzyme [151]. As illustrated in Figure 22, substratebased 

inhibitors such as the highly potent peptidomimetic 95 [151a] show well-defined hydrophobic 

pockets for the P 3-P 1' side chains as well 


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as hydrogen-bonding to the backbone of the inhibitor that exists in β-sheet type extended conformation. 

Structure-based design strategies of renin inhibitors have focused on systematic transformation of its 

substrate (angiotensinogen). Noteworthy examples which illustrate topographical designed include 

inhibitors 96 [152], 97 [153], 98 [154], and 99 [155], of which the latter macrocyclic inhibitor is quite 

novel in terms of having two D-aromatic amino acids and lacking a “transition state” bioisostere 

replacement at the P 1-P 1' site. 

In contrast to renin, the discovery of HIV protease inhibitors provides a high degree of synchronization 

of x-ray crystallography studies with iterative structure-based drug design efforts as well as the 

identification of nonpeptide ligands from mass screening (e.g., coumarins and pyrones) or 3D 

computational searching (e.g., haloperidol) to advance what has become a milestone achievement in 

rational drug design [113]; and for reviews on HIV protease see Reference 156 and the first chapter of 

this book). Representative of the scope of the many contributions to the discovery of both 

peptidomimetic and nonpeptide inhibitors of HIV protease (Figure 23) are the de novo designed C 2symmetric 


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Figure 22 

Protease 3D structural models: renin-inhibitor complex and drug design 


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Figure 23 

Protease structure-based drug design: HIV protease-targeted peptidomimetics and nonpeptides 


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inhibitors 100 [157] and 101 [158]; the nonsymmetric peptidomimetics 44 [50] and 102–106 [159–163, 

respectively]; and a series of nonpeptides derived originally from either 3-D computational searching 

107 [164] or high-throughput sceening 108–110 [165–168], respectively. Of these compounds, FDA 

approval has been recently granted to 102 (Indinivar), 103 (Ritonavir), and 44 (Saquinavir). 

Currently, it is believed that there exists well over 150 x-ray crystallographic structures of HIV proteaseinhibitor 

complexes, not including mutated forms of the target enzyme that have also been determined to 

be important to develop inhibitors which will be effective in so-called HIV resistant strains. The initial 

series of x-ray crystallographic structures of HIV protease included the apoprotein [169] and enzymeinhibitor 

complexes derived from substrate-based analogues having P 1-P 1' substitutions by 

N1eΨ[CH 2NH]N1e [170], LeuΨ[CH(OH)CH 2]Val [171], PheΨ[CH(OH)CH 2N]Pro [172], and 

LeuΨ[CH(OH)]Gly or statine [173]. As illustrated in Figure 24, the first reported HIV protease-inhibitor 

complex [170] with the pseudopeptide 111 provided a high-resolution map of the active site of the 

enzyme as formed in a C 2-symmetric fashion by the homodimer, and the “flaps” of each monomeric 

subunit (i.e., residues 35–57) were shown to make intermolecular interactions with the backbone of the 

inhibitor by both direct hydrogen-bonding and through a structural water molecule (W301). Relative to 

the C 2-symmetry of the target enzyme, the discovery of C 2-symmetric inhibitors was successfully 

achieved by the design of PheΨ[CH(OH)]gPhe- and PheΨ[CH(OH)CH(OH)]gPhe-modified 

peptidomimetics (gPhe refers to gem-diamino-Phe in which the Cα-CO 2H moiety is replaced by Cα- 

NH 2) as exemplified by 101 [157] Among the plethora of other structure-based drug design strategies 

focused on HIV protease inhibitor discovery it is also noteworthy to highlight the nonpeptide leads 100 

and 108–111 as they displace a key structural water (i.e., W301) and, as opposed to all previously 

discovered substrate-based inhibitors, are capable of direct hydrogen-bonding interactions to the HIV 

protease flap regions (Figure 24). These discoveries provide impetus for molecular design strategies to 

consider tightly bound water molecules as possible secondary “ligands” in either de novo design or 

iterative structure-based design of novel peptidomimetic or nonpeptide lead compounds. Previous 

studies [174] on biotinstrepavidin and an x-ray crystallographic structure of the complex showed that 

structural water molecule displacement (relative to the apoprotein) by key functional groups of the 

ligand (biotin) was possible. It is noted, however, that HIV protease is unique from other members of the 

aspartyl protease family with respect to the role of structural water W301 role in substrate/ inhibitor 

binding. The catalytic water, which is critical to the mechanism of substrate cleavage for all aspartyl 

proteases, has been a key feature in the design of a plethora of so-called “transition state” modified 

inhibitors that incorporate a tetrahedral 


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Figure 24 

Protease 3D structural models: HIV protease-inhibitor complex and drug design 


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hydroxymethyl substituent within various types of nonhydrolyzable surrogates of the scissile amide 

bond (e.g., Ψ[CH(OH)], Ψ[CH(OH)CH 2], and Ψ[CH(OH) CH 2N]; see above). Nevertheless, there exist 

examples of highly potent inhibitors of renin e.g., the Ψ[CH 2NH]-modified 41 [47] and macrocylic 

peptide 99 [155] and HIV protease e.g., the pyrone-based series 108–111 [165–168] respectively) that 

do not possess a tetrahedral CH(OH) moiety per se. 

Serinyl Proteases 

The serinyl proteases include trypsin, chymotrypsin-A, elastase, thrombin, kallikrein, cathepsins-A, G, 

and R, Factor VII, Factors IXa-XIIa, and tissue plasminogen activator. High-resolution x-ray 

crystallographic structures of this protease family have been determined for thrombin (for a review see 

[175]; also refer to Table II [176–182]), Factor Xa [183], trypsin [184], kallikrein-A [185], and elastase 

[186–190]. As illustrated in Figure 25, a substrate-based inhibitor of thrombin having a boronic acid, 

B(OH) 2, substitution for the scissile amide was determine by x-ray crystallography to form a covalent 

bond to the active site Ser-195 residue [176]. The N-terminal Ac-D-Phe-Pro moiety of this inhibitor 

binds in a β-sheet type extended conformation that involves hydrogen-bonding contacts to the enzyme 

and well-defined hydrophobic and aromatic-aromatic (edge-to-face) stacking interactions. The inhibitor 

Arg side chain binds in an extended conformation and the guanidino moiety forms bidentate hydrogenbonding 

interactions with an Asp189 residue at the base of the S 1 “specificity” pocket as well as 

additional hydrogen-bonds to the enzyme, one of which is mediated through a structural water. Relative 

to the substrate-based peptidomimetic inhibitors of thrombin having C-terminal electrophilic groups 

(e.g., aldehyde, ketone, and boronic acid), the discovery and structure-based design of nonpeptide 

inhibitors not having P 1 electrophilic functionalization has also been extremely successful as represented 

by 52 [70], 60 [71], and 113 [182]. As shown in Figure 25, the design of a highly potent and selective 

amidinopiperidine-based thrombin inhibitor 113 was derived from analysis of the x-ray crystallographic 

structures of thrombin complexed with inhibitors 52 and 60. The latter two compounds showed different 

trajectories of their P 1 side chains (i.e., guanidinoalkyl and amidinophenyl, respectively) into the S 1 

pocket to account for the observed opposite chirality preferences at the Cα-position of the P 1 amino acid 

residues. Also, the C-terminal cycloalkyl moieties of both 52 and 60 were observed to bind to the socalled 

inhibitor “P-pocket” (i.e., the P 2 substrate pocket), thus explaining that these compounds were not 

binding in a substrate-like conformation such as the peptidomimetic inhibitors Ac-D-Phe-Pro-boroArg- 

OH as described above. Thus, the design of the novel amidinopiperidine-based inhibitor 113 illustrates a 

“transposition” of the P-pocket binding group to an N-substituted Gly-β-Asp scaffold. 


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Figure 25 

Protease 3D structural models: thrombin-inhibitor complex and drug design 


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For other serinyl proteases, such as Factor Xa, kallikrein and elastase, the availability of x-ray 

crystallographic structure of the enzymes (apo/complexes) provides further examples in which structurebased 

drug design is being advanced (Table 2). In particular, elastase-targeted drug discovery is 

highlighted here as it illustrates substrate-based peptidomimetic inhibitor design strategies that have 

focused on key P 2–P 3 side chain and backbone hydrogen-bonding interactions with the enzyme (for 

reviews see Reference [191]). Several x-ray crystallographic structures have been determined for 

pancreatic elastase [187,188,189b,190,191] and leukocyte elastase [186,187,189a], including complexes 

with peptide substrate-based inhibitors having P 1 electrophilic functionalities such as benzoxazole, [188] 

trifluoromethyl ketone [56,187,189], and α-,α-difluoro-β-ketoamide [190]. Recently, the design of 

peptidomimetic inhibitors incorporating nonpeptidyl P 2-P 3 replacements has resulted in the discovery of 

highly potent compounds [56,187]. Specifically, a lead series of highly potent trifluoromethylketonebased 

inhibitors of human leukocyte elastase which incorporate a N-carboxymethyl-3-amino-6-arylpyridone 

template (50, 116; see Figure 26) was developed and shown by x-ray crystallography to 

provide backbone hydrogen-bonding and a novel trajectory of a P 2 group from the pyridone ring to the 

enzyme. Further modification of the pyridone ring to give the bicyclic pyridopyrimidine derivative 117 

was predicted from molecular modeling studies to provide additional hydrogen-bonding to the enzyme 

as well as another site on the bicyclic heteroaromatic ring system for tethering various hydrophobic or 

hydrophilic groups. Finally, a series of novel dipeptide-based inhibitors (e.g., trifluoromethylacetyl-Leu- 

Phe-p-isoproylanilide and a peptidomimetic derivative) are particularly intriguing because they bind 

“backwards” as based on analysis of the x-ray crystallography structures of their complexes with 

pancreatic elastase [191]. In this binding mode the trifluoromethylacetyl moiety is proximate to the 

active site Ser residue and the ligand backbone and side chain substructures make hydrogen bonding and 

hydrophobic contacts with the enzyme, respectively. 

Cysteinyl Proteases 

The cysteinyl proteases include papain; calpains I and II; cathepsins B, H, and L; proline endopeptidase; 

and interleukin-converting enzyme (ICE) and its homologs. The most well-studied cysteinyl protease is 

likely papain, and the first x-ray crystallographic structures of papain [193] and a peptide 

chloromethylketone inhibitor-papain complex [194] provided the first high resolution molecular maps of 

the active site. Pioneering studies in the discovery of papain substrate peptide-based inhibitors having P 1 

electrophilic moieties such as aldehydes [195], ketones (e.g., fluoromethylketone, which has been 

determined [196] to exhibit selectivity for cysteinyl proteases versus serinyl proteases), semicarbazones, 

and nitriles are noteworthy since 13C-NMR spectro- 


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Figure 26 

Protease 3D structural models: elastase-inhibitor complex and drug design 


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scopic studies provided direct evidence that the active site Cys25 of papain forms a reversible covalent 

bond with such electrophiles (see review Reference 142). The x-ray crystallographic structures of 

cathepsin-B [197] and picornaviral 3C protease [198] have also been determined, but only in the 

apoprotein form. Recently, high-resolution x-ray crystallographic structures of ICE-inhibitor complexes 

have been determined [199,200]. This enzyme is structurally unique relative to other cysteinyl proteases 

(for a review see Reference 201) in that it has a heterodimeric architecture in which two subunits form 

the catalytically active enzyme site (actually, two p10/p20 heterodimers apparently create a tetrameric 

form of the competent protease). As shown in Figure 27, an x-ray crystallographic structure of ICE 

complexed with a substrate peptide-based chloromethylketone inhibitor (118 [200]) that is irreversibly 

bonded to the active site Cys285 shows the P 1 Asp specificity pocket to be comprised of two Arg 

residues that lie at the base of the S 1 binding pocket. Relative to other side chain binding pockets, a 

hydrophobic “channel” type S 4 site exists for the P 4 Tyr of the inhibitor, whereas the P 3-P 2 Val-Ala side 

chains are well exposed to solvent. With respect to the peptide backbone of the inhibitor, hydrogen 

bonding interactions between the P 3 Val (both NH and CO) and the P 1 Asp (NH) and the P 10 monomer 

are predicted. 

The x-ray crystallographic structure of ICE complexed with the inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde 

(119 [202]), supports structure-activity studies [202] that employed a systematic analysis of N-methylamino 

acid substitutions in which hydrogen bonding interactions between inhibitor and enzyme tolerated 

only N-Me-Ala replacement at the P 2 site. Furthermore, C-terminal modification of P 1 Asp by 

irreversible alkylating groups, such as the aryloxymethyl ketone analog 120 [203], have led to the first 

reported peptidomimetic inhibitor of ICE (121 [204]). Noteworthy in the structure of this 

peptidomimetic inhibitor is that a pyridone template provided an effective P 2-P 3 replacement, similar to 

that for the elastase inhibitor design (see above). It is likely that the backbone hydrogen bonding 

network between 121 and ICE is conserved as compared to the x-ray crystallographic structure of the 

substrate peptide-based inhibitor 118 complexed to the target enzyme. Finally, the x-ray crystallographic 

structure of the ICE homolog referred to as apopain, or CPP32, as a complex with a peptide-aldehyde 

inhibitor has been recently determined [205] and provides additional insight as the specificity of 

substrate recognition at both the S 1 (P 1 Asp) and S 4 (P 4 Asp) subsites. Thus, the opportunity for iterative 

structure-based drug design exists to advance novel peptidomimetic and nonpeptide inhibitors of ICE 

and/or its homologs. 

Metalloproteases 

The metalloproteases include both exopeptidases (e.g., angiotensin-converting enzyme, aminopeptidase- 

M, and carboxypeptidase-A) and endopeptidases (e.g., 


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Figure 27 

Protease 3D structural models: ICE-inhibitor complex and drug design 


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(e.g., thermolysin, endopeptidase 24.11 or NEP, collagenase, gelatinase and stromelysin). Historically, 

the most well-studied metalloprotease is thermolysin (for a review see [206]), and the first x-ray 

crystallographic structures of thermolysin [207] and several structurally distinct peptide inhibitors (see 

Table 2 [208–212]) provided the first high-resolution molecular maps of the active site and insight into 

the mechanistic roles of the metal ion for substrate hydrolysis. Specifically, the binding interactions of 

P 1–P 1' “transition state” amide bond isosteres (e.g., ψ[P=O(OH)NH]) as well as metal-chelating 

functionalities (e.g., hydroxamates, carboxylates, and sulfhydryls), introduced as N-substitutions on 

P 1–'P 2' peptide scaffolds [208–212] have been determined. Moreover, the structural and mechanistic 

information derived from studies on thermolysin have provided insight into the design of inhibitors of 

the therapeutically relevant target, angiotensin-converting enzyme or ACE (for reviews see Reference 

142). This has been particularly significant since the 3D structure of ACE has not yet been determined. 

However, it is noted that x-ray crystallographic structures of carboxypeptidase, a related metalloprotease 

of the exopeptidase group, for both the apoprotein and a Glyψ[P=O(OH)NH]Phe-modified inhibitor 

complex have been reported [213]. As illustrated in Figure 28, the x-ray crystallographic structure of the 

Pheψ[P=O(OH)NH]Leu-modified peptidomimetic inhibitor 122 complexed with thermolysin show the 

Zn 2+ coordination and P 1'-P 2' (or P 1-P 2' “collected product”) mode of binding. Relative to this structure 

the predicted molecular interactions between ACE and its inhibitors (e.g. Captopril [51], Enalaprilat 

[52], and Fosfinoprilat [214] as well as an emerging class of “dual specific” inhibitors of ACE and NEP 

(e.g., 123 [215]) may be envisaged. 

The ability to design specific inhibitors of several matrix metalloproteases (MMPs) is rapidly 

developing (for reviews see Reference 216). Such MMP targets include fibroblast collagenase (MMP-1), 

gelatinase-A (MMP-2), and stromelysin (MMP-3). Both x-ray crystallography and NMR spectroscopy 

have provided 3D structural information for several MMPs as well as MMP-inhibitor complexes (see 

Table 2; [217–226]). In the example of collagenase, “first generation” substrate-based inhibitor design 

strategies have focused on modifying the P 1–P 1' cleavage site (e.g., Gly-Phe, Ala-Tyr, and Ala-Phe) by 

N-terminal functionalities capable of Zn 2+ coordination (e.g., sulfhydryl, carboxyl, phosphonoalkyl, and 

hydroxamate [227–229]). As shown in Figure 29, an x-ray crystallographic structure of the Nhydroxamate-modified 

peptide 124 complexed to fibroblast collagenase provides a molecular map of 

both its hydrogen-bonding interactions to the enzyme active site and binding to bound Zn 2+ [219]. 

Potent MMP-1 inhibitors have been designed that tether the P 2' side chain to the inhibitor's C-terminus 

as macrocylic rings [53,230]. In the example of gelatinase-A, potent inhibitors have been designed (e.g., 

48, Figure 11 [54]) by N-terminal hydroxamate and P 1' extended aromatic side chain modifications 


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Figure 28 

Protease 3D structural models: thermolysin-inhibitor complex ACE inhibitor drug design 


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of a P 1'–P 3' peptide scaffold [54]. To date, no 3D structural information is available for MMP-2 with 

respect to either the apoprotein catalytic domain or inhibitor complexes thereof. Finally, in the example 

of stromelysin-1, potent inhibitors have been designed (e.g., 49, Figure 11 [55]) by N-terminal 

carboxyalkylamino functionalization that includes a P 1 substituent. An x-ray crystallographic structure 

of a related MMP-3 inhibitor 125 shows (Figure 29) the hydrogen-bonding interactions at the active site 

and carboxylate coordination with the Zn 2+ [223]. Finally, a recently determined x-ray crystallographic 

structure of an Pheψ[P=O(OH)CH 2]Ala-modified peptide inhibitor 126 complexed with astacin (Figure 

29) shows the extensive hydrogen-bonding network between inhibitor, enzyme, Zn 2+, and a structural 

water [226]. It is expected that iterative structure-based design of inhibitors of the MMP family will 

enable the discovery of novel compounds with superior binding affinities and/or selectivities. 

C. Signal-Transduction Protein Targets 

Beyond proteases the opportunity for structure-based drug design is being realized in the rapidly 

developing area of signal-transduction research (e.g., intracellular protein and nucleic acid targets). Both 

x-ray crystallography and/or NMR spectroscopy have significantly contributed to a wide-scope database 

of 3D structural information for various catalytic and noncatalytic signal-transduction protein targets 

(see Table 3). These include tyrosine kinases (e.g., growth factor receptor kinases and Src family 

kinases; for reviews see Reference 231), serine/threonine and “dual specificity” kinases (e.g., mitogenactivated 

protein kinases and CDK2 and cAMP-dependent protein kinases; for reviews see Reference 

232), phosphotyrosine phosphatases (e.g., PTP1B and Syp; for reviews see Reference 233), 

phosphoserine/phosphothreonine and “dual specificity” phosphatases (e.g., VH1 and CDC25; for 

reviews see Reference 234), noncatalytic “adapter” proteins (e.g., Crk, Grb2, Shc, and IRS-1; for 

reviews see Reference 85), transferases (e.g., Ras farnesyl transferase; for reviews see Reference 81), 

proline cis-trans isomerases (e.g., FKBP-12 and cyclophilin A; for reviews see Reference 235), and GTPbinding 

proteins (e.g., p21 Ras and α-β/γ heterotrimeric G-protein for GPCR superfamily; for reviews 

see References 236 and 237, respectively). The diversity of targets, mechanistic relationships (e.g., 

enzyme–substrate or regulatory protein–protein interaction), and potential therapeutic opportunities has 

created great impetus for focused research in the area of signal transduction (for reviews see References 

265–267). 

Src Homology-2 and Homology-3 Domains 

The identification of noncatalytic regulatory domains referred to as Src homology (SH) domains has 

been rapidly advanced over recent years as a critical link 


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Figure 29 

Protease 3D structural models: matrix metallo protease-inhibitor complexes 

and drug design 


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Table 3 Some Known 3D Structures of Signal-Transduction Proteins (Apo/Complexes) 

Protein Target Apo/Complex Resolution Reference 

Src homology domains 

Abl SH2 —(Apo) NMR 238 

Src SH2 —(Apo) 2.5 Å 239 

phosphopeptide 2.7 Å 239 

phosphopeptide NMR 240 

PLC (C) SH2 phosphopeptide NMR 241 

Shc SH2 phosphopeptide NMR 242 

Lck SH2 phosphopeptide 2.2 Å 243a 

p85 (N) SH2 phosphopeptide 2.0 Å 244 

Syp SH2 phosphopeptide 2.0 Å 245 

Grb2 SH2 phosphopeptide 2.1 Å 246 

Syk SH2 phosphopeptide NMR 247 

Zap70 SH2-SH2 phosphopeptide (tandem) 1.9 Å 248 

Src SH3 —(Apo) NMR 249a 

peptide NMR 249b 

Abl SH3 peptide 2.0 Å 250 

Crk SH3 peptide 1.5 Å 251 

Grb2 SH3 peptide NMR 252 

Grb2 SH3-SH2-SH3 —(Apo) 3.1 Å 253 

Tyr and Ser/Thr kinases 

Insulin receptor Tyr kinase —(Apo) 2.1 Å 254 

cAMP-dep. protein kinase —(Apo) 3.9 Å 255 


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(Ser/Thr) peptide 2.9 Å 255 

Cell cycle-dep. protein —(Apo) 2.4 Å 256a 

kinase (CDK2) protein (p27 Kip1 inhibitory domain) 2.3 Å 256b 

Mitogen-activated protein kinase 

(MAPK) 

Tyr and Ser/Thr phosphatases 

—(Apo) 2.3 Å 257 

BH-PTP (Tyr) —(Apo) 2.2 Å 258a 

PTP1B (Tyr) —(Apo) 2.8 Å 258b 

peptide (C215S mutant enzyme) 2.6 Å 258c 

VH1 (Tyr and Ser/Thr”) —(Apo) 2.1 Å 259 

PP-1 (Ser/Thr) —(Apo) 2.1 Å 260 

pTyr binding domains 

IRS-1 PTB —(Apo) 1.95 Å 261 

peptide 1.8 Å 261 

IRS-1 PTB peptide NMR 262 

Pro cis-trans isomerases 

Cyclophilin peptide (cyclosporin-A) 2.8 Å 263 

FKBP-12 FK-506 (macrolide antibiotic) 1.7 Å 264 


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in deconvoluting both enzyme-substrate and regulatory protein-protein interactions for a number of 

signal-transduction pathways (for reviews see Reference 268). This emerging “superfamily” of proteins 

includes SH2 and SH3 domains, the so-called “choreographers of multiple signalling pathways,” and 

include very intriguing new therapeutic targets [85]. The SH2 domains have been determined to bind 

cognate phosphotyrosine (pTyr) containing proteins in a sequence-dependent manner relative to the 

amino acids contiguous to the C-terminal side of the pTyr residue (e.g., for Src SH2 a preferred 

sequence is ~pTyr-Glu-Glu-Ile~ versus ~pTyr-Tyr-Asn-Tyr for Grb2 [87,269]. The SH3 domains have 

been determined to specifically bind Pro-rich sequences of cognate proteins. Interestingly, as a result of 

the pseudosymmetrical nature of the SH3 domains there is the possibility of binding both N rarrow.gif 

C and C rarrow.gif N directions (e.g., for Src SH3 preferred sequences are ~Arg-Ala-Leu-Pro-Pro-Leu- 

Pro-Arg-Tyr and Ala-Phe-Ala-Pro-Pro-Leu-Pro-Arg-Arg, wherein Arg binds to a site 3 pocket [249b]). 

With respect to SH2 domain structure-based drug design, the first x-ray crystallographic structures of 

pTyr-containing peptide ligands complexed with Src SH2 domain [239] have been utilized to design the 

first peptidomimetic antagonists [89]. As illustrated in Figure 30, a molecular map of the tetrapeptide 

sequence ~pTyr-Glu-Glu-Ile~ complexed with Src SH2 [239] shows the pTyr binding pocket and a 

second binding site for the P +3 Ile residue. As previously described, a prototypic peptidomimetic AcpTyr-Glu-D-Hcy-NH 

2 (66) was first discovered by a peptide scaffold design strategy (see Figure 16; 

[77]) that took into account the x-ray crystallographic structure of Src SH2-phosphopeptide (Glu-Pro- 

Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu, 127) complex. Further structure-based drug design 

modifications have led to the discovery a series of potent peptidomimetics having novel C-terminal 

functionalization (e.g., “transposed” side chain of the P +1 Glu or conformational constraint using a 

pyrrolidine ring; see Figure 30) as represented by 128–130 [89,270]. Studies focused on Src SH2 [88] 

have shown that the phosphate ester of pTyr is particularly critical for molecular recognition, and that 

significant loss in binding occurs by replacement with sulfate, carboxylate, nitrosyl, hydroxy, and 

amino. However, backbone modifications of pTyr which replace its acylated amino functionality with 

aromatic rings designed to form π-cation type interactions with the Arg-αA2 were effective substitutions 

[271]. 

Recently, high-resolution 3D structures have been described for the noncatalytic “adapter” protein Grb2 

with respect to the apoprotein (SH3-SH2-SH3 [153]) as well as the individual SH2 and SH3 domains 

[246 and 252, respectively]. In the case of the SH2 domain of Grb2 an x-ray crystallographic structure 

of a phosphopeptide complex provided insight to the molecular basis of the specificity of Grb2 SH2 

binding of ~pTyr-Xxx-Asn-Yyy~ sequences. As illustrated in Figure 31, the binding interactions of Lys- 

Pro-Phe-pTyr-Val-Asn- 


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Figure 30 

SH2 domain 3D structural models: pp60Src SH2 domain antagonists 


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Figure 31 

SH2 domain 3D structural models: Grb2 SH2-SH3 domain antagonists 


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Val (131) showed that the phophopeptide adopts a β-turn conformation about the P-P +3 residues and that 

the P +2 Asn side chain carboxamide moiety is extensively hydrogen bonded to the protein. In contrast to 

the well-defined binding pocket for the P +3 Ile of 127 to bind Src SH2, the P +3 Val of 131 engages in 

limited surface hydrophobic interactions because the Trp121 residue of Grb2 SH2 sterically blocks the 

phosphopeptide from attaining a similar binding mode. In the case of the SH3 domain, the binding of a 

cognate Pro-rich peptide sequence ~Pro-Pro-Pro-Val-Pro-Pro-Arg-Arg~ shows distinct pockets which 

recognize the Pro, Val, and Arg residues as illustrated in Figure 31. The peptide adopts a left-handed 

polyproline type-II helical conformation which projects the three aforementioned residues to their 

complementary binding pockets in the Grb2 SH3 domain. 

Tyrosine Phosphatases and Phosphotyrosine Binding Domains 

Two other types of signal-transduction proteins that recognize phosphotyrosine-containing sequences 

are tyrosine phosphatases (e.g., PTP1B, Syp, and CD45) and proteins that contain a noncatalytic motif 

referred to as a phosphotyrosine binding (PTB) domain. Although tyrosine phosphatases and PTB 

domains are structurally quite different from each other they both are similar with respect to the binding 

of pTyr-containing sequences with preference to the amino acids N-terminal to the pTyr residue. In 

other words, the tyrosine phosphatase PTP1 binds EGF receptor (pTyr 992)-based phosphopeptide 

substrates Asp-Ala-Asp-Glu-pTyr-Leu-NH 2 and Asp-Ala-Asp-Glu-pTyr-Leu-Ile-Pro-Gln-Gln-Gly 

equally [272], and the substitution of pTyr by nonhydrolyzable F 2Pmp in the hexapeptide derivative has 

been reported [273] to give a highly potent inhibitor (see compound 69, Figure 14). In the case of PTB 

domains, ~Asn-Pro-Xxx-pTyr~ (where Xxx is variable) has been determined [274] as the cognate 

sequence for several PTB-domain-containing proteins such as Shc and the insulin receptor substrate-1 

(IRS-1). The preference for amino acids N-terminal to the pTyr residue in binding to either tyrosine 

phosphatases or PTB domains is, therefore, opposite of that known for SH2 domains [275]. 

Among the tyrosine phosphatase superfamily, PTP1B was the first to be discovered and structurallydetermined 

by x-ray crystallography, as the apoprotein catalytic domain [258b]. The first x-ray 

crystallographic structure of PTP1B complexed with a phosphopeptide has also been very recently 

determined [258c] using a catalytically inactive Cys215 rarrow.gif Ser PTP1B mutant and the 

phosphopeptide Asp-Ala-Asp-Glu-pTyr-Leu-NH 2 (133). As illustrated in Figure 32, the molecular 

interactions between the tyrosine phosphatase and the phosphopeptide are dominated by electrostatic 

(i.e., the pTyr, P -1 Glu, and P -2 Asp residues) and hydrogen bonding contacts to key amide functionalities 

of the backbone of 133. The P +1 Leu side chain forms hydrophobic contacts with 


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Figure 32 

PTP and PTB 3D structural models: PTP1B inhibitor and PTB antagonists 


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several PTP1B residues at the surface proximate to the well-defined pTyr binding pocket. Such 3D 

structural information provides insight for the design of peptidomimetic inhibitors of PTP1B. 

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In the case of the IRS-1 PTB domain, x-ray crystallographic studies of PTB complexed with a pTyrcontaining 

peptide (134) complex have shown (Figure 32) that the phosphopeptide forms a type-I β-turn 

within the Asn-Pro-Ala-pTyr sequence, and the peptide backbone is extensively hydrogen bonded to the 

PTB domain from the P +1-P -7 residues of 134 [261]. The pTyr binding pocket provides both electrostatic 

and multiple hydrogen bonding contacts to the phosphate ester moiety, and hydrophobic interactions 

exist for the P -1-P -3 side chains of the peptide. As in the case of PTP1B, such 3D structural information 

provides the opportunity for structure-based drug design to discover potent inhibitors which may be used 

for further exploration in cellular studies. 

V. Future Perspectives 

The impact of structure-based drug design on both peptidomimetic and nonpeptide drug discovery has 

been significant over the past few years. The integration of sophisticated computational chemical 

technologies, structural biology (x-ray crystallography and NMR spectroscopy), molecular diversity and 

high-through-put screening, and targeted biological testing are expected to provide invaluable guidance 

to drug discovery. These “technological tools” are contributing to an emerging “3D structure-activity 

database” that is the essence of rational drug design. Certainly, this intriguing area of peptidomimetic 

and nonpeptide drug discovery and design is providing tremendous insight to our understanding of 

molecular recognition and biochemical mechanisms. With particular regard to molecular diversity, the 

generation of new leads from combinatorial chemistry focused on synthetic peptide, peptidomimetic, or 

nonpeptide-type libraries will provide new opportunities (and challenges) for structure-based drug 

design strategies (for reviews see Reference 276). Novel scaffolds and templates will continue to be 

advanced and iteratively modified using “randomized” or “targeted” substructural replacements, and 

such work is exemplified in this review. Of particular significance to the field of peptidomimetic and 

nonpeptide drug discovery will be the rational use of D-amino acids, Nα-alkyl or Cα-alkyl amino acids, 

N-substituted Gly, XxxΨ[Z]Yyy (dipeptide isosteres), benzodiazepines, and amino-benzoic acids in 

structure-based drug design. Without question, structure-based drug design will be a decisive factor in 

drug discovery efforts ranging from de novo design to iterative structural optimization of 

peptidomimetic and nonpeptide lead compounds. 


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I wish to acknowledge my colleagues at Parke-Davis for their critical review of this manuscript as well 

as for their collaborative contributions to structure-based drug design research in the several areas, 

including HIV protease inhibitor discovery, Ras farnesyl transferase inhibitor discovery, Src homology- 

2 domain antagonist discovery, interleukin-converting enzyme inhibitor discovery, and melanocortin 

receptor modeling development to probe MSH agonist and antagonists binding. I especially thank Mark 

Plummer, Elizabeth Lunney, Charles Stankovic, Kim Para, Aurash Shahripour, Vara Prasad, Carrie 

Haskell-Luevano, Daniel Ortwine, Daniele Leonard, Wayne Cody, and Christine Humblet in this regard. 

I also very much thank Pandi Veerapandian for the opportunity to contribute a chapter to this book, and 

for his critical review of this manuscript and excellent editorial suggestions. 

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Index 

A 

Acquired immunodeficiency virus (AIDS), 41, 56, 62, 65, 70 

Active site, 45, 48, 50, 54, 55, 56, 59, 61, 62, 63, 64, 65 

Acyclovir diphosphate, 154, 160, 164 

Alcohol dehydrogenase (ADH) 202-203 

catalytic mechanism, 233-234 

inhibitors, 231-232 

mutations, 234 

NADPH cofactor binding, 232-233 

relation to diabetic complications, 229-231 

structure 

active site, 233-235 

NADPH-bound form, 231-233 

ternary complex with zopolrestat, 235-239 

α-ketoamide transition state, 282 

Page 635 

Activate platelets, 247 

Activity, 41, 48, 50, 55, 56, 64, 65, 67, 68, 69, 70 

Addison's disease, 195 

Aldo-keto reductase superfamily, 240 


Aldose reductase (ALR2)

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replacements, 563-565 

1-amidinopiperidine, 256, 257 

Amino acids 

C alpha, 128 

N-methyl, 128 

side chain constraints, 124-128 

converting enzyme, 120 

Animal models 

thrombosis of, 267 

Anthopleurins, 297, 300, 301, 302, 312, 313, 314 

Antibody-neuraminidase complexes, 470 

Anticoagulation, 247 

Anticoagulant protein, 257 

ALR2 (see Aldose reductase) 

Amantidine, 

462 

Amide bond 

Aminoalcohols 326 

Amphipathic alpha-helices (see Leucine zipper) 


Angiotensin, 321

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Antirhinoviral agents 

capsid-binding compounds, 497-500 

clinical trials, 517-518 

drug sensitivity groups, 503 

resistance, 514-517 

structure-activity relationships, 502-514 

WIN compounds, 498-500 

Antithrombin III, 248 

eflornithine, 365-367 

melarsoprol, 365-366 

pentamidine, 365 

suramin, 365-366 

Argatroban, 251, 255 

Arginine 

boronate esters, 250 

guanidinium, 251 

Arrhythmias, 296, 298 

Aspartic proteinases 

Page 636 

Anti-influenza drugs, 462, 477-480 

Antitrypanosomal agents 

Antiviral 

agent, 41, 67 

Apparent mineralocorticoid excess (AME), 191 


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inhibitor binding, 323, 332 

inhibitors, 323 

mechanism, 328 

specificity, 333 

structure, 322 

transition state analogs, 323 

Atherosclerosis 395, 398 

Autoimmune 

disease, 395,398 

atherosclerosis, 395, 398 

insulin-dependant diabetes, 395, 398 

myasthenia gravis, 398 

rheumatoid arthritis, 395, 398 

systemic lupus erythematosus, 398 

disorders, 152 

Available chemicals database (ACD), 381 

B 

Bacteriorhodopsin, 131 

BCX-34, 166, 167 

Benzodiazepinone peptidomimetic, 571 

β-barrels, 248 

β-turn, 122, 124, 126, 127 


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Bicyclic peptidomimetic, 251 

Bidentate hydrogen bond, 252 

Binding 

cleft, 45, 48, 61, 65 

pocket, 56, 58, 60 

Bivalent inhibitors, 257 

Blood clot formation, 247 

Boroarginine, 261 

Boronate esters, 251 

Boronic acid analog, 250 

Bovine trypsin inhibitor mutants 

C 

phage display, 287 

positional requirements for Fxa inhibition, 286-288 

random mutagenesis, 287 

site specific mutagenesis, 285 

structure of complexes, 285 

Calcium regulators, 296 

cAMP generators, 296 

Cancer, 395 

Carbonyl reductase, 199 

Cardiotonic activity, 301, 304, 305, 306, 314 

Catalytic 


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site, 52, 55, 61 

triad, 247, 275 

Catechol O-methyltransferase 

active site, 349-350, 355-356 

catalysis, 345, 350-351 

inhibition mechanism, 356-358 


kinetics, 346 

physiological role, 344-345 

structure 

AdoMet (see S-Adenosyl-L-methionine) 

crystal structure, 347-350 


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[Catechol O-methyltransferase] drug complexes, 354-355 

Mg++ ion, 349-350 

S-adenosyl-L-methionine, 349 

sequence, 345, 347 

substrates, 345,346 

Cation-p site, 277, 282 

Ceriamide, 473 

Charge calculation, 381 

CHARMm, 133 

databases, 536 

selection algorithm, 536 

systems integration, 536 

virtual library production, 536 

Chemotherapy, 66 

Chimeric receptor, 126 

Chymotrypsin serine protease family, 248 

Coagulation 

cascade, 247, 265 

Page 637 

Chemical 

library, 142 

Chemi-informatics, 526, 535, 537 

Circular dichroism (CD), 438, 444 


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factors, 247 

Combination therapy, 62, 68, 69 

approach, 550-552 

chemistry, 141, 525, 537 

combinatorial explosion, 552 

drug lead source, 528, 529 

focusing, 530 

parallel synthesis, 528, 532 

refinement, 530 

robotic instrumentation, 528, 532 

scaffolds, 530, 532 

structure-activity relationship (SAR), 529 

pruning, 552 

Compound selection, 531 

chemi-informatics, 536 

drug properties, 535 

receptor fit, 536 

similarity, 536 

structure-activity relationship (SAR) models, 533, 536 

virtual libraries, 533, 536 

Computer programs 

BIOGRAF, 381 


Combinatorial

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CHARMM, 542-544 

computational combinatorial ligand design (CCLD), 550-552 

DELPHI, 383 

DOCK, 379-383 

MCSS, 542-544 

Conformational 

change, 59, 60, 61, 70 

degrees of freedom, 252 

protein, 235-237 

Congestive heart failure, 295, 296, 298, 301, 314 

Cortiso, 193 

Cortisone, 193 

Cross linking, 137-138 


cocktail soak approach, 377-379 

Crystal structure, 45, 54, 56, 59, 61, 64 

aldose reductase, 231-239 

other aldo-keto reductases, 240-241 

glyceraldehyde-3-phosphate dehydrogenase, 374-375 

phosphoglycerate kinase, 376-377, 388 

triosephosphate isomerase, 371-373 

Cutaneous T-cell lymphoma, 167 

Cyclic template, 252 


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Cyclotheonamide A (CtA), 254 


Document 

Cytokines, 395-398, 412, 420 

alpha-beta, 396 

atherosclerosis, 398 

autoimmune disease and, 395, 98 

beta sandwich 396 

beta strands, 402 

beta-trefoils, 396 

4-helix bundle, 396 

growth hormone, 398 

immune system, 398 

immunomodulation, 398 

insulin-dependant diabetes, 398 

myasthenia gravis, 398 

network, 396 

nuclear magnetic resonance (NMR) and, 396 

production of, 395 

prolactin, 398 

receptors, 396 

rheumatoid arthritis, 398 

signalling by, 396 

structures, 396 



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D 

ddI, 152 

tumour necrosis factor, 398 

x-ray crystallography and, 396 

9-deazaguanine, 161, 163, 164, 165, 167 

Diabetic complications, 229-231 

Digoxin,295, 296, 297, 300 

Dihydropteridine reductase, 197, 202 

Diversity 

directed, 536 

of candidate ligands, 542,555 

dNTP binding, 56, 61 

Drug, 43, 45, 53, 55, 56, 60, 62, 63, 65, 66, 67, 68, 69, 70 

binding affinity prediction, 555 

combinatorial, 550-552 

design, 55, 66, 70, 402 

docking, 241-242, 379-383, 542-544 

fragment approach, 541 

inhibition, 70 

lead 

discovery, 377-384 

optimization, 384-387 

linked-fragment approach, 378 


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resistance, 56, 66, 68, 70 

scoring, 382-383 

solvation effects, 544-547 

Drug properties, 527, 535 

absorption, 535 

excretion, 535 

metabolism, 535 

refinement, 535, 536, 537 

structure-based design, 527 

DTic (tetrahydroisoquinnoline carboxylic acid), 124 

E 

Energy 

F 

minimization, 132-133 

with CHARMM, 542-544 

Fab, 45, 49, 51, 56, 58, 59, 69 

Factor Xa 

active site blocked (DEGR-Xa), 267, 269 

active site substrate sequences, 271 

modeled inhibitor complexes 

amidinoaryls, 279 


DUP714, 250

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antistasin peptides, 281 

cyclotheonamide, 284 

dansyl-Glu-Glu-Arg-CMK, 280 

DX-9065a, 276 

SEL-2711, 283 

natural inhibitors of 

AcAP's, 273-274 

antistasin, 266, 268, 270, 271-272 

ecotin, 268, 270, 274, 288 

tick anticoagulant peptide (TAP), 266, 268, 270, 272-273 


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structure and function, 267-269, 274 

synthetic inhibitors of 

amidinoaryls, 277-279 

antistasin peptides, 280-282 

bisamidines, 277 

BPTI mutants, 285-288 

cyclotheonamide, 282 

dansyl-Glu-Glu-Arg-CMK, 280 

DX-9065a, 275-277 

peptidyl argininals, 288 

PPACK, 275 

SEL-2711, 282 

Factor XIII, 247 

Factor XIIa, 119 

Feline immunodeficiency virus (FIV), 441 

Fibrin, 247 

Fibrinogen, 247 

Fibrinopeptide A, 250, 261 

Flavonoid, 473 

Fluoroketone analogs, 327 

Fragment approach (see Computer programs, MCSS, CCLD) 

Page 639 

[Factor Xa] TFPI, 270-271, 285, 287 


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fragment-based programs, 541-542 

Fourier maps, 154, 161, 166 

G 

Geminal diol analogs, 327 

Genomic data, 525, 527 

GG167 (see Neu5Ac2en,4-guanidino) 

Glucopyranoside 

peptidomimetic, 571 

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 

catalysis, 372, 374 

crystal structure, 374-375 

human, 374-375 

inhibitors, 381-382, 384-387 

Leishmania mexicana, 375-376, 385 

sequence, 374 

Glucocorticoid 193 

G-protein coupled receptors (GPCR), 592-594 

H 

Heart attack, 247 

Hemagglutinin, 459, 460, 462, 463, 464, 477 


Glycol analogues 326 

GRID maps, 475 

Hematophageous organisms

Document 

Ancylostoma caninum, 273 

Haementeria officinalis, 266 

Ornithidorous moubata, 266 

Hemopexin domain, 172-174 

Hemostasis, 247 

Heparin, 248 cofactor II, 248 

High throughput screening, 530 

Hirudin, 251 

HOE 140, 127-128 

Homology 

model building, 275-276,278 

models, 527 

scaffold development, 532 

structure-based design, 527 

sequence, 176 

structure, 176-177 

Hormone therapy, 206 

Human immunodeficiency virus (HIV), 441 

protease 

cleavage sites, 7 

flexibility, 7-8 

inhibitors, 586-587 AG1284, 22-27 


Document 

[Human immunodeficiency virus] 

cyclic ureas, 21-22 

hydroxycoumarins, 27-28 

indinavir, 9, 15-17, 28-29 

inversion of binding mode, 24 

nelfinavir, 17-21, 29-30 

nonpeptidic, 17-28 

peptidic, 9-10 

peptidomimetic, 10-19 

ritonavir, 13-16, 28-29 

saquinavir, 10-13, 28-29 

symmetric binding, 13-15, 21-22 

mutations, 28-32 

resistance 

primary mutations, 29-30 

secondary mutations, 30-32 

sequential passage, 28 

vitality factor, 32 

structure 


three dimensional, 3-5 

Wat 301, 6, 7, 10, 13, 16, 17, 21, 22, 24, 27 


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integrase 

amino acid sequence, 90-91 

amino terminal domain, 92, 102 

biochemical properties, 85-88 

biophysical properties, 88, 92 

carboxyl terminal domain, 89, 92 

structure of, 102-103 

catalytic core domain, 88-89 

biophysical properties of, 92-93 

mutation of hydrophobic residues of, 102-103 

structure of, 93-102 

comparison to toher 

polynucleotidyl 

transferases, 96-100 

conserved acidic residues in 


dimer, 100-102 

domain structure, 88-92 


3'-azido-3'-deoxy-thymidine (AZT), 107-108 

common pharmacophore of, 104-107 

curcumin, 107, 111 

design of, 110-112 


Document 

overview, 103-104, 108-109 

rationale for, 85 

Human prothrombin fragment F1, 261 

Hydroxamate, 172, 182-184 

Hydroxysteroid dehydrogenases, 191 

7α, 197, 199 

11β, 191-194, 203 

17β, 193-199, 205-207 

20β, 197, 199 

Hypertension, 193 

I 

Inflammation, 395 

Influenza virus 

antigenic variation, 462, 468-470 

classification, 459 

drug resistance, 478, 479 

inhibition, 478 

replication cycle, 461 

vaccines, 463 

Inhibition, 50, 54, 60, 61, 65, 69, 70 

mechanism, 65 

Inhibitors, 41, 43, 45, 48, 50, 55, 56, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70 


Hydrophobic collapse, 252

Document 

design, 358-359 


Document 

[Inhibitors] 

2-((3,4-dihydroxy-2-nitrophenyl)vinyl)phenyl-ketone, 352-353, 356 

entacapone, 352-353, 360 

first generation inhibitors, 351-352 

tolcapone, 352-353, 360 

Insulin-dependent diabetes 395,398 

Interactions 

electrostatic, 544 

hydrophobic, 545 

van der Waals, 545 

Interferon 

α, 435, 439, 440 

β, 435 

activity, 442 

cytotoxicity, 439, 443 

receptor, 441 

structural studies, 443, 444 

clinical uses, 436 

definition, 435 

γ, 435 

activity, 448, 449 

antibody studies, 445 


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receptor, 446, 447, 450 

intron A, 436 

roferon, 436 

side effects, 436 

sub types, 435, 436 

synthetic peptide studies, 448 

signal transduction, 451 

structural studies, 449-451 

synthetic peptide studies, 446, 448 

τ, 435, 439, 440, 

activity, 441-443 

antibody studies, 442 

expression, 442, 443 

receptor, 441 

signal transduction, 443 

structural studies, 443, 44 

synthetic peptide studies, 441, 442, 444 

ω, 435, 440 

Interleukin-1 

accessory protein, 398, 401 

affinity for receptors, 401 

α, 398, 399, 401, 402, 406, 420, 421, 423, 427, 428 

alternatively spliced, 399 


Document 

antagonistic activity, 416 

auto-antibodies and, 401 

autoimmune disease and, 401 

barrel, 402, 404 

β, 398, 399, 401, 402, 420, 421, 423, 427, 428 

barrel, 406, 409 

bulge, 415-416 

converting enzyme (ICE), 399, 402, 412 

hairpins, 405 

strands and, 402 

beta-trefoil fold, 402, 403, 404, 407 

binding, 412-416 

catalytic 

affinity, 421 

epitope, 410 

pocket, 412 

activity of, 412 

diad, 412 

cysteine protease, 399, 412 

enzyme mechanism, 412 

epitopes of, 405, 413-416, 418 

expression of, 399 

fibroblast growth factors, 402 


Document 

gene duplication, 398 

glycosylation of, 399, 409 

GTPase proteins and, 401 


Document 

[Interleukin-1] 

homology with CED-3 protein, 412 

hydrophobic patch, 406 

hydrophobic residues, 404 

immunoglobulin superfamily and, 399 

Kunitz family, 402 

leukemia, 395 

location of, 399 

low molecular weight antagonists, 421-427 

monoclonal antibodies and, 421 

monocyte phagocytes by, 399 

mutational studies of, 407, 409 

N-terminal extension 

nuclear magnetic resonance (NMR), 402, 404 

overexpression of, 419 

peptide fragments, 416 

peptomimetics, 412 

precursor, 399 

form, 399 

receptor, 398, 402, 420 

antagonist, 398, 399, 401, 407, 420, 421 

binding, 404-405, 406, 407-410, 412-416 


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recombinant, 421 

sequence identity of, 398 

signal transduction and, 401 

site-directed mutagenesis, 412-416 

structure of, 401-412, 419 

synthesis of, 395, 419 

substrate, 399 

specificity, 412 

subunits of, 412 


therapeutic strategies and, 412 

tumor necrosis factor and, (TNF) 421 

type I, 399, 401, 420 

type 2, 399, 401, 420 

x-ray crystallography, 401-412 

Ion-channel modulators, 296, 297, 301 

Ischemia, 296, 297 

K 

Kinins, 119 

turn propensity of, 123 

bradykinin, 119 

Kampo drug, 473 

Ketomethylene pseudo peptide bond, 260 


Document 

Kininogen, 119 

high molecular weight, 119 

low molecular weight, 119 

Kinin receptors, 120 

agonist binding site, 131-133 

antagonists of, 124 

antagonist site, 137-138 

B1 subtype, 120 

B2 subtype, 119, 120 

chimeras and, 138, 139 

mutagenesis of, 133-134 

Kallidin, 119 

molecular dynamics of, 123 

pharmacology of, 121 

solution conformation of, 121 

Kunitz domains, 271, 273, 282, 285 

Kyte-Doolittle, 131 

L 

Lactam amide, 222-223 

Leucine zipper 

propane functional map, 545-546 

stereo view, 546 


Document 

yeast transcriptional activator protein GCN4, 545-546 

Leydig cells, 193 

Licorice, 193, 195-196 

Ligand docking, 133 

Lock-and-key, 159 

M 

Malignant 

tissue, 214 

transformation, 214 


Document 

Matrix-metalloproteinase (MMP), 171-186 

fibroblast collagenase, 171-173, 176, 179, 183-184 

gelatinase, 171-173, 184 

matrilysin, 171-175, 182-184 

neutrophil collagenase, 171-175, 183-184 

stromelysin, 171-173, 184 

Medicinal leech, 257 

Metal requirement, 88, 89 

Mineralocorticoid receptor, 193 

Molecular 

fragments 

aliphatic, 542 

aromatic, 542 

charged, 542 

polar, 542 

modelling, 182-183 

MuA transposase 

structural comparison to integrase, 97-100 

Mutation, 62, 67, 68, 69 

Myristic acid, 214 

N 


Page 643 

Myocardial infarction, 247

Document 

NAD, 199, 201 

NADP, 199, 201 

NADPH, 199-201 


carbohydrate structure, 467 

dimensions, 465 

enzyme function, 464, 473 

inhibitors, 472-473, 476 

molecular weight, 465 

morphology, 464 

topology, 466 

Neu5Ac2en, 472, 473, 474, 477, 478, 480 

4-guanidino, 475, 476, 477, 479 

4-amino, 475, 476 

Nonnecleoside ingibitor binding pocket (NNIBP), 56, 58, 59, 60, 61, 62, 63, 66, 67 

NAPAP, 255, 261 

Napthalenesulfonyl, 257 

Neuraminidase 

Nonnucleoside reverse transcriptase inhibitors (NNRTI), 41, 45, 48, 49, 50, 56, 58, 59, 60, 61, 62, 63, 

64, 65, 66, 67, 68, 69, 70 

Nonnucleoside, 41, 45, 56 

inhibitor, 45, 56 

inhibitor binding pocket, 56 


Document 

RT inhibitor, 41, 56 

Nonpeptide 

angiotensin agonist, 586-587 

antagonist 

angiotensin, 586-587 

cholecystokinin, 586-587 

endothelin, 586-587 

gastrin-releasing hormone, 586-587 

glucagon, 586-587 

neurokinin, 586-587 

neuropeptide Y, 588-589 

neurotensin, 586-587 

oxytocin, 586-587 

drug discovery, 559, 570, 586-587 

vasopressin, 586-587 

NPC 17731, 124 

NPC 18325, 137 

NPC 567, 124 

Nuclear magnetic resonance (NMR), 402, 404, 407 

Nucleic acid, 48, 51, 54, 64, 65, 69 

Nucleophile, 221 

Nucleoside, 41, 50, 52, 53, 56 

reverse transcriptase (RT) inhibitor, 41, 50, 52, 53, 54, 55, 56, 65 


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Nucleotide, 51, 53, 54, 55, 69 

O 

binding domain, 200 

Oxyanion hole, 248, 250 

P 

Parkinson's disease 

Peptides 

disease, 359 

therapy, 359-360 

synthetic 

structure-function studies in, 437, 438, 440-442, 444, 446, 448 

Peptidomimetic 

drug discovery, 591-598, 613 

Page 644 

Octahydroindole carboxylic acid (Oic), 124 

(see also Protease targets, Receptor targets, Signal transduction protein targets, Structure-based 

drug design) 

Peptidomimetics, 251 

peptide and nonpeptide models, 593-594 

copper complexes as integrase inhibitors, 107 


PGK (see 

Phosphoglycerate 

kinase) 

Pharmacophore, 225, 560 

Phenanthroline

Document 

Phosphoglycerate kinase (PGK) 

catalysis, 376-377 

crystal structure, 376-377, 388 

human, 377 

sequence, 377 

Trypanosoma brucei, 376-377 

Phosphorylysis, 151 

Phosphotransfer, 222 

Platelet aggregation, 247 

active site, 45, 48, 50, 54, 55, 56, 59, 61, 62, 63, 64 

catalytic site, 52, 55, 61 

Polymerization, 41, 48, 50, 55, 61, 67, 70 

mechanism, 56 

Polynucleotidyl transferases, 96-100 

Positive 

inotropes, 295, 296, 297, 298, 300, 309, 310, 314 

inotropy, 295, 297, 298, 305, 314 

PPACK, 250, 261 

Primer grip, 48, 61 

Proliferative diseases 

asthma, 214 

Phosphostatine analogs, 327 

Polymerase, 41, 45, 48, 50, 52, 54, 55, 56, 59, 61, 62, 63, 64, 65, 67, 69 


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atherosclerosis, 214 

fibrosis, 214 

osteo arthritis, 214 

psoriasis, 214 

restenosis, 214 

rheumatoid arthritis, 214 

septic shock, 214 

Protein C, 247 

Protease targets 

angiotensin-converting enzyme (ACE) inhibitors, 576, 607, 610 

aspartyl proteases, 595-596 

classes, 567-568 

cysteinyl proteases, 605 

inhibitors 

elastase, 576, 597, 606 

gelatinase, 576 

human immunodeficiency virus (HIV) protease, 576-578, 595-596, 598, 600-602 

interleukin-converting enzyme (ICE), 576, 597, 607-608 

stromelysin, 576, 596, 609-612 

thermolysin, 597, 610 

thrombin, 576, 578-579, 596, 604 

metalloproteases, 597-598, 607, 609-612 


Document 

[Protease targets] 

serinyl proteases, 603 

x-ray structures 

apoprotein and complexes, 595-598 

Protein data bank, 173 

activation loop, 218, 219 

calmodulin dependent, 219 

caseine (CK-1), 218, 220, 222, 224 

catalytic 

core, 214, 219-221 

loop, 217, 221 

cyclin dependent (CDK-2), 214, 215, 218-219 

EGFR, 223 

insulin receptor (IRK), 214-216, 218-220, 222 

MAP, 214, 218-219 

phosphorylase, 218-220, 222, 224 

protein A (cAPK) 214-216, 218-220, 222-225 

protein C, 224 

phosphorylation, 217, 218 


Page 645 

Protein 

kinase

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