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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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