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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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