Design of protease inhibitors on the basis of substrate stereospecificity.

The substrate stereospecificity in enzymic reactions, which is one of characteristics of enzymes along with the substrate and regiospecificity can provide a basis for the rational design of inhibitors. This has been demonstrated using alpha-chymotrypsin, a prototypic serine protease as a model enzyme. On the basis of the structure-activity relationships for substrates as well as inhibitors and mechanism of the enzymic reaction, a schematic three-dimensional model of the S1 subsite of alpha-chymotrypsin is constructed. It was envisioned from the three-dimensional active site model that 2-benzyl-3,4-epoxybutanoic acid methyl ester (1) having a (2S)-configuration would bind the enzyme with its oxirane ring being rested at the catalytic site, in which the oxirane ring is subject to a nucleophilic attack by the Ser-195 hydroxyl to form a ether linkage. Kinetic analysis of the enzymic reaction in the presence of the potential inhibitors showed that (2S,3R)-1 inactivates alpha-chymotrypsin, while (2S,3S)-1 inhibits the enzyme competitively. The lack of inactivating activity in the case of (2S,3S)-1 may be due to the unfavorable alignment of the C3-O bond with respect to the hydroxyl of Ser-195 for the SN2-type ring cleave reaction of the oxirane moiety. When the design protocol was applied to papain, a representative cysteine protease, (2S,3S)-1 inhibited the enzyme irreversibly, while (2S,3R)-1 inhibited reversibly. On the basis of the stereospecificity shown in the inactivation of the enzymes, it was inferred that in the case of alpha-chymotrypsin, the nucleophilic attack of the Ser-195 hydroxyl at the scissile carbonyl carbon of substrates occurs in a si fashion, while the thiolate of Cys-25 in papain attacks the substrate amide bond in a re fashion. The inhibitor design protocol may be applied to other proteases.