E64 S-EPOXYSUCCINYL-L-LEUCYLAMIDO-(4-GUANIDINO)BUTANE] ANALOGS AS INHIBITORS OF CYSTEINE PROTEINASES - INVESTIGATION OF S-2 SUBSITE INTERACTIONS

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl) an d benzyl amides (HO-Eps-AA-NHBzl) (where AA represents amino acid) wer e synthesized as analogues of E64, a naturally occurring inhibitor of cysteine proteinases. These inhibitors were designed to evaluate if se lectivity for cathepsin B could be achieved by varying the amino acid on the basis of known substrate specificity. Contrary to the situation with substrates, it was found that variation of the amino acid in the E64 analogues does not lead to major changes in the kinetic parameter k(inac.)/K-i and that the specificity of these analogues does not par allel that observed for substrates. This is particularly true in the c ase of the benzyl ester derivatives where the deviation from substrate -like behaviour is more important than with the benzyl amide derivativ es. The results suggest that the amide proton of the benzyl amide grou p in HO-Eps-AA-NHBzl interacts in the S-2 subsite in both cathepsin B and papain and contributes to increase the potency of these inhibitors . The kinetic data also suggest that differences in the orientation of the C alpha-C beta bond of the side chain in the S-2 subsite of the e nzyme might explain the differences between substrate and E64 analogue specificities. This hypothesis is supported by the fact that the orde r of inactivation rates with chloromethane inhibitors (which are belie ved to be good models of enzyme-substrate interactions) is indeed very similar to that observed with the corresponding amidomethylcoumarin s ubstrates. In conclusion, the information available from S-2-P-2 inter actions with substrates cannot be used to enhance the selectivity of t he E64 analogues in a rational manner.