E-64 Analogs as Inhibitors of Cathepsin L and Cathepsin S: Importance of the S2-P2 Interactions for Potency and Selectivity1

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl, 1) in which the amino acid (AA) had been systematically varied were tested as inhibitors of cathepsins L and S. These E-64 analogs were designed to investigate whether selectivity for cathepsin L or cathepsin S could be attained by varying the amino acid bound to the essential epoxide ring which induces inhibition by alkylating the active site thiol of the cysteine proteases. The results indicate that the specificity of these analogs does not parallel that observed for substrates. This is possibly due to the fact that the direction of the peptide portion of these analogs, which is the reverse of that found for the substrate-like chloromethyl ketones (if the situation is analogous to papain), leads to differences in the orientation of the inhibitor side chain when bound in the S2subsite compared to substrates. The greatest selectivity was obtained with HO-Eps-Arg-OBzl which exhibited an 89-fold preference for cathepsin L over cathepsin S. A change from the L to the D stereochemistry for the phenylalanine analogs resulted in a 19-fold drop in k2/Ki for cathepsin L and a 14-fold drop for cathepsin S. Both E-64 and Cbz-Phe-Ala-CH2Cl form two hydrogen bonds with Gly 66 in the active site of papain. With the benzyl esters (HO-Eps-AA-OBzl) one of these hydrogen bonds is necessarily absent. In order to evaluate the importance of this hydrogen bond, three benzyl amide derivatives (HO-Eps-AA-NHBzl, 2) were synthesized. In all cases the potency of the inhibitor was increased and indeed the HO-Eps-Phe-NHBzl analog is 64-fold more potent than the corresponding benzyl ester. For cathepsin L, there is also a 237-fold preference for L-Phe over D-Phe in the benzyl amide analog. In conclusion, although the information available from S2-P2 interactions with substrates cannot be used to enhance the selectivity of the E-64 I analogs in a rational manner, the hydrogen-bonding interaction between the amide proton of the benzyl amid group in HO-Eps-AA-NHBzl and the S2 subsite for both cathepsins L and S contributes to increase the potency of these types of inhibitors.