E-64 ANALOGS AS INHIBITORS OF CATHEPSIN-L AND CATHEPSIN-S - IMPORTANCE OF THE S-2-P-2 INTERACTIONS FOR POTENCY AND SELECTIVITY

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl, 1) in which the amino acid (AA) had been systematically varied were test ed as inhibitors of cathepsins 1, and S. These E-64 analogs were desig ned to investigate whether selectivity for cathepsin 1, or cathepsin S could be attained by varying the amino acid bound to the essential ep oxide ring which induces inhibition by alkylating the active site thio l 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 substrat e-like chloromethyl ketones (if the situation is analogous to papain), leads to differences in the orientation of the inhibitor side chain w hen bound in the SZ subsite compared to substrates. The greatest selec tivity was obtained with HO-Eps-Arg-OBzl which exhibited an 89-fold pr eference for cathepsin 1, over cathepsin S. A change from the L to the D stereochemistry for the phenylalanine analogs resulted in a 19-fold drop in k(2)/K-i 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) on e 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-f old 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 S-2-P-2 interactions with substrates cannot be used to enhance the sel ectivity of the E-64 I analogs in a rational manner, the hydrogen-bond ing interaction between the amide proton of the benzyl amid group in H O-Eps-AA-NHBzl and the S-2 subsite for both cathepsins L and S contrib utes to increase the potency of these types of inhibitors.