PEPTIDE ALPHA-KETO ESTER, ALPHA-KETO AMIDE, AND ALPHA-KETO ACID INHIBITORS OF CALPAINS AND OTHER CYSTEINE PROTEASES

A series of dipeptidyl and tripeptidyl alpha-keto esters, alpha-keto a mides, and alpha-keto acids having leucine in the P2 position were syn thesized and evaluated as inhibitors for the cysteine proteases calpai n I, calpain II, cathepsin B, and papain. In general, peptidyl alpha-k eto acids were more inhibitory toward calpain I and II than alpha-keto amides, which in turn were more effective than alpha-keto esters. In the series Z-Leu-AA-COOEt, the inhibitory potency decreased in the ord er: Met (lowest K(I)) > Nva > Phe > 4-Cl-Phe > Abu > Nle (highest K(I) ) with calpain I, while almost the reverse order was observed for calp ain II. Extending the dipeptide alpha-keto ester to a tripeptide alpha -keto ester yielded significant enhancement in the inhibitory potency toward cathepsin B, but smaller changes toward the calpains. Changing the ester group in the alpha-keto esters did not substantially decreas e K(I) values for calpain I and calpain II. N-Monosubstituted alpha-ke to amides were better inhibitors than the corresponding alpha-keto est ers. Alpha-Keto amides with hydrophobic alkyl groups or alkyl groups w ith an attached phenyl group had the lower K(I) values. N,N-Disubstitu ted alpha-keto amides were much less potent inhibitors than the corres ponding N-monosubstituted peptide alpha-keto amides. The peptide alpha -keto acid Z-Leu-Phe-COOH was the best inhibitor for calpain I (K(I) = 0.0085 muM) and calpain II (K(I) = 0.0057 muM) discovered in this stu dy. It is likely that the inhibitors are transition-state analogs and form tetrahedral adducts with the active site cysteine of cysteine pro teases and form hydrogen bonds with the active site histidine and poss ibly another hydrogen bond donor in the case of monosubstituted amides . Several inhibitors prevented spectrin degradation in a platelet memb rane permeability assay and may be useful for the treatment of disease s which involve neurodegeneration.