Peptidyl epoxides as selective protease inactivators

Peptidyl epoxides were developed as a mechanistic probe to distinguish between serine and cysteine proteases, with the expectation of selectively inactivating only the latter. Stereoselective schemes for the synthesis of erythro and threo peptidyl epoxides were developed. Peptidyl epoxides were found to be stable under various conditions, including in human serum. Indeed, peptidyl epoxides did not inhibit serine proteases, while erythro peptidyl epoxides exhibited time- and concentration-dependent inhibition of cysteine proteases. Selectivity within the family of cysteine proteases was achieved by varying the peptide sequence, in good correlation with sequences of known substrates and other inhibitors. Inhibition is due to the formation of a covalent equimolar enzyme-inhibitor complex. The peptidyl epoxide alkylates the enzyme active-site thiol by its 'exo' epoxidic methylene. The alkylation reaction depends on a native conformation of the enzyme and is 10⁸ faster than a bimolecular model reaction. These results suggest that peptidyl epoxides are mechanismbased inhibitors of cysteine proteases and bear mechanistic implications regarding the catalytic activity of this family proteases. (C) 2000 Wiley-Liss, Inc.