Peptidyl epoxides as selective protease inactivators

Peptidyl epoxides were developed as a mechanistic probe to distinguish betw een serine and cysteine proteases, with the expectation of selectively inac tivating on ly the latter. Stereoselective schemes for the synthesis of ery thro and three peptidyl epoxides were developed. Peptidyl epoxides were fou nd 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 cystein e proteases. Selectivity within the family of cysteine proteases was achiev ed by varying the peptide sequence, in good correlation with sequences of k nown substrates and other inhibitors. Inhibition is due to the formation of a covalent equimolar enzyme-inhibitor complex. The peptidyl epoxide alkyla tes the enzyme active-site thiol by its "exo" epoxidic methylene. The alkyl ation reaction depends on a native conformation of the enzyme and is 10(8) faster than a biomolecular model reaction. These results suggest that pepti dyl epoxides are mechanism-based inhibitors of cysteine proteases and bear mechanistic implications regarding the catalytic activity of this family of proteases. (C) 2000 Wiley-Liss, Inc.