Further Evidence for the Importance of Free Carboxylate in Epoxysuccinate Inhibitors of Thiol Proteases

Abstract

Analogs of Ep-475 (2a), designed to explore the role played by the carboxylate in epoxysuccinate thiol protease inhibitors, have been synthesized and tested as inhibitors of papain and cathepsin B. Papain and cathepsin B are rapidly inactivated by carboxylates 2a and 6a, but are inactivated much more slowly by 2b-2f, 6c, and 6f, in which the carboxylate is absent or replaced by an amide, ester, or ketone. This order of reactivity contrasts with the inherent reactivity of substituted epoxides toward a non-enzymatic thiolate, previously shown to decrease in the order: COCH3 > CO2CH3 > CONH2 > H > CO2H. The results suggest that electrostatic attraction between the carboxylate of the inhibitor and protonated His159 of papain facilitates docking of the inhibitor in the active site of the enzyme, a conclusion reached previously from X-ray crystallographic structures of epoxysuccinates bound to papain. The most reactive isoleucine analog, 6a, was significantly less reactive than leucine-containing Ep-475 (2a), while the less reactive isoleucine derivatives, 6c and 6f, were similar in reactivity to the corresponding leucine derivatives, 2c and 2f, respectively.