FURTHER EVIDENCE FOR THE IMPORTANCE OF FREE CARBOXYLATE IN EPOXYSUCCINATE INHIBITORS OF THIOL PROTEASES

Analogs of Ep-475 (2a), designed to explore the role played by the car boxylate in epoxysuccinate thiol protease inhibitors, have been synthe sized and tested as inhibitors of papain and cathepsin B. Papain and c athepsin B are rapidly inactivated by carboxylates 2a and 6a, but are inactivated much more slowly by 2b-2f, 6c, and 6f, in which the carbox ylate is absent or replaced by an amide, ester; or ketone. This order of reactivity contrasts with the inherent reactivity of substituted ep oxides toward a non-enzymatic thiolate, previously shown to decrease i n the order: COCH3 > CO2CH3 > CONH2 > H > CO2H. The results suggest th at electrostatic attraction between the carboxylate of the inhibitor a nd 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. T he most reactive isoleucine analog, 6a, was significantly less reactiv e than leucine-containing Ep-475 (2a), while the less reactive isoleuc ine derivatives, 6c and 6f, were similar in reactivity to the correspo nding leucine derivatives, 2c and 2f, respectively.