From Poor Substrates to Good Inhibitors:  Design of Inhibitors for Serine and Thiol Proteases

Serine and thiol proteases react with peptide substrates to form an acyl-enzyme. We have synthesized inhibitors which are pseudo-substrates and react with the proteases to generate acyl-enzymes which hydrolyze slowly. This is achieved by incorporating an electron-donating group near the carbonyl group of inhibitors I [Ac-Phe−C(O)NH−NH−C(O)X] and II [benzyl-O-C(O)-ΨAla-Leu-ArgOMe]. The acyl-enzymes derived from the reaction of I with papain and II with chymotrypsin hydrolyze with t 1/2 of 12 and 1 h, respectively. The increased electron density on the carbonyl group of the inhibitor also reduces the rate of acyl-enzyme formation. Components were incorporated into the inhibitor which interact with the leaving group binding site (S‘ subsite) and which accelerate the rate of reaction of inhibitor with enzyme. For inhibitor I, X = NH(CH3), k on < 0.13 M-1 s-1 for the reaction with papain, but if X = ΨLeu(CH3)2, k on = 105 M-1 s-1. Similar results were obtained with II and chymotrypsin. Concomitant with acyl-enzyme formation, X is released and a slowly hydrolyzing acyl-enzyme remains.