SYNTHESIS AND INHIBITING ACTIVITIES OF 1-PEPTIDYL-2-HALOACETYL HYDRAZINES TOWARD CATHEPSIN-B AND CALPAINS

Twenty-four 1-peptidyl-2-haloacetyl hydrazines which can be considered azapeptide halomethanes were synthesized and tested as models of cathepsin B, calpain I and calpain II inhibitors. Reagents designed for cathepsin B inactivation include Z-Tyr, Z-Tyr(I) and Z-Leu-Leu attached to an alpha-azaglycine or alpha-azaalanine unit in P1. By use of kinetic analysis, they proved to irreversibly inactivate cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants in the range 725-306 000 M-1s-1 were found for cathepsin B inactivation, with no more than 7 500 M-1s-1 for calpain II. K(I) for the reversible EI adducts ranged from 11 to 0.06 muM for cathepsin B. Structure of the possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. I-Peptidyl-2-haloacetyl hydrazines designed for calpain inactivation include Boc-Val-(N(epsilon)-carbomethoxy)Lys-Leu, Boc-Val-Lys(N(epsilon)-Z)-Leu, Boc-Val-Lys(N(epsilon)-Tos)-Leu and Z-Leu-Leu attached to an alpha-azatyrosine unit in P1. They gave poor results. Title compounds proved to be selective for cysteine proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration. Relatively low aspecific alkylating properties were also demonstrated in tests using glutathione as the nucleophile.