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 cath epsin B, calpain I and calpain II inhibitors. Reagents designed for ca thepsin 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 kinet ic 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 inactivatio n, with no more than 7 500 M-1s-1 for calpain II. K(I) for the reversi ble EI adducts ranged from 11 to 0.06 muM for cathepsin B. Structure o f the possible reversible EI complex is proposed and used to discuss t he effects of structural variation of the inhibitors on the kinetic pa rameters 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 an d Z-Leu-Leu attached to an alpha-azatyrosine unit in P1. They gave poo r results. Title compounds proved to be selective for cysteine proteas es, since no inhibiting activity could be detected toward trypsin, chy motrypsin and porcine pancreatic elastase at 0.1 mM concentration. Rel atively low aspecific alkylating properties were also demonstrated in tests using glutathione as the nucleophile.