INHIBITION OF CRUZIPAIN VISUALIZED IN A FLUORESCENCE QUENCHED SOLID-PHASE INHIBITOR LIBRARY ASSAY - D-AMINO-ACID INHIBITORS FOR CRUZIPAIN, CATHEPSIN-B AND CATHEPSIN-L

A PEGA-resin was derivatized with a 3:1 mixture of hydroymethyl benzoi c acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO2)KLR FSKQK(Abz)-PEGA was assembled on the lysine using the active ester app roach. Following esterification of the hydroxymethyl benzoic acid with Fmoc-Val-OH a library XXX-k/r-XXXV containing approximately 200,000 b eads was assembled by split synthesis. The resulting 'one bead, two pe ptides' library was subjected to extensive hydrolysis with cruzipain. One hundred darker beads were isolated and the 14 most persistently da rk beads were collected and sequenced. The putative inhibitor peptides and several analogues were synthesized and found to be competitive mu M to nM inhibitors of cruzipain in solution. The inhibitory activity was found to be unspecific to cruzipain when compared with cathepsins B and L and specific when compared with kallikrein. One of the inhibit ors was docked into the active site of cathepsin B and was found most probably to bind to the enzyme cavity in an unusual manner, owing to t he inserted D-amino acid residue. (C) 1998 European Peptide Society an d John Wiley & Sons, Ltd.