USE OF PAPAIN AS A MODEL FOR THE STRUCTURE-BASED DESIGN OF CATHEPSIN-K INHIBITORS - CRYSTAL-STRUCTURES OF 2 PAPAIN-INHIBITOR COMPLEXES DEMONSTRATE BINDING TO S'-SUBSITES

Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexe s and the rational design of novel cathepsin K inhibitors. Unlike prev iously known crystal structures of papain-inhibitor complexes, our pap ain structures show ligand binding extending deep within the S'-subsit es. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leuci nal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, wer e refined to 2.2- and 2.5-Angstrom resolution with R-factors of 0.190 and 0.217, respectively. The S'-subsite interactions with the inhibito rs are dominated by an aromatic-aromatic stacking and an oxygen-aromat ic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yiel ded a novel, symmetric inhibitor selective for cathepsin K. Simultaneo us exploitation of both S- and S'-sites provides a general strategy fo r the design of cysteine protease inhibitors having high specificity t o their target enzymes.