CHARACTERIZATION OF ENGINEERED HEPATITIS-C VIRUS NS3 PROTEASE INHIBITORS AFFINITY SELECTED FROM HUMAN PANCREATIC SECRETORY TRYPSIN-INHIBITOR AND MINIBODY REPERTOIRES

Given the extent of hepatitis C virus (HCV) infection as a worldwide h ealth problem and the lack of effective treatment, the development of anti-HCV drugs is an important and pressing objective. Previous studie s have indicated that proteolytic events mediated by the NS3 protease of HCV are fundamental to the generation of an active viral replicatio n apparatus, as unequivocably demonstrated for flaviviruses. As a resu lt, the NS3 protease has become a major target for discovering anti-HC V drugs. To gain further insight into the biochemical and biophysical properties of the NS3 enzyme binding pocket(s) and to generate biologi cal tools for developing antiviral strategies, we decided to engineer macromolecular ligands of the NS3 protease domain. Phage-displayed rep ertoires of minibodies (''minimized'' antibody-like proteins) and huma n pancreatic secretory trypsin inhibitor were sampled by using the rec ombinant NS3 protease domain as a ligate molecule. Two protease inhibi tors were identified and characterized biochemically. These inhibitors show marked specificity for the viral protease and potency in the mic romolar range but display different mechanisms of inhibition. The impl ications for prospective development of low-molecular-weight inhibitor s of this enzyme are discussed.