Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors (Ingallinella et al. (1998) Biochemistry 37, 8906-8914). Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the Ssite, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex. Hepatitis C virus (HCV) 1 is the major etiologic agent of transfusion associated and sporadic non-A non-B hepatitis, with the estimation of infected individuals being more than 1% of the world population (1). Neither a generally effective therapy nor a vaccine has been developed so far, and much effort is currently devoted to small-molecule drug discovery to eliminate or control HCV infection. In this respect, the inactivation of virally encoded enzymes that are essential for viral replication is generally accepted as the preferential strategy. The N-terminal third of HCV NS3 protein contains a chymotrypsin-like serine protease (henceforth called NS3 protease) that accomplishes four out of the five processing We follow the nomenclature of Schechter and Berger (53 )i n designating the cleavage sites as P6-P5-P4-P3-P2-P1...P1'-P2'- P3'-P4' etc., with the scissile bond between P1 and P1' and the C-terminus of the substrate on the prime site. The binding sites on the enzyme corresponding to residues P6-P5-P4-P3-P2-P1...P1'-P2'- P3'-P4' are indicated as S6-S5-S4-S3-S2-S1...S1'-S2'-S3'-S4' etc. 13844