Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

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Protease inhibitors are potent antiviral agents against human immunodeficiency virus type 1. As with reverse transcriptase inhibitors, however, resistance to protease inhibitors can develop and is attributed to the appearance of mutations in the protease gene. With the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS, 350-to 1,500-fold-resistant variants have been selected in vitro and were found not only to contain mutations in the protease gene but also to contain mutations in Gag precursor p1/p6 and/or NC (p7)/p1 cleavage sites. Mutations in cleavage sites give rise to better peptide substrates for the protease in vitro and to improved processing of p15 precursors in drug-resistant clones. Importantly, removal of cleavage site mutations in resistant clones leads to a decrease or even an absence of viral growth, confirming their role in viral fitness. Therefore, these second-locus mutations indicate that cleavage of p15 is a rate-limiting step in polyprotein processing in highly resistant viruses. The functional constraint of p15 processing also suggests that additional selective pressure could further compromise viral fitness and maintain the benefits of antiviral therapies.

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced linebroadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and proteasebound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors. Hepatitis C virus (HCV)1 infection is an important cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure worldwide (1). Approved therapies with proven benefit for patients with chronic hepatitis C include various drug regimens of interferon-␣. These therapies have limited efficacy with a low sustained response rate and frequent side effects (1). Therefore, there is an urgent need for the development of new therapies for the treatment of HCV infections.HCV is a small enveloped virus containing a single-stranded RNA genome of positive polarity, which encodes a unique polyprotein of approximately 3000 amino acids (for reviews see Refs. 2 and 3). This polyprotein is the precursor of four structural and six nonstructural (NS) proteins (4 -10). The structural proteins are proteolytically processed by host signal peptidases, whereas two virally encoded proteases within the NS2 and NS3 regions process the remaining nonstructural proteins.The NS3 serine protease domain (20 kDa), located within the N-terminal portion of the NS3 protein, mediates the proteolysis at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions (6 -11). We and others have recently reported that N-terminal cleavage products of peptide substrates are competitive inhibitors of NS3 protease activity (12, 13), which has served as the basis for designing substrate-based inhibitors (14, 15). To date, there have been no reports in the literature on the structure of substrates or inhibitors when bound to NS3 protease, which would certainly be valuable for inhibitor design efforts. However, x-ray crystal structures have been determined for NS3 protease alone (16) and for NS3 protease in the presence of an NS4A peptide cofactor (17,18). These structures show that NS3 protease adopts a chymotrypsin/trypsin-like fold.In this report we applied NMR methods to study the structure of peptides and inhibitors, based on N-terminal cleavage products of peptide substrates when bound to the NS3 protease domain of HCV. Transferred NOESY experiments were used to determine the conformation of ligands when bound to the protease, and differential line-broadening experiments were used to identify which segments of the ligands contact the protease. EXPERIMENTAL PROCEDURESPurification of NS3 Protease-A modification of a previously published procedure (19) was used ...

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