Solution Structure of Substrate-based Ligands When Bound to Hepatitis C Virus NS3 Protease Domain

Abstract

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 ...

Dissection Study on the Severe Acute Respiratory Syndrome 3C-like Protease Reveals the Critical Role of the Extra Domain in Dimerization of the Enzyme

“…Our results clearly indicate that only the entire enzyme is capable of interacting with the substrate peptide S1. Furthermore, to our surprise, unlike other proteases such as hepatitis C virus N3 protease, which only binds to the N-terminal part after the substrate cleavage (38), the SARS 3CL protease showed binding interactions with both the N-and C-terminal parts of the cleaved substrate peptide S1. These observations indicate that the interaction interface between the SARS 3CL protease and its in vivo substrates might be very large, and the enzymatic activity and specificity are under regulation by many factors.…”

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

“…The structural data for the complex of compound 1 and the NS3 protease domain-NS4A cofactor NS4A were taken from the solution structure of the corresponding complex in the absence of NS4A (9). Additional information was taken from the cocrystal structures of NS3-NS4A with ketoacid inhibitors (13) and the NMR structure of tripeptide inhibitors published by the Boehringer group (26,27). All calculations were carried out with the program BatchMin and the molecular modeling package InsightII/Discover (Biosym Technologies Inc., San Diego, Calif.).…”

Sensitivity of NS3 Serine Proteases from Hepatitis C Virus Genotypes 2 and 3 to the Inhibitor BILN 2061

“…The suspension obtained from E. coli BL21(DE3) pLysS cells was processed in a Dounce homogenizer, supplemented with 20 mM MgCl 2 and 10 g of DNase I/ml, and incubated for 20 min on ice, while the suspension obtained from E. coli BL21(DE3) cells was homogenized by using a microfluidizer. Following a brief sonication, the extract was clarified by a 30-min centrifugation at 21,200 ϫ g. Then the purification was performed essentially as described previously (13). Briefly, the protease domains were purified from the soluble fraction by using SP-Sepharose, heparin, and Superdex 75 columns (Amersham Biosciences).…”