An NS3 Serine Protease Inhibitor Abrogates Replication of Subgenomic Hepatitis C Virus RNA

Abstract

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-␣ (IFN-␣), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-␣ showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-␣ also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection. HCV1 as a member of the Flaviviridae family is the major etiological agent of non-A, non-B viral hepatitis and an important cause of chronic liver disease leading to cirrhosis and hepatocellular carcinoma in humans (1, 2). An estimated 170 million people worldwide are infected with HCV, and end stage liver disease associated with this virus is now the leading cause of liver transplantation in the western world (3). Many patients treated with IFN-␣ alone or with a combination of IFN-␣ plus ribavirin fail to show a sustained virologic response and currently have no other treatment option. Given the high prevalence of the infection, HCV has become the focus of intensive research. Originally cloned in 1989 (1), the viral RNA genome is now well characterized. The ϳ9600-nucleotide genome is of positive polarity that encodes a ϳ3000-amino acid polyprotein, which is the precursor of at least 10 mature viral proteins: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C is the nucleocapsid protein that binds and encapsulates the viral RNA genome (4), E1 and E2 are the virion glycoproteins, and p7 is of unknown function (5). The NS2 to NS5B proteins inclusively are thought to comprise nonstructural proteins involved in replication and polyprotein processing (6). The individual proteins are processed from the polyprotein by a combination of host and viral proteases. Host signal peptidases are responsible for the cleavages between C, E1, E2, p7, and NS2. The cleavage between NS2 and NS3 is performed in an autoproteolytic manner by the metal-dependent protease NS2/3 (7, 8). The proteolytic release of NS4A, NS4B, NS5A, and NS5B is catalyzed by the multifunctional NS3 enzyme, which in conjunction with the mature NS4A cofactor mediates efficient processing (for a review, see Ref. 9). The large polyprotein open...

Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3

“…This is further proof of the high specificity of the inhibition. Earlier studies demonstrated that compound A inhibits proteolytic activity of NS3 in genotype 1b HCV (51). Our data show that compound A is also efficient in inhibiting genotype 1a NS3.…”

“…The NS3 protease is competitively inhibited by specific penta-or hexapeptides derived from the aminoterminal NS3 cleavage products (69,79). One potent and highly specific inhibitor of the NS3 protease activity (compound A) was shown to reduce replication of a subgenomic serotype 1b HCV RNA to undetectable levels in Huh7 cells (51).…”

Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

“…HCV is an RNA virus and, in many cases, is difficult to eradicate from infected individuals even with an intensive antiviral therapy that utilizes IFN and ribavirin (7,14,15,25,30,41). Although a number of other antiviral compounds, including inhibitors against the NS3 protease and the RNA-dependent RNA polymerase of NS5B (1,4,8,39), are currently being tested for their therapeutic applicability, such attempts have not always been promising.…”

Hepatitis C Virus Subgenomic Replicons in the Human Embryonic Kidney 293 Cell Line

“…Whole-cell extracts obtained by lysing cells in Laemmli buffer were resolved by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and then blocked in 5% nonfat milk in Tris-buffered saline-Tween buffer. Blots were incubated with either E1A monoclonal antibody (NeoMarkers), ␣-fetoprotein (AFP) polyclonal antibody (NeoMarkers), albumin polyclonal antibody (Sigma), NS3 polyclonal antibody (27), or NS5A monoclonal antibody (Maine Biotech Services). Membranes were then incubated with the appropriate mouse-or rabbit-specific antibodies conjugated with horseradish peroxidase, developed by use of LumiLight Western blotting solution (Roche), and exposed to film (Hyperfilm; Amersham Pharmacia Biotech).…”

“…Approximately 10 5 cells were grown on LabTek 4-well chamber slides (Nalge Nunc International) that were precoated with 150 g of poly-D-lysine per ml. At 1 day postseeding, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then incubated with either a polyclonal antibody against NS3 protein (27) or a monoclonal antibody against NS5A (Maine Biotech Services), and bound antibodies were detected with AlexaFluor 488 goat-anti-rabbit or AlexaFluor 488 goat anti-mouse antibody, respectively (Molecular Probes). Cells were visualized with a fluorescence detection microscope (Olympus).…”