Synergy of Small Molecular Inhibitors of Hepatitis C Virus Replication Directed at Multiple Viral Targets

Abstract

Chronic hepatitis C virus (HCV) infection isChronic hepatitis C virus (HCV) infection is a major worldwide health problem; in the United States, an estimated 3 million persons are chronically infected (4). Estimates of the health care burden of chronic HCV infection predict a drastic increase in hospitalizations and medical costs related to complications such as cirrhosis and hepatocellular carcinoma over the next 1 to 2 decades (3). Effective and better-tolerated therapy for HCV could effectively stem this tide (7).Current interferon-based therapy for chronic HCV infection results in sustained responses in roughly 55% of patients and is accompanied by significant toxicity. Genotype 1 HCV, the most prevalent genotype in the United States, responds less well to therapy with pegylated interferon plus ribavirin, with response rates of 42 to 46% (11, 23). These limitations have spurred an intense drug discovery effort, resulting in a number of promising compounds (8).Hepatitis C virus replication takes place in the cytoplasm, with the replication complex being tightly associated with lipid membranes (1). Key components of the replication complex include several promising antiviral targets, including the NS3/4A protease and the NS5B RNA-dependent RNA polymerase. A number of candidate protease inhibitors (PIs) which have excellent potency in vitro have been developed (2,17,20); several of these compounds have also been evaluated in phase I/II trials, with encouraging results (15,16,29,36). Resistance to this class of inhibitors has been described, with some mutations conferring cross-resistance to several compounds (17,18,21,34,35).The NS5B RNA polymerase is also essential for viral replication, and a number of nucleoside inhibitors and nonnucleoside inhibitors (NNIs) of the HCV polymerase with potent activity in vitro and in early clinical trials have been described (5,12,13,27,30). Resistance to both nucleoside and nonnucleoside inhibitors in vitro has been described (22,24,26).We have assessed a number of combinations of HCV inhibitors with several molecular targets currently in development, using an HCV genotype 1 replicon-based luciferase reporter system.Replicon constructs. The BM4-5 replicon is a subgenomic HCV genotype 1b replicon which contains a deletion of a serine in NS5A and has been previously described (14). The firefly luciferase gene was inserted in the BM4-5 replicon, in a manner previously described (33), to create a luciferase/neomycin phosphotransferase fusion protein (FEO) and the replicon (BM4-5 FEO). Briefly, the Photinus pyralis luciferase gene was amplified using primers coding for the AscI restriction site. Following amplification, both the BM4-5 plasmid and luciferase PCR product were restriction digested with AscI. Ligation was then carried out to insert the luciferase gene in phase with the neomycin phosphotransferase gene, creating the desired BM4-5 FEO replicon. The sequence of the replicon was verified by DNA sequencing.Cell culture. Human hepatoma Huh-7.5.1 cells (a kind gift from Fra...

Synergy of a Hepatitis C Virus (HCV) NS4A Antagonist in Combination with HCV Protease and Polymerase Inhibitors

“…Cell culture and luciferase compound assays were performed as previously described in detail (9,27). Briefly, 10,000 BM4-5 FEO cells/ well were seeded into 96-well plates and incubated for 4 h. Medium was then aspirated and replaced with 100 l of complete medium containing a single compound or combinations at the desired concentration(s).…”

“…The BM4-5 replicon is a subgenomic HCV genotype 1b replicon which contains a deletion of a serine in NS5A (10). The firefly luciferase gene was inserted into the BM4-5 replicon, in a manner we and others have previously described (26,27), to generate the BM4-5 FEO replicon. The sequence of the replicon was verified by DNA sequencing.…”

The Octadecyloxyethyl Ester of ( S )-9-[3-Hydroxy-2-(Phosphonomethoxy) Propyl]Adenine Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication in Genotype 1A, 1B, and 2A Replicons

“…The HCV genotype 1B replicon BM4-5 FEO has been previously described (26) and contains a firefly luciferase-neomycin phosphotransferase fusion protein. The SGR-JFH FEO replicon, based on the full-length JFH-1 genotype 2A HCV virus (25), was created by insertion of the PacI-KpnI fragment of the BM4-5 FEO plasmid into the PacI-KpnI-digested SGR-JFH1/Luc plasmid (Mighty Mix; Takara).…”

“…In vitro transcription and generation of cell lines stably expressing the BM4-5 FEO and SGR-JFH-1 FEO replicons were accomplished by electroporation of human hepatoma Huh-7.5.1 cells (a kind gift from Francis Chisari, Scripps Research Institute, La Jolla, CA) and selection with 500 g/ml of G-418 as we have previously described (26).…”

“…Compound activity assays were carried out as previously described using 10,000 (BM4-5-or JFH-1-based) FEO replicon cells per well on 96-well plates (26). Cells and compounds were incubated for 48 to 72 h with all conditions run in triplicate.…”