The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-␣ results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections. Hepatitis C virus (HCV)1 is the most common blood-borne infection and a major cause of chronic liver disease and liver transplantation in industrialized countries. The prevalence of HCV infection is estimated to be ϳ5-fold greater than HIV infection and ranges from 1-5% in most developed countries (1). Current therapy is both poorly tolerated and has limited efficacy, with less than 50% response rates among patients infected with the most prevalent virus genotype (1b) (1). Currently approved drugs for the treatment of hepatitis C are interferon-␣ and ribavirin, neither of which appears to act directly on the virus, and their antiviral effects appear to be mediated by multiple, indirect mechanisms. Therefore, there is a need for more efficient and better tolerated anti-HCV agents.The success of antiviral therapies based on chemotherapeutic agents targeting viral polymerases has prompted intense efforts to develop inhibitors of HCV NS5B, the virally encoded RNA-dependent RNA polymerase (RdRp). Studies with HIV reverse transcriptase validate the clinical utility of two distinct classes of viral polymerase inhibitors, nucleoside and non-nucleoside inhibitors. Nucleoside inhibitors function as competitive substrate analogs that prevent RNA chain elongation when incorporated by the viral enzyme, resulting in premature chain termination (2, 3). HIV reverse transcriptase non-nucleoside inhibitors bind to a site residing outside the enzyme active site and inhibit catalysis by an allosteric mechanism (4, 5). Several putative allosteric binding sites on the surface of HCV NS5B have been suggested based on recent structural studies (6 -8), and several chemical classes of NS5B non-nucleoside inhibitors have ...

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2-substituted nucleosides as inhibitors of HCV replication. The 5-triphosphates of 2-C-methyladenosine and 2-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2-O-methylcytidine inhibit HCV RNA replication. The 5-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2-C-methyladenosine triphosphate than 2-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication. Hepatitis C virus (HCV)1 infection is the leading cause of sporadic, post-transfusion, non-A non-B hepatitis (1, 2). One hundred seventy million people worldwide are thought to be infected with hepatitis C virus of which an estimated 4 million reside in the United States (3). Approximately 80% of infected individuals progress to chronic infection. Long term chronic HCV infection can lead to liver cirrhosis and to hepatocellular carcinoma (4 -6). Currently, the recommended therapy is treatment with a combination of interferon ␣2b and ribavirin, which results in a sustained viral response in 40% of patients (7,8). Investigational therapies using a combination of pegylated interferon and ribavirin have lead to an sustained viral response in 54% of patients, but the response rate (42%) of patients harboring HCV genotype 1 is lower (9, 10). Consequently, additional therapies for HCV infection are needed.Antiviral chemotherapies based on administration of analogs of deoxynucleosides have been widely successful as treatment for HIV, herpes virus, and hepatitis B infection (11,12). Intracellular phosphorylation of the nucleoside analog to the triphosphate creates the active form of the inhibitor that then serves as a substrate for the viral polymerase. Generally, incorporation of the nucleotide analog at the 3Ј-end of the replicating viral DNA causes termination of DNA synthesis, owing to the lack of the 3Ј-hydroxyl required for extension. These successes suggest that an investigation of ribonucleoside analogs as inhibitors of HCV replication would be worthwhile.The HCV NS5B protein, the RNA-dependent polymerase responsible for the synthesis of the viral RNA geno...

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2-C-methyl-adenosine and 2-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.Hepatitis C virus (HCV) chronically infects about 3% of the human population, causing a slowly evolving liver disease that leads to cirrhosis, liver failure, and occasionally hepatocellular carcinoma (39). Given the size of the HCV epidemic and the limited efficacy of the present therapy based on alpha interferon (16), the development of new, safer, and more effective drugs is of paramount importance and is presently an area of intensive research. The strategy most widely applied for developing novel anti-HCV therapeutics aims at identifying small molecule inhibitors of viral enzymes. The nonstructural protein 5B RNA-dependent RNA polymerase (NS5B RdRp) is an important target of drug discovery activities largely because it is essential for viral replication and also due to the clinical successes of inhibitors of other viral polymerases. In addition, the extensive structural and biochemical characterization of this enzyme provides the basis for drug design efforts as well as for elucidating the mechanism of action of inhibitors and for rapidly optimizing their potency.The NS5B protein was initially identified as an RdRp based on the presence of the signature GDD (Gly-Asp-Asp) motif characteristic for this class of enzymes (11). Its function was confirmed when an active form of the full-length protein was purified from baculovirus-infected insect cells (5). Subsequently, attempts to improve solubility, stability, and activity lead to the expression of C-terminal-truncated forms lacking the hydrophobic membrane anchor contained within the last 21 amino acids (1,17,25,33,35). In vitro, the enzyme shows little, if any, specificity for the HCV genome and can catalyze the synthesis of RNA by using a variety of homo-or heteropolymeric RNA templates both with and without a primer. In the absence of primer, NS5B can initiate RNA synthesis either by using the 3Ј-termi...