Hepatitis C virus (HCV) is one of the most important Flaviviridae infections in humans and is responsible for the second most common cause of viral hepatitis. Presently, nearly 2% of the U.S. population, and an estimated 170 million people worldwide, are HCV carriers (2). The only approved therapy for chronic hepatitis C is alpha interferon (IFN-␣), either alone or in combination with ribavirin. Anemia is the most common adverse effect associated with ribavirin treatment, and neuropsychiatric adverse effects of IFN-␣ lead to premature cessation of therapy in 10 to 20% of patients (9, 13).As additional treatment options are urgently needed, there is an ongoing search for more potent antiviral compounds with fewer adverse effects. However, the search for improved antiviral agents is hampered by the limited and cumbersome propagation of HCV in vitro (4). Therefore, surrogate models such as the HCV RNA replicon that replicates in the human hepatoma cell line Huh7 have been developed (6,29). Improved versions of these HCV replicons contain adaptive mutations (25), and their use has facilitated the evaluation of candidate anti-HCV drugs.Bovine viral diarrhea virus (BVDV) is one of the best characterized members of the Flaviviridae family and has one of the largest RNA genomes (12.5 kb) in this family (8). This virus has the remarkable property of existing as noncytopathic and cytopathic (cpBVDV) biotypes, with cpBVDV strains showing insertions or viral genome rearrangements at the junction site between nonstructural protein 2 (NS2) and NS3 (32). BVDV may provide a surrogate model for HCV, both for the molecular study of viral proteins (33) and for the evaluation of antiviral compounds (3,7,47).In the search for therapeutic agents, any element that is essential for viral (or replicon) RNA replication may be considered a drug discovery target. Such elements can be either viral proteins (NS2-NS3 protease, NS3-NS4A serine proteinase, NS3 RNA helicase, or RNA-dependent RNA polymerase [3,24,34,36] [18,31,41,43]). Current knowledge of the human genome, combined with array technology and pathogen infection models, will likely lead to more defined host-pathogen-related targets for future drug design (17, 23). Today, however, the most successful classes of antiviral compounds with clinical utility in combat against other human viral pathogens (human immunodeficiency virus type 1 [HIV-1], hepatitis B virus [HBV], herpes simplex virus [HSV], and cytomegalovirus) are the protease, the nonnucleoside analogue, and the nucleoside analogue inhibitors. As the latter class of compounds is crucial in controlling herpesvirus, HIV-1, and HBV infections, it is likely and anticipated that

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.

) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K m of 81 M and a k cat of 0.007 s ؊1 , but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K i ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 M. Similar results were obtained with 2-C-methyladenosine triphosphate (K i ‫؍‬ 1.5 M) and 2-C-methylcytidine triphosphate (K i ‫؍‬ 1.6 M). NS5B with the S282T mutation, which is known to confer resistance to 2-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.Hepatitis C virus (HCV) is an RNA virus which possesses a single-stranded positive-sense RNA as the viral genome. This viral RNA plays important roles during viral replication, as it serves as an mRNA for viral protein synthesis, a template for RNA replication, and a nascent RNA genome for a newly formed virus (17). HCV NS5B RNA-dependent RNA polymerase is a key enzyme in viral RNA replication. This enzyme, which does not require a primer for initiation of RNA synthesis, catalyzes de novo RNA synthesis (8, 11). Nucleoside analogs have been used to treat viral infections, such as herpes simplex virus, human immunodeficiency virus, and hepatitis B virus infections (5,6,21). These drugs are designed to inhibit viral polymerases by a process called chain termination, in which DNA synthesis is quenched by incorporating the triphosphate forms of these drugs, which lack the 3Ј-hydroxyl group on the sugar moiety. In order for nucleoside analogs to inhibit a viral polymerase, they must be transported into the cell and converted to the active 5Ј-triphosphate form by cellular kinases. 2Ј-C-Methylnucleosides have been investigated as anti-HCV agents targeting HCV NS5B RNA polymerase (2, 20). 2Ј-C-Methyladenosine (2Ј-C-Me-A) and 2Ј-C-methylguanosine (2Ј-C-Me-G) showed potent anti-HCV activities in a cell-based replicon assay, and their triphosphate forms inhibited replicase and NS5B RNA polymerase in vitro (20). In addition, 2Ј-C-Me-A exhibited significant activity against HCV in a cell culture system which involves complete HCV replication and which produces infectious HCV (16). A resistant replicon has been selected by passage of HCV in the...

The antiviral activity of 2'-C-MeC against strains of two different NoV genogroups and the low EC(50) suggest that this nucleoside analogue may be effective against the more prevalent GII NoVs. In the absence of a vaccine, antiviral agents could be an effective intervention to control the spread of human NoV in populations at a high risk for NoV disease.