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...

We report that residues Lys-16 and Asp-l19 play critical roles in the guanine nudeotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala'19] Comparisons of amino acid sequences suggested two peptide regions of Ha-ras protein (Ha) p21 that might be involved in nucleotide interactions. Ha p21 sequence 5-20 is homologous to sequences present in a large series of nucleotide-binding proteins, with the sequence Gly-Lys that occurs at positions 15-16 being invariant (14). Structural studies of adenylate kinase have implicated the involvement of this region with binding to the phosphate groups of the nucleotide (15). The sequence Asn-Lys-Xaa-Asp that occurs at positions 116-119 is less universal but occurs in the known sequences for G proteins [e.g., EF-Tu, EF-G, and a-transducin (16, 17)].To examine the role of these residues in Ha-ras protein function, we investigated the biochemical properties of Ha proteins having amino acid substitutions at positions 16, 117, and 119. The biological properties of these proteins were determined in both mammalian and yeast cells. MATERIALS AND METHODSProtein Biochemistry. Oligonucleotide mutagenesis (11) of the genes for Ha p21 and [Val12,Thr59Ha was used to create ras variants, with all mutations confirmed by nucleotide sequencing. Mutant proteins were expressed in Escherichia coli strain HB101 and purified by chromatography on DEAESephacel and Sephadex G-75 in buffer A (50 mM sodium Hepes, pH 7.5/1 mM sodium EDTA/1 mM sodium EGTA/ 1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/0.01% n-octyl glucoside), as described (11). For [Ala'19]Ha, 5 mM MgCl2 and 10 ,M GDP were included in buffer A through the DEAE-Sephacel chromatography (4°C), and then purification was continued in buffer A by highperformance liquid chromatography (LKB) on a column of Superose 12 (Pharmacia) at 22°C, followed by immediate chilling. Protein determinations and other assays were as described (11).In trypsin-digestion experiments, pure Ha proteins (2-4 ,ug) were incubated at 4°C in 10 ,u of 50 mM sodium Hepes, pH 7.5/10 mM MgCl2/1 mM dithiothreitol with nucleotides (Sigma) for 15 min prior to the addition of 1 ug of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). Reactions were terminated by the addition of 3 ,ug of soybean trypsin inhibitor (Sigma).Equilibrium-dialysis measurements of nucleotide binding utilized 4-mm dialysis tubing with a 12-to 14-kDa cutoff (Spectra/Por 2, Spectrum Medic...

Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.

We previously identified a series of compounds which specifically inhibited the transcription of influenza A and B viruses (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J. Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994). The compounds, 4-substituted 2,4-dioxobutanoic acids, selectively targeted the cap-dependent endonuclease activity of the transcriptase complex. Additionally, several of these compounds effectively inhibited the replication of influenza virus but not other viruses in cell culture assays. Here, we report on the anti-influenza virus activities of other potent derivatives of the series evaluated in both in vitro and in vivo infectivity assays. These compounds inhibited the replication of influenza virus in yield reduction assays, with 50% inhibitory concentrations ranging from 0.18 to 0.71 microM. These 50% inhibitory concentrations were similar to those observed for inhibition of in vitro transcription (0.32 to 0.54 microM). One selected compound also elicited a dose-dependent inhibition of influenza virus replication in mice following an upper respiratory tract challenge. These studies demonstrate the antiviral efficacy of this inhibitor class and thereby establish the utility of influenza virus endonuclease as a chemotherapeutic target.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human An essential step in the replicative cycle of human immunodeficiency virus type 1 (HIV-1) is the synthesis, catalyzed by the virally encoded reverse transcriptase (RT), of a DNA copy of the viral RNA. Accordingly, the development of RT inhibitors has been the central focus of numerous anti-HIV-1 therapeutic research programs. Over the past several years, a chemically diverse class of RT inhibitors has been described. These compounds have been designated the nonnucleoside RT inhibitors to distinguish them from the nucleoside analogs. The class includes the pyridinone derivatives L-697,661 and L-696,229 as well as BI-RG-587 and the TIBO derivative R82913 (7,8,14,16,18,22). These compounds are potent inhibitors of HIV-1 infection in cell culture.