Ribavirin 5′-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5′-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5′-triphosphate and guanosine 5′-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP.

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Pyrimidine metabolism is a major route for therapeutic intervention against malaria. Here we report inhibition and structural studies on the deoxyuridine nucleotidohydrolase from the malaria parasite Plasmodium falciparum (PfdUTPase). We have identified a series of triphenylmethane derivatives of deoxyuridine with antimalarial activity in vitro which inhibit specifically the Plasmodium dUTPase versus the human enzyme. A 2.4 Angstrom crystal structure of PfdUTPase in complex with one of these inhibitors reveals an atypical trimeric enzyme in which the triphenylmethane derivative can be seen to select for PfdUTPase by way of interactions between the trityl group and the side chains of residues Phe46 and Ile117. Immunofluorescence microscopy studies of parasitized red blood cells reveal that enzyme concentrations are highest during the trophozoite/schizont stages, suggesting that PfdUTPase has a major role in DNA replication. Taken together the data show that PfdUTPase may be considered as an antimalarial drug target.

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2-␣-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of ␣-hydroxy moieties. 2-Deoxy-2-␤-fluoro-4-azidocytidine (RO-0622) and 2-deoxy-2-␤-hydroxy-4-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC 50 ‫؍‬ 171 ؎ 12 nM and 24 ؎ 3 nM for RO-9187 and RO-0622, respectively; CC 50 >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4-azidocytidine) or 2-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC 50 values 8 -150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2-␣-deoxy-4-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection. Hepatitis C virus (HCV)3 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation. Current treatment options available to HCV-infected persons have limitations with regard to efficacy and tolerability. Only about 50% of individuals infected with HCV genotype 1 achieve sustained virological response when treated with a combination of pegylated interferon ␣ and ribavirin (1, 2). In addition, high viral load, age, body weight, co-infection with human immunodeficiency virus, and cirrhosis negatively affect the probability of achieving sustained virological response (3, 4). Therefore, there is an urgent need to develop new and more effective therapies for the treatment of HCV infection. A number of new antiviral candidates are currently being evaluated in clinical studies, the majority targeting either the HCV protease or HCV polymerase enzymes, which are essential for viral replication (5). The HCV RNA-dependent RNA polymerase, NS5B, contains the active site responsible for viral RNA synthesis and functions as part of a membrane-associated replicase complex. Nucleoside and non-nucleoside inhibitors of HCV polymerase h...

This paper describes a structure-activity study to identify novel, small-molecule inhibitors of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) from parasitic protozoa. The successful synthesis of a variety of analogues of dUMP is described in which the substituents are introduced at the 3'- and 5'-positions, together with variation in the heteroatom at the 5'-position. The compounds were assayed against recombinant Plasmodium falciparum and Leishmania major enzymes and the human enzyme to give a measure of selectivity. The compounds were also tested in vitro against the intact parasites P. falciparum and L. donovani. A number of potent and selective inhibitors of the P. falciparum dUTPase that show drug-like properties and represent good leads for future development were identified. The best inhibitors included the compounds 5'-tritylamino-2',5'-dideoxyuridine (2j) (Ki = 0.2 microM) and 5'-O-triphenylsilyl-2',3'-didehydro-2',3'-dideoxyuridine (5h) (Ki = 1.3 microM), with selectivity greater than 200-fold compared to the human enzyme. Structural features important for antiplasmodial activity were determined. The correlation observed between the inhibition of the enzyme and the inhibition of the parasite growth in vitro demonstrates that the P. falciparum dUTPase constitutes a valid and attractive novel target for the development of much-needed new antimalarial drugs.