2-Deoxy-2-fluorocytidine (FdC) is a potent inhibitor of the hepatitis C virus RNA replicon in culture, and FdC-5-triphosphate is an effective inhibitor of the NS5B polymerase. Dynamic profiling of cell growth in an antiviral assay showed that FdC caused cytostasis due to an S-phase arrest. These observations demonstrate that FdC treatment is affecting both a viral target and a cellular target.Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in the United States, with sequelae including fibrosis, cirrhosis, and hepatocellular carcinoma (1). In vivo, HCV replication occurs mainly in the cytoplasm of infected hepatocytes, but it has been difficult to demonstrate replication in vitro. Replicon-based systems have now been developed that sustain efficient replication of HCV RNA in cell culture. Initially, subgenomic replicons that expressed only nonstructural proteins were constructed; however, recent reports described replicons that can express the entire HCV polyprotein (5, 7).In addition to the currently approved standard treatment options for HCV infections that use interferon and ribavirin, several new antiviral agents are in preclinical or clinical development. Similar to the case with human immunodeficiency virus type 1 treatment, multiple drug targets (e.g., protease, helicase, polymerase, and entry) may be needed to limit the emergence of drug-resistant variants. The HCV subgenomic replicon provides an excellent system for evaluating HCV antiviral agents in cell culture (3,5,6,10,16,18). We report here the antiviral activity of 2Ј-deoxy-2Ј-fluorocytidine (FdC) (Fig. 1) measured in the HCV subgenomic replicon system and in the bovine viral diarrhea virus (BVDV)-Madin-Darby bovine kidney (MDBK) cell system. HCV-replicon RNA-containing Huh-7 cells (Clone A cells; Apath, LLC, St. Louis, Mo.) were kept in exponential growth as described previously (16). Antiviral assays were performed in medium without G418. Cells were seeded in a 96-well plate at 1,000 cells per well, and test compounds were added immediately after seeding. After 96 h of incubation, total cellular RNA was isolated (Rneasy 96 kit; Qiagen, Valencia, Ca.), and HCV replicon RNA and an internal control (TaqMan rRNA Control Reagents; Applied Biosystems, Foster City, Ca.) were amplified in a single-step multiplex reverse transcription-PCR protocol. FdC (obtained from the Pharmasset compound library) was tested in a concentration range of 0.1 to 200 M, and a 90% effective concentration (EC 90 ) for reducing the intracellular HCV replicon RNA levels of 5.0 M was found ( Fig. 2A). FdC was found to be more potent than ribavirin (EC 90 , ϳ100 M) and comparable in potency to -D-N 4 -hydroxycytidine (NHC) (EC 90 ϭ 5 M) (16). The cellular toxicity against Huh-7 and HepG2 cells was measured after 96 h of incubation by using the CellTiter 96 AQ ueous One solution cell proliferation assay (Promega, Madison, Wis.), and the concentration resulting in 50% reduction in cell growth (CC 50 ) was found to be greater than 100 M. This resul...
A new approach to the synthesis of 2',3'-didehydro-2',3-dideoxynucleosides was described in excellent yield through unusual olefin formation by PhSe-F trans-elimination.
Nucleic acids U 0700Unusual Olefin Formation by PhSe-F trans-Elimination. -The title elimination leads to product (V) rather than the expected 3'-F analogue (some yields not given).-(DU*, J.; SHI, J.; CHUN, B.-K.; HOBBS, A.; HOLLECKER, L.; WATANABE, K. A.; Nucleosides, Nucleotides Nucleic Acids 24 (2005) 9, 1289-1292; Pharmasset, Inc., Tucker, GA 30084, USA; Eng.) -K. Woydowski 08-185
Synthesis of 2-Deoxy-2-fluoro-2-C-methyl-D-ribofuranoses. -The synthesis of novel carbohydrate building blocks for base-modified 2'-fluoronucleosides is described. Key step is the fluorination of the tertiary center of 2-C-methyl-β-D-arabinofuranoside (II). -(CLARK*, J. L.; MASON, J. C.; HOBBS, A. J.; HOLLECKER, L.; SCHINAZI, R. F.; J.
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