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Fw: NATAP/Resist Wksp: New HCV Polymerase Drug

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  • Alley Pat
    NATAP http://natap.org/ ... New HCV Polymerase Inhibitor is in Early Development; resistance at 282 is associated with polymerase drug resistance, but PSI-6130
    Message 1 of 1 , Jul 2, 2006
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      New HCV Polymerase Inhibitor is in Early Development; resistance at 282 is associated with polymerase drug resistance, but PSI-6130 may remain active when mutation is present

      Reported by Jules Levin
      XV Intl Resistance Workshop
      June 13-17, 2006, Sitges, Spain

      "Characterization of resistant HCV Replicon-containing cells selected with PSI-6130"

      P Furman1, L Stuyver1, T McBrayer1, C Niu1,
      M Keilman1, E Murakami1, H Bao1, A De La Rosa1,
      W-R Jiang2, J Symons2 and M J Otto1
      1Pharmasset, Inc., Princeton, NJ, USA;
      2Roche, Palo Alto, CA, USA

      BACKGROUND: Hepatitis C virus (HCV), is an important member of the Flaviviridae family of viruses with 2% of the US population and an estimated 170 million people world-wide being HCV carriers. The current standard of care is pegylated interferon plus ribavirin. However, there is still a need for more potent
      anti-HCV compounds with fewer adverse effects. -betaD-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of HCV in Huh-7 replicon cells (EC90 = 4.6uM) with its 5'-triphosphate inhibiting the HCV NS5B RNA-directed-RNA polymerase (RdRP).

      In vitro resistance characterized by an S to T change in position 282 of the NS5B gene, has been reported for related 2'-C-Methyl purine nucleosides.

      METHODS: PSI-6130 was tested for activity in the replicon assay using a replicon containing the S282T mutation generated by passaging in the presence of 2'-C-methyladenosine. The 5'-triphosphate of PSI-6130 was tested for activity against the cloned HCV RdRp. To determine if PSI-6130 could select for a resistant replicon, passaging experiments were performed. Resistant replicons were tested for their sensitivity to PSI-6130, 2'-C-methyladenosine, and 2'-C-methylcytidine, and their NS5B genes cloned and sequenced. In addition, the expression and activity of cellular dCK was also assessed.

      RESULTS:
      The S282T replicon showed a 6.5 fold increase in EC90 whereas the EC90 value for 2'-C-methyladenosine and 2'-C-methylcytidine was >100 _M. Similar results were seen with a transient transfection assay. In enzyme assays with the S282T RdRp the fold resistance for PSI-6130 5_-triphosphate was 3 fold whereas the fold resistance for 2'-C-methyladenosine and 2'-C-methylcytidine was 150 and 12 fold, respectively. Sequencing the RdRp from resistant replicons generated by passaging in the presence of PSI-6130 did not identify the S282T or any other signature mutation. However, it was found that there was a reduction in cellular dCK activity in these resistant replicons.

      CONCLUSION: The HCV NS5B S282T mutation, which is associated with resistance to several 2'-Cmethyl analogs, remained sensitive to PSI-6130 in both replicon and RdRp assays. Sequencing the RdRp of resistant replicons isolated by passaging in the presence of PSI-6130 failed to identify resistant mutations. However, reduced cellular dCK activity was observed.




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