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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
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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. _-D-2_-Deoxy-2_-fluoro-2_-C-methylcytidine (PSI-6130) is a potent specific inhibitor of HCV in Huh-7 replicon
cells (EC90 = 4.6_M) 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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