Mechanism of activation of β-D-2′-deoxy-2′-fluoro-2′-C-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5'-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K-m of 81 mu M and a k(cat) of 0.007 s(-1), but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K-i) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 mu M. Similar results were obtained with 2'-C-methyladenosine triphosphate (K-i = 1.5 mu M) and 2'-C-methylcytidine triphosphate (K-i = 1.6 mu M). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.