title: Mechanism of activation of beta-D-2'-deoxy-2'-fluoro-2'-c-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase.
abstract: 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 Km of 81 microM and a kcat 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 (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 microM. Similar results were obtained with 2'-C-methyladenosine triphosphate (Ki=1.5 microM) and 2'-C-methylcytidine triphosphate (Ki=1.6 microM). 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.
authors.affiliation: Pharmasset, Inc., 303A College Road East, Princeton, NJ 08640, USA. eisuke.murakami@pharmasset.com
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
authors.forename: Haiying
authors.initials: H
authors.lastname: Bao
authors.forename: Mangala
authors.initials: M
authors.lastname: Ramesh
authors.forename: Tamara R
authors.initials: TR
authors.lastname: McBrayer
authors.forename: Tony
authors.initials: T
authors.lastname: Whitaker
authors.forename: Holly M
authors.initials: HM
authors.lastname: Micolochick Steuer
authors.forename: Raymond F
authors.initials: RF
authors.lastname: Schinazi
authors.forename: Lieven J
authors.initials: LJ
authors.lastname: Stuyver
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Obikhod
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Otto
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Furman
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects / Q000378:metabolism*; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D012367:RNA, Viral / Q000187:drug effects / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C504520:2'-deoxy-2'-fluoro-2'-C-methylcytidine; D003841:Deoxycytidine; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase