title: Mechanism of hepatitis C virus RNA polymerase inhibition with dihydroxypyrimidines.
abstract: We studied the biochemical mechanisms associated with inhibition and resistance to a 4,5-dihydroxypyrimidine carboxylate that inhibits the hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B. On the basis of the structure of the pharmacophore, it has been suggested that these compounds may act as pyrophosphate (PP(i)) mimics. We monitored nucleotide incorporation events during the elongation phase and showed that the polymerase activity of wild-type NS5B was inhibited by the dihydroxypyrimidine at a 50% inhibitory concentration (IC(50)) of 0.73 muM. Enzymes with the G152E or P156L mutation, either of which confers resistance to this compound, showed four- to fivefold increases in IC(50)s. The inhibitor was competitive with respect to nucleotide incorporation. It was likewise effective at preventing the PP(i)-mediated excision of an incorporated chain terminator in a competitive fashion. In the absence of the dihydroxypyrimidine, the reaction was not significantly affected by the G152E or P156L mutation. These data suggest that the resistance associated with these two mutations is unlikely due to an altered interaction with the pyrophosphate-mimicking domain of the compound but, rather, is due to altered interactions with its specificity domain at a region distant from the active site. Together, our findings provide strong experimental evidence that supports the notion that the members of this class of compounds can act as PP(i) mimics that have the potential to mechanistically complement established nucleoside and nonnucleoside analogue inhibitors.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada H3A 2B4.
authors.forename: Megan H
authors.initials: MH
authors.lastname: Powdrill
authors.forename: Jerome
authors.initials: J
authors.lastname: Deval
authors.forename: Frank
authors.initials: F
authors.lastname: Narjes
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: Matthias
authors.initials: M
authors.lastname: Götte
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D012367:RNA, Viral / Q000096:biosynthesis / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase