title: Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase.
abstract: T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits replication of the influenza virus in vitro and in vivo. T-705 has been shown to be converted to T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) by intracellular enzymes and then functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRp) of the influenza virus. To elucidate these inhibitory mechanisms, we analyzed the enzyme kinetics of inhibition using Lineweaver-Burk plots of four natural nucleoside triphosphates and conducted polyacrylamide gel electrophoresis of the primer extension products initiated from (32)P-radiolabeled 5'Cap1 RNA. Enzyme kinetic analysis demonstrated that T-705RTP inhibited the incorporation of ATP and GTP in a competitive manner, which suggests that T-705RTP is recognized as a purine nucleotide by influenza virus RdRp and inhibited the incorporation of UTP and CTP in noncompetitive and mixed-type manners, respectively. Primer extension analysis demonstrated that a single molecule of T-705RTP was incorporated into the nascent RNA strand of the influenza virus and inhibited the subsequent incorporation of nucleotides. These results suggest that a single molecule of T-705RTP is incorporated into the nascent RNA strand as a purine nucleotide analog and inhibits strand extension, even though the natural ribose of T-705RTP has a 3'-OH group, which is essential for forming a covalent bond with the phosphate group.
authors.affiliation: Research Laboratories, Toyama Chemical Co., Ltd., Toyama, Japan.
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Sangawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Komeno
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishikawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yoshida
authors.forename: Kazumi
authors.initials: K
authors.lastname: Takahashi
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Nomura
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Furuta
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000577:Amides / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001667:Binding, Competitive; D003570:Cytidine Triphosphate / Q000378:metabolism; D004285:Dogs; D057075:Enzyme Assays; D006160:Guanosine Triphosphate / Q000378:metabolism; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000235:genetics; D007700:Kinetics; D061985:Madin Darby Canine Kidney Cells; D011485:Protein Binding; D011719:Pyrazines / Q000494:pharmacology*; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000577:Amides; D000998:Antiviral Agents; D011719:Pyrazines; D012367:RNA, Viral; D014764:Viral Proteins; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012324:RNA-Dependent RNA Polymerase; C462182:favipiravir; D014544:Uridine Triphosphate
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