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Antimicrob Agents Chemother. Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase
[Source: Antimicrobial Agents Chemotherapy, full page: (LINK). Abstract, edited.]
Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase
Hidehiro Sangawa, Takashi Komeno, Hiroshi Nishikawa, Atsushi Yoshida, Kazumi Takahashi, Nobuhiko Nomura and Yousuke Furuta
Author Affiliations: Research Laboratories, Toyama Chemical Co., Ltd., 2-4-1 Shimookui, Toyama, Japan
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 herein 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 <SUP>32</SUP>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 non-competitive 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.
FOOTNOTES
Corresponding author. Mailing address: Research Laboratories, Toyama Chemical Co., Ltd., 2-4-1 Shimookui, Toyama 930-8508, Japan. Phone: +00-81-76-431-8268. Fax: +00-81-76-431-8208. hidehiro_sangawa@toyama-chemical.co.jp
Copyright ? 2013, American Society for Microbiology. All Rights Reserved.
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Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase
Hidehiro Sangawa, Takashi Komeno, Hiroshi Nishikawa, Atsushi Yoshida, Kazumi Takahashi, Nobuhiko Nomura and Yousuke Furuta
Author Affiliations: Research Laboratories, Toyama Chemical Co., Ltd., 2-4-1 Shimookui, Toyama, Japan
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 herein 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 <SUP>32</SUP>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 non-competitive 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.
FOOTNOTES
Corresponding author. Mailing address: Research Laboratories, Toyama Chemical Co., Ltd., 2-4-1 Shimookui, Toyama 930-8508, Japan. Phone: +00-81-76-431-8268. Fax: +00-81-76-431-8208. hidehiro_sangawa@toyama-chemical.co.jp
Copyright ? 2013, American Society for Microbiology. All Rights Reserved.
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