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.

Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters

“…Both direct inhibition of the polymerase and induction of lethal mutagenesis have been described (23,24,47,49). In the T-705-treated hamsters inoculated with HMPV, viral genomes were detected in all animals even when the animals were treated at high doses.…”

“…Intracellular host enzymes act upon T-705, converting it to its active form, T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) (20). T-705RTP functions as a purine nucleotide analog that selectively inhibits the RNA-dependent RNA polymerase (RdRp) or causes lethal mutagenesis upon incorporation into the viral RNA (21)(22)(23)(24). T-705 is presently in clinical development as an influenza virus inhibitor in Japan (new drug application filed) and the United States (phase 3 clinical trial) (18).…”

Characterization of Susceptibility Variants of Influenza Virus Grown in the Presence of T-705

“…T-705 inhibits viral RNA-dependent RNA polymerase by inhibiting elongation at the incorporate site as a purine analog (2). This mechanism does not produce mutation in the replicating genome, and accordingly we failed to isolate resistant mutants but did find variants with less susceptibility to T-705.…”

“…In contrast, we sequenced the cloned replication-competent virus and observed one transversion (T®G) in 33 substitutions (3%) in our variants, indicating that these substitutions might be due to a naturally occurring transition and not induced by T-705. Moreover, T-705 ribosyl triphosphate terminates chain elongation at the incorporated site and thereby inhibits RNA synthesis without inducing a mismatched substitution (2,3). Therefore T-705 would not be a mutagen for viral RNA.…”

Backs against the Wall: Novel and Existing Strategies Used during the 2014-2015 Ebola Virus Outbreak

“…Favipiravir (developed by Toyama Chemical, Japan), a pyrazinecarboxamide derivative, is well characterized as a drug against influenza virus infections and acts by selectively inhibiting the activity of the viral RNA-dependent RNA polymerase (29,30). Favipiravir has completed phase III clinical trials for the treatment of influenza (31), and several studies have supported it as a broadspectrum antiviral against a panel of arenaviruses and bunyaviruses (32), West Nile virus (33), yellow fever virus (34), and others, which have been summarized in a review (35).…”