The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5′-Triphosphate towards Influenza A Virus Polymerase

Abstract

T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3′-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus.

Post-exposure treatments for Ebola and Marburg virus infections

“…The mechanism of action is thought to be due to selective inhibition of viral RNA-dependent RNA polymerase 167 , although it has also been suggested that favipiravir induces lethal RNA transversion mutations 168 . There have been no published preclinical studies in NHPs using favipiravir as a post-exposure treatment or therapeutic.…”

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

“…The HCV polymerase activity was measured as the incorporation of radiolabeled nucleotide monophosphates into acid-insoluble RNA products using HCV NS5B and complementary internal ribosome entry site (IRES)-derived RNA templates as described previously (14), with the following modifications: HCV polymerase reaction mixtures contained 50 nM 5=-untranslated region (UTR) RNA template, 1 M tritiated CTP (18. Chain termination in the presence of the next correct nucleotides. After the NS5B EC with the 9-mer RNA was assembled in the extension and pause reaction, 20 M CTP containing 33 nM 33 P-radiolabeled CTP was added to the reaction and reacted for 30 s at 30°C to generate the EC containing a 10-mer RNA with [␣- 33 P]CMP at the 3= end. The 10-mer EC then was incubated with either 100 M UTP for 40 s or 100 M UTP analog for 5 min to generate an 11-mer RNA with the U analog at the 3= end.…”

“…After the NS5B EC with the 9-mer RNA was assembled in the extension and pause reaction, 33 nM [␣-33 P]CTP was added to the reaction and reacted for 30 s at 30°C to generate the EC containing a 10-mer RNA with [␣- 33 P]CMP at its 3= end. The complex was isolated by centrifugation as described above.…”

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

“…The incorporation efficiencies of the different rNTP analogs were evaluated by measurement of the K 1/2 and the corresponding discrimination values (39,40). K 1/2 calculations.…”