The ambiguous base-pairing and high substrate efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate towards influenza A virus polymerase

• Published in: PloS one Vol. 8; no. 7; p. e68347
• Main Authors: Jin, Zhinan, Smith, Lucas K, Rajwanshi, Vivek K, Kim, Baek, Deval, Jerome
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.07.2013; Public Library of Science (PLoS)
• Subjects: Adenosine; Adenosine triphosphate; Amides - metabolism; Amides - pharmacology; Analogs; Analysis; Animals; Antimetabolites - metabolism; Antimetabolites - pharmacology; Antiviral agents; Antiviral Agents - metabolism; Antiviral Agents - pharmacology; Base Pair Mismatch - drug effects; Base Pair Mismatch - physiology; Base Pairing - drug effects; Biology; Care and treatment; Developmental stages; Discrimination; DNA-Directed DNA Polymerase - drug effects; DNA-Directed DNA Polymerase - metabolism; Drug resistance; Elongation; Enzyme kinetics; Enzymes; Fever; Genomes; GTP; Guanosine; Guanosine triphosphate; Health aspects; Humans; Influenza; Influenza A; Influenza A virus - enzymology; Influenza viruses; Kinetics; Medicine; Metabolism; Metabolites; Mutagenesis; Mutation; Nucleotide analogs; Pandemics; Polymerase; Polyphosphates - metabolism; Polyphosphates - pharmacology; Pyrazines - metabolism; Pyrazines - pharmacology; Ribavirin - analogs & derivatives; Ribavirin - pharmacology; Ribonucleic acid; RNA; RNA synthesis; Sf9 Cells; Spodoptera; Substrate inhibition; Substrate Specificity; Swine flu; Transcription; Viruses; West Nile virus; United States > US; South San Francisco California; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0068347

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.