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. 2013 Jul 10;8(7):e68347.
doi: 10.1371/journal.pone.0068347. Print 2013.

The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate Towards Influenza A Virus Polymerase

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Free PMC article

The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate Towards Influenza A Virus Polymerase

Zhinan Jin et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

Competing Interests: ZJ, LKS, VKR and JD are current or former employees of Alios BioPharma, Inc. BK is collaborating with Alios BioPharma, Inc. Alios BioPharma funded this study. T-705 is developed by Toyama Chemical Co., LTD. Co-author BK is a PLOS ONE Editorial Board member. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Inhibition of the influenza virus RNP complex by 3′dGTP and T-705 RTP.
(A) Chemical structures of the obligate chain terminator 3′dGTP and the base-modified T-705 ribofuranosyl 5′-triphosphate (T-705 RTP). (B) Polyacrylamide gel electrophoresis (6%) showing the decrease in radiolabeled viral RNA product from the enzymatic reaction in the presence of increasing concentrations of 3′dGTP. Concentrations of inhibitor are as follows: lane 1 (0), lane 2 (0.023), lane 3 (0.069), lane 4 (0.21), lane 5 (0.62), lane 6 (1.9), lane 7 (5.6), lane 8 (16.7), lane 9 (50), and lane 10 (150 µM). (C) same as (B), with the same concentration range of T-705 RTP as inhibitor.
Figure 2
Figure 2. Inhibition of recombinant IAVpol using a 50-mer RNA template.
(A) Principle of the reaction. Recombinant IAVpol (PA/PB1/PB2) was incubated in the presence of a 50-mer RNA template sequence derived from the 3′-end of the PA gene of the NanChang strain . The 15-nt 5′vRNA oligo that is partially complementary to the 3′vRNA is needed as promoter for the enzyme. The 5′-pApG dinucleotide primer is extended and allows for multiple incorporation events of α-33P-GMP used as tracer (star). (B) Representative curves of inhibition potency of 3′dGTP and T-705 RTP against IAVpol RNA synthesis activity. IC50s were determined by adding increasing concentrations of each inhibitor, and quantitative analysis of the amount of remaining full length RNA product is expressed as % inhibition (see Materials and Methods). Each experiment was conducted at least twice to calculate the average value and standard deviation. (C) The inhibition percentage was measured in the presence of a saturating concentration of T-705 RTP (100 µM), and either low (3 µM) or high (300 µM) concentration of one of the two purines GTP or ATP. (D) The same experiment as (C) instead with either low (3 µM) or high (300 µM) of pyrimidine UTP or CTP.
Figure 3
Figure 3. GMP and T-705 RMP incorporation opposite C on template.
(A) A 14-mer RNA template sequence (t14-1) was designed to favor the extension of the 5′-pApG dinucleotide primer (AG) to a 9-mer in the presence of CTP and UTP, or a 14-mer full-length product (FL) in the presence of the additional GTP that can be specifically incorporated at position +10. (B) Example of a polyacrylamide gel electrophoresis result showing the products of primer extension. In addition to 25 µM CTP and UTP, 100 µM of GTP (lane 4), T-705 RTP (lane 5), ATP (lane 6), or 3′dGTP (lane 7) were added to the enzymatic reaction. The 9-mer and 14-mer product sequences were chemically synthesized, radiolabeled, and used as molecular size markers during the gel migration (lane 1 and 2). (C) Natural GTP was added to the primer extension reaction at increasing concentrations up to 10 µM in the presence of 25 µM CTP and UTP. (D) Quantitative analysis of GMP incorporation, based on the extension of the 9-mer RNA product obtained in Fig. 3C. The percentage of the extended products from 9-mer was plotted against GTP concentration and the data was fitted to a hyperbolic equation (see Materials and Methods) to derive the K app for GTP incorporation. The inset shows the same plot on semi-log scale. Each experiment was conducted at least twice to calculate the average value and standard deviation (see Table 2).
Figure 4
Figure 4. AMP and T-705 RMP incorporation opposite U on the template.
(A) A 14-mer RNA template sequence (t14-2) was designed to favor the extension of the 5′-pApG dinucleotide primer (AG) to a 9-mer in the presence of CTP and UTP, or a 14-mer full-length product (FL) in the presence of additional ATP that can be specifically incorporated at position +10. (B) Polyacrylamide gel electrophoresis result showing the product of primer extension in the presence of 100 µM of either ATP (lane 4), T-705 RTP (lane 5), or GTP (lane 6). The 9-mer and 14-mer product sequences were chemically synthesized, radiolabeled, and used as molecular size markers (lane 1 and 2). (C) Natural ATP was added to the reaction at increasing concentrations up to 10 µM. (D) Quantitative analysis of AMP incorporation, based on the extension of the 9-mer RNA product obtained in (C). The percentage of the extended products from 9-mer was plotted against ATP concentration, and the data was fit to a hyperbolic equation (see Materials and Methods) to derive the K app for ATP incorporation. The inset shows the same plot on semi-log scale. Each experiment was conducted at least twice to calculate the average value and standard deviation (see Table 3).
Figure 5
Figure 5. Effect of multiple incorporations of T-705 RMP on RNA synthesis.
(A) A 14-mer RNA template sequence (t14-3) was designed to favor the extension of the 5′-pApG dinucleotide primer (AG) to a 9-mer in the presence of CTP and UTP, or a 10-mer product in the presence of additional ATP that can be specifically incorporated at position +10. (B) Polyacrylamide gel electrophoresis result showing the product of primer extension in the presence of 100 µM of ATP (lane 4) or T-705 RTP (lane 5). The 10-mer and 14-mer, representing the full length product (FL), RNA sequences were chemically synthesized, radiolabeled, and used as molecular size markers (lane 1 and 2).
Figure 6
Figure 6. Schematic of ambiguous base pairing of T-705 opposite cytidine and uridine on RNA template.
Enzyme kinetics of single nucleotide incorporation combined with competitive inhibition experiments demonstrated that T-705 RTP is able to base pair with a cytidine and a uridine prior to being incorporated by influenza virus polymerase with low discrimination against natural GTP or ATP.

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This research is supported by Alios BioPharma Inc. (www.aliosbiopharma.com). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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