Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence of favipiravir-RTP.

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

Favipiravir tautomerism: a theoretical insight.

There is no experimental information about the tautomerism of Favipiravir (T-705). Therefore, its tautomeric state was predicted by using density functional theory in gas phase and in solution (toluene, acetonitrile and water). The results have shown that, in neutral state, the enol form is strongly dominating in both gas phase and solution. The carboxamide group is easily protonated in the presence of acid, which leads to shift of the tautomeric equilibrium toward the keto tautomer. In order to validate the theoretical predictions, 2-hydroxy pyridine and 2-hydroxy pyrazine were also included in the set of studied compounds. The available experimental data about their tautomerism are in very good agreement with the theoretical predictions, which validate the conclusions made for T-705.

Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir.

Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks taking a considerable toll in human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic, and emerging RNA virus infections in humans, suggesting applicability to control some viral outbreaks. Treatment with favipiravir was shown to reduce the infectivity of Rift Valley fever virus both in cell cultures and in experimental animal models, but the mechanism of this protective effect is not understood. In this work, we show that favipiravir at concentrations well below the toxicity threshold estimated for cells is able to extinguish RVFV from infected cell cultures. Nucleotide sequence analysis has documented RVFV mutagenesis associated with virus extinction, with a significant increase in G to A and C to U transition frequencies and a decrease of specific infectivity, hallmarks of lethal mutagenesis.

Viral polymerase inhibitors T-705 and T-1105 are potential inhibitors of Zika virus replication.

Since 2015, 69 countries and territories have reported evidence of vector-borne Zika virus (ZIKV) transmission. Currently, there are no effective licensed vaccines or drugs available for the treatment or prevention of ZIKV infection. We tested a series of compounds for their ability to inhibit ZIKV replication in cell culture. The compounds in T-705 (favipiravir) and T-1105 were found to have antiviral activity, suggesting that these compounds are promising candidates for further development as specific antiviral drugs against ZIKV.

T-705 (Favipiravir) suppresses tumor necrosis factor α production in response to influenza virus infection: A beneficial feature of T-705 as an anti-influenza drug.

Influenza virus infection induces the production of various cytokines, which play important roles in the pathogenesis of infection. Among the cytokines induced by influenza, tumor necrosis factor α (TNF-α) production has been correlated with the severity of lung lesions. We investigated the effects of T-705 (Favipiravir, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) on cytokine production due to influenza virus infection in vitro and in vivo, compared with oseltamivir or GS 4071, an active form of oseltamivir. TNF-α production in mouse macrophage-derived P388D1 cells infected with the influenza virus was lower following treatment with T-705 at concentrations of 0.3 to 100 µg/ml than treatment with GS 4071 at the same concentrations. The effect of treatment with T-705 on the cytokine production induced by the influenza virus infection was investigated in mouse influenza virus infection model. At 48 h post-infection (p.i.) T-705 significantly suppressed the viral load in the lungs and TNF-α production in the airways of infected mice even when viral loads were high. Furthermore, T-705 suppressed only TNF-α production from the early phase of infection. In this study, T-705 showed the antiviral activity of reducing pulmonary viral load compared with oseltamivir, thereby suppressing the TNF-α production. This feature of T-705 is benefit against severe influenza infection.

Efficacy of Favipiravir (T-705) in Rabies Postexposure Prophylaxis.

Rabies is a fatal encephalitis caused by rabies virus (RABV), and no antiviral drugs for RABV are currently available. We report for the first time the efficacy of favipiravir (T-705) against RABV in vitro and in vivo. T-705 produced a significant, 3-4 log10 reduction in the multiplication of street and fixed RABV strains in mouse neuroblastoma Neuro-2a cells, with half-maximal inhibitory concentrations of 32.4 µM and 44.3 µM, respectively. T-705 significantly improved morbidity and mortality among RABV-infected mice when orally administered at a dose of 300 mg/kg/day for 7 days, beginning 1 hour after inoculation. T-705 significantly reduced the rate of virus positivity in the brain. Furthermore, the effectiveness of T-705 was comparable to that of equine rabies virus immunoglobulin for postexposure prophylaxis. Collectively, our results suggest that T-705 is active against RABV and may serve as a potential alternative to rabies immunoglobulin in rabies postexposure prophylaxis.

Comparison of Routes of Administration, Frequency, and Duration of Favipiravir Treatment in Mouse and Guinea Pig Models of Ebola Virus Disease.

Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.

HAND2 suppresses favipiravir efficacy in treatment of Borna disease virus infection.

Borna disease virus (BoDV-1) is a bornavirus prototype that infects the central nervous system of various animal species and can cause fatal encephalitis in various animals including humans. Among the reported anti-BoDV-1 treatments, favipiravir (T-705) is one of the best candidates since it has been shown to be effective in reducing various bornavirus titers in cell culture. However, T-705 effectiveness on BoDV-1 is cell type-dependent, and the molecular mechanisms that explain this cell type-dependent difference remain unknown. In this study, we noticed a fact that T-705 efficiently suppressed BoDV-1 in infected 293T cells, but not in infected SH-SY5Y cells, and sought to identify protein(s) responsible for this cell-type-dependent difference in T-705 efficacy. By comparing the transcriptomes of BoDV-1-infected 293T and SH-SY5Y cells, we identified heart- and neural crest derivatives-expressed protein 2 (HAND2) as a candidate involved in T-705 interference. HAND2 overexpression partly attenuated the inhibitory effect of T-705, whereas HAND2 knockdown enhanced this effect. We also demonstrated an interaction between T-705 and HAND2. Furthermore, T-705 impaired HAND2-mediated host gene expression. Because HAND2 is an essential transcriptional regulator of embryogenesis, T-705 may exhibit its adverse effects such as teratogenicity and embryotoxicity through the impairment of HAND2 function. This study provides novel insights into the molecular mechanisms underlying T-705 interference in some cell types and inspires the development of improved T-705 derivatives for the treatment of RNA viruses.

A novel LC-MS/MS method for the determination of favipiravir ribofuranosyl-5'-triphosphate (T-705-RTP) in human peripheral mononuclear cells.

Favipiravir is a broad-spectrum antiviral that is metabolised intracellularly into the active form, favipiravir ribofuranosyl-5'-triphosphate (F-RTP). Measurement of the intracellular concentration of F-RTP in mononuclear cells is a crucial step to characterising the pharmacokinetics of F-RTP and to enable more appropriate dose selection for the treatment of COVID-19 and emerging infectious diseases. The described method was validated over the range 24 - 2280 pmol/sample. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and lysed using methanol-water (70:30, v/v) before cellular components were precipitated with acetonitrile and the supernatant further cleaned by weak anion exchange solid phase extraction. The method was found to be both precise and accurate and was successfully utilised to analyse F-RTP concentrations in patient samples collected as part of the AGILE CST-6 clinical trial.

Evaluation of a novel severe combined immunodeficiency mouse model for antiviral drug evaluation against Chandipura virus infection.

Chandipura virus (CHPV) is a negative-sense single-stranded RNA virus known to cause fatal encephalitis outbreaks in the Indian subcontinent. The virus displays tropism towards the pediatric population and holds significant public health concerns. Currently, there is no specific, effective therapy for CHPV encephalitis. In this study, we evaluated a novel C.B-17 severe combined immunodeficiency (SCID) mouse model which can be used for pre-clinical antiviral evaluation. Inoculation of CHPV developed a lethal infection in our model. Plaque assay and immunohistochemistry detected increased viral loads and antigens in various organs, including the brain, spinal cord, adrenal glands, and whole blood. We further conducted a proof-of-concept evaluation of favipiravir in the SCID mouse model. Favipiravir treatment improved survival with pre-symptomatic (days 5-14) and post-symptomatic (days 9-18) treatment. Reduced viral loads were observed in whole blood, kidney/adrenal gland, and brain tissue with favipiravir treatment. The findings in this study demonstrate the utility of SCID mouse for in vivo drug efficacy evaluation and the potential efficacy of favipiravir against CHPV infection.

Antiviral agents and disinfectants for foot­and­mouth disease (Review).

Fluorouracil, 5-azacytidine, 6-azauridine, ribavirin, favipiravir (T-705) and its derivative (T-1105) exhibit anti-foot-and-mouth disease virus (FMDV) effects. In particular, T-1105 exhibits promising results when administered to guinea pigs orally, and pigs in their feed. FMDV is excreted in the early stages of infection in aerosols and oral or nasal droplets from animals. T-1105 along with the FMDV vaccine can be used to combat foot-and-mouth disease (FMD) epidemics. Several studies have shown that sodium hypochlorous solutions are widely used to inactivate viruses, including FMDV. However, these solutions must be stored under cool and dark conditions to maintain their virucidal effects. Interestingly, a study indicated that the virucidal activity of a calcium bicarbonate solution with a mesoscopic structure (CAC-717) did not decrease after storage at room temperature for at least four years outside direct sunlight. Numerous lessons acquired from the 2010 FMD outbreak in Japan are relevant for the control of COVID-19. However, the widespread use of chlorite can cause environmental issues. Chlorite can be combined with nitrogen to produce chloramine or N-nitrosodimethylamine, which plays a role in carcinogenesis. Therefore, risk assessments should be conducted in aquatic environments. Moreover, there is a need to develop nonchlorine disinfectants that can be used during epidemics, including FMD. The approach of 'One Health' should be shared between the public health and veterinary fields to improve the management of viral outbreaks, including those due to FMD.

T-705-Derived Prodrugs Show High Antiviral Efficacies against a Broad Range of Influenza A Viruses with Synergistic Effects When Combined with Oseltamivir.

Emerging influenza A viruses (IAV) bear the potential to cause pandemics with unpredictable consequences for global human health. In particular, the WHO has declared avian H5 and H7 subtypes as high-risk candidates, and continuous surveillance of these viruses as well as the development of novel, broadly acting antivirals, are key for pandemic preparedness. In this study, we sought to design T-705 (Favipiravir) related inhibitors that target the RNA-dependent RNA polymerase and evaluate their antiviral efficacies against a broad range of IAVs. Therefore, we synthesized a library of derivatives of T-705 ribonucleoside analogues (called T-1106 pronucleotides) and tested their ability to inhibit both seasonal and highly pathogenic avian influenza viruses in vitro. We further showed that diphosphate (DP) prodrugs of T-1106 are potent inhibitors of H1N1, H3N2, H5N1, and H7N9 IAV replication. Importantly, in comparison to T-705, these DP derivatives achieved 5- to 10-fold higher antiviral activity and were non-cytotoxic at the therapeutically active concentrations. Moreover, our lead DP prodrug candidate showed drug synergy with the neuraminidase inhibitor oseltamivir, thus opening up another avenue for combinational antiviral therapy against IAV infections. Our findings may serve as a basis for further pre-clinical development of T-1106 prodrugs as an effective countermeasure against emerging IAVs with pandemic potential.

Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106.

The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.

Analysis of the responsible site for favipiravir resistance in RNA-dependent RNA polymerase of influenza virus A/PR/8/34 (H1N1) using site-directed mutagenesis.

Favipiravir (T-705, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits the replication of influenza virus in vitro and in vivo. Favipiravir is converted to favipiravir-4-ribofuranosyl-5-triphosphate (favipiravir RTP) by intracellular enzymes and functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRP) of influenza virus. Our previous experiments failed in an attempt to obtain a favipiravir-resistant influenza virus in vitro using influenza virus A/PR/8/34(H1N1). Conversely, Goldhill et al. reported a favipiravir-resistant influenza virus generated by in vitro passage of influenza virus A/England/195/2009 (H1N1), an early isolate from the 2009 H1N1 pandemic (pdm09), in the presence of favipiravir with K229R mutation in PB1. This study focused on K229R mutation near the NTP cross-linked region in PB1 based on the above conflicting findings to confirm whether K229R mutation brings favipiravir resistance to influenza virus A/PR/8/34. Thirty PB1 mutants generated by site-directed mutagenesis of the NTP cross-linked region were evaluated using an influenza virus A/PR/8/34 replicon system. Among the 30 mutants, 10 possessed but 20 lost replicon activity. When susceptibility to favipiravir in 10 mutants was further assessed, the PB1 E491D mutant was five times more sensitive than the wild-type (WT), while only the PB1 K229R mutant was resistant to favipiravir. Results suggested that the evaluated region was essential for polymerase activity, and K229 mutation was responsible for polymerase inhibition of favipiravir in the influenza virus A/PR/8/34. Interestingly, the tested K229X series mutants entirely lost replicon activity, except for K229R. This suggested that the amino acid at position 229 in PB1 of influenza virus may play a pivotal role in polymerase activity. Moreover, this lysine residue is highly conserved among positive- and negative-sense single-stranded RNA viruses, in which favipiravir showed potent activity, suggesting that this mutation may determine the characterization of the in vitro broad-spectrum activity of favipiravir. Additionally, this mutation acquisition greatly influences the viral replication and the susceptibility to favipiravir.

Favipiravir for the treatment of COVID-19 in elderly patients-what do we know after 2 years of COVID-19?

Since the appearance of coronavirus disease 2019 (COVID-19), numerous studies have been conducted to find effective therapeutics. Favipiravir (FVP) is one of the repurposed drugs which has been authorized in a few countries on an emergency basis to treat COVID-19. Elderly individuals especially 65 years or older are more prone to develop severe illness. We aim to provide a short summary of the current knowledge of the antiviral efficacy of favipiravir with respect to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected elderly patients. We found that it is rather controversial whether favipiravir is effective against SARS-CoV-2 infection. Data regarding patients 65 years or older is not sufficient to support or reject the usage of favipiravir for COVD-19 treatment. Further studies would be advisable to elicit the efficiency of favipiravir in elderly COVID-19 patients.

In vitro and in vivo efficacy of a novel nucleoside analog H44 against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus that causes fever, hemorrhage, and multi-organ failure, with an average fatality rate of ∼40% in humans. Currently, there are no available vaccines or drugs for the treatment of Crimean-Congo hemorrhagic fever (CCHF). Favipiravir (T-705), a nucleoside analog, protects against CCHFV infection in animal models. Here, we evaluated the anti-CCHFV efficacy of several nucleoside analogs, including some well-known compounds such as remdesivir (GS-5734), EIDD-1931 and its prodrug molnupiravir (EIDD-2801), as well as a novel T-705-derived compound H44. T-705, H44, and EIDD-1931 inhibited CCHFV infection in vitro while GS-5734 had no inhibitory effect. All three nucleoside analogs functioned at the "post-entry" stage of virus infection. However, EIDD-2801 failed to protect type I interferon receptor knockout (IFNAR)-/- mice from CCHFV infection. H44, similar to T-705, conferred 100% protection to IFNAR-/- mice against lethal CCHFV challenge, even with delayed administration. This study provided in vitro and in vivo data regarding the anti-CCHFV efficacy of different nucleosides and identified a novel compound, H44, as a promising drug candidate for the treatment of CCHFV infection in vivo.

The mechanism of action of T-705 as a unique delayed chain terminator on influenza viral polymerase transcription.

Favipiravir (T-705) has been developed as a potent anti-influenza drug and exhibited a strong inhibition effect against a broad spectrum of RNA viruses. Its active form, ribofuranosyl-triphosphate (T-705-RTP), functions as a competitive substrate for the RNA-dependent RNA polymerase (RdRp) of the influenza A virus (IAV). However, the exact inhibitory mechanisms of T-705 remain elusive and subject to a long-standing debate. Although T-705 has been proposed to inhibit transcription by acting as a chain terminator, it is also paradoxically suggested to be a mutagen towards IAV RdRp by inducing mutations due to its ambiguous base pairing of C and U. Here, we combined biochemical assay with molecular dynamics (MD) simulations to elucidate the molecular mechanism underlying the inhibitory functions exerted by T-705 in IAV RdRp. Our in vitro transcription assay illustrated that IAV RdRp could recognize T-705 as a purine analogue and incorporate it into the nascent RNA strand. Incorporating a single T-705 is incapable of inhibiting transcription as extra natural nucleotides can be progressively added. However, when two consecutive T-705 are incorporated, viral transcription is completely terminated. MD simulations reveal that the sequential appearance of two T-705 in the nascent strand destabilizes the active site and disrupts the base stacking of the nascent RNA. Altogether, our results provide a plausible explanation for the inhibitory roles of T-705 targeting IAV RdRp by integrating the computational and experimental methods. Our study also offers a comprehensive platform to investigate the inhibition effect of antivirals and a novel explanation for the designing of anti-flu drugs.

Antiviral Effect of Favipiravir (T-705) against Measles and Subacute Sclerosing Panencephalitis Viruses.

Subacute sclerosing panencephalitis (SSPE) is a late-onset, intractable, and fatal viral disease caused by persistent infection of the central nervous system with a measles virus mutant (SSPE virus). In Japan, interferon-α and ribavirin are administered intracerebroventricularly to patients with SSPE. However, as the therapeutic effect is insufficient, more effective drugs are needed. Favipiravir, which is clinically used as an anti-influenza drug, demonstrates anti-viral effects against RNA viruses. In this study, the antiviral effect of favipiravir against measles virus (Edmonston strain) and SSPE virus (Yamagata-1 strain) was examined in vitro. The 50% effective concentration (EC50) of favipiravir (inhibiting viral plaque formation by 50%) against Edmonston and Yamagata-1 strains were 108.7 ± 2.0 µM (17.1 ± 0.3 µg/mL) and 38.6 ± 6.0 µM (6.1 ± 0.9 µg/mL), respectively, which were similar to those of ribavirin. The antiviral activity of favipiravir against the SSPE virus was demonstrated for the first time in this study.

Favipiravir Does Not Inhibit Chikungunya Virus Replication in Mosquito Cells and Aedes aegypti Mosquitoes.

Favipiravir (T-705) is a broad-spectrum antiviral drug that inhibits RNA viruses after intracellular conversion into its active form, T-705 ribofuranosyl 5'-triphosphate. We previously showed that T-705 is able to significantly inhibit the replication of chikungunya virus (CHIKV), an arbovirus transmitted by Aedes mosquitoes, in mammalian cells and in mouse models. In contrast, the effect of T-705 on CHIKV infection and replication in the mosquito vector is unknown. Since the antiviral activity of T-705 has been shown to be cell line-dependent, we studied here its antiviral efficacy in Aedes-derived mosquito cells and in Aedes aegypti mosquitoes. Interestingly, T-705 was devoid of anti-CHIKV activity in mosquito cells, despite being effective against CHIKV in Vero cells. By investigating the metabolic activation profile, we showed that, unlike Vero cells, mosquito cells were not able to convert T-705 into its active form. To explore whether alternative metabolization pathways might exist in vivo, Aedes aegypti mosquitoes were infected with CHIKV and administered T-705 via an artificial blood meal. Virus titrations of whole mosquitoes showed that T-705 was not able to reduce CHIKV infection in mosquitoes. Combined, these in vitro and in vivo data indicate that T-705 lacks antiviral activity in mosquitoes due to inadequate metabolic activation in this animal species.

PAMAM and polyester dendrimers as favipiravir nanocarriers: a comparative study using DFT method.

The electronic sensitivity and reactivity of polyamidoamine (PAMAM) and polyester dendrimers toward favipiravir (T705) were inspected using density functional theory method. The T705 drug is adsorbed on the surface of PAMAM and polyester dendrimers with the binding energy of -27.26 and -26.80 kcal mol-1, respectively, in the solvent phase. The energy gap of PAMAM and polyester dendrimers reduced by about 32% and 27%, indicating that the electrical conductance of carriers become 8.16 × 1023 and 4.41 × 1022 times higher, upon T705 adsorption. The work function (Φ) value of PAMAM and polyester is changed about 1.53 and 0.71 eV, respectively. Thus, PAMAM dendrimer is about 2.5 times stronger Φ-type sensor than polyester dendrimer. The recovery time for T705 desorption from the PAMAM and polyester surface is predicted to be 9.2 × 103 and 4.2 × 103 s, respectively, at physiological environment.