Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Favipiravir induces HuNoV viral mutagenesis and infectivity loss with clinical improvement in immunocompromised patients.

Chronic human norovirus (HuNoV) infections in immunocompromised patients result in severe disease, yet approved antivirals are lacking. RNA-dependent RNA polymerase (RdRp) inhibitors inducing viral mutagenesis display broad-spectrum in vitro antiviral activity, but clinical efficacy in HuNoV infections is anecdotal and the potential emergence of drug-resistant variants is concerning. Upon favipiravir (and nitazoxanide) treatment of four immunocompromised patients with life-threatening HuNoV infections, viral whole-genome sequencing showed accumulation of favipiravir-induced mutations which coincided with clinical improvement although treatment failed to clear HuNoV. Infection of zebrafish larvae demonstrated drug-associated loss of viral infectivity and favipiravir treatment showed efficacy despite occurrence of RdRp variants potentially causing favipiravir resistance. This indicates that within-host resistance evolution did not reverse loss of viral fitness caused by genome-wide accumulation of sequence changes. This off-label approach supports the use of mutagenic antivirals for treating prolonged RNA viral infections and further informs the debate surrounding their impact on virus evolution.

Favipiravir, remdesivir, and lopinavir: metabolites, degradation products and their analytical methods.

Severe acute respiratory syndrome 2 (SARS-CoV-2) caused the emergence of the COVID-19 pandemic all over the world. Several studies have suggested that antiviral drugs such as favipiravir (FAV), remdesivir (RDV), and lopinavir (LPV) may potentially prevent the spread of the virus in the host cells and person-to-person transmission. Simultaneously with the widespread use of these drugs, their stability and action mechanism studies have also attracted the attention of many researchers. This review focuses on the action mechanism, metabolites and degradation products of these antiviral drugs (FAV, RDV and LPV) and demonstrates various methods for their quantification and discrimination in the different biological samples. Herein, the instrumental methods for analysis of the main form of drugs or their metabolite and degradation products are classified into two types: optical and chromatography methods which the last one in combination with various detectors provides a powerful method for routine and stability analyses. Some representative studies are reported in this review and the details of them are carefully explained. It is hoped that this review will be a good guideline study and provide a better understanding of these drugs from the aspects investigated in this study.

Identification of dihydroorotate dehydrogenase inhibitor, vidofludimus, as a potent and novel inhibitor for influenza virus.

Influenza A virus (IAV) infection causes respiratory disease. Recently, infection of IAV H5N1 among mammals are reported in farmed mink. Therefore, to discover antivirals against IAV, we screened a compound library by using the RNA-dependent RNA polymerase (RdRp) assay system derived from H5N1 IAV including a drug-resistant PA mutant (I38T) and a viral polymerase activity enhancing PB2 mutant (T271A). Upon screening, we found vidofludimus can be served as a potential inhibitor for IAV. Vidofludimus an orally active inhibitor for dihydroorotate dehydrogenase (DHODH), a key enzyme for the cellular de novo pyrimidine biosynthesis pathway. We found that vidofludimus exerted antiviral activity against wild-type and drug-resistant mutant IAV, with effective concentrations (EC50 ) of 2.10 and 2.11 µM, respectively. The anti-IAV activity of vidofludimus was canceled by the treatment of uridine or cytidine through pyrimidine salvage synthesis pathway, or orotic acid through pyrimidine de novo synthesis pathway. This indicated that the main target of vidofludimus is DHODH in IAV RdRp expressing cells. We also produced recombinant seasonal IAV H1N1 virion and influenza B virus (IBV) RdRp assay system and confirmed vidofludimus also carried highly antiviral activity against seasonal IAV and IBV. Vidofludimus is a candidate drug for the future threat of IAV H5N1 infection among humans as well as seasonal influenza virus infection.

Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial.

In early symptomatic COVID-19 treatment, high dose oral favipiravir did not accelerate viral clearance. BACKGROUND: Favipiravir, an anti-influenza drug, has in vitro antiviral activity against SARS-CoV-2. Clinical trial evidence to date is inconclusive. Favipiravir has been recommended for the treatment of COVID-19 in some countries. METHODS: In a multicentre open-label, randomised, controlled, adaptive platform trial, low-risk adult patients with early symptomatic COVID-19 were randomised to one of ten treatment arms including high dose oral favipiravir (3.6g on day 0 followed by 1.6g daily to complete 7 days treatment) or no study drug. The primary outcome was the rate of viral clearance (derived under a linear mixed-effects model from the daily log10 viral densities in standardised duplicate oropharyngeal swab eluates taken daily over 8 days [18 swabs per patient]), assessed in a modified intention-to-treat population (mITT). The safety population included all patients who received at least one dose of the allocated intervention. This ongoing adaptive platform trial was registered at ClinicalTrials.gov (NCT05041907) on 13/09/2021. RESULTS: In the final analysis, the mITT population contained data from 114 patients randomised to favipiravir and 126 patients randomised concurrently to no study drug. Under the linear mixed-effects model fitted to all oropharyngeal viral density estimates in the first 8 days from randomisation (4,318 swabs), there was no difference in the rate of viral clearance between patients given favipiravir and patients receiving no study drug; a -1% (95% credible interval: -14 to 14%) difference. High dose favipiravir was well-tolerated. INTERPRETATION: Favipiravir does not accelerate viral clearance in early symptomatic COVID-19. The viral clearance rate estimated from quantitative measurements of oropharyngeal eluate viral densities assesses the antiviral efficacy of drugs in vivo with comparatively few studied patients.

Assessment of toxicological effects of favipiravir (T-705) on the lung tissue of rats: An experimental study.

This study aimed to present new data on the side effects of favipiravir on healthy lung tissue and the respiratory system. In the study, two different durations (5 and 10 days) were preferred to determine the effect of favipiravir treatment due to clinical improvement rates of approximately 5 and 10 days during the use of favipiravir in COVID-19 patients. In addition, after 10 days of favipiravir treatment, animals were kept for 5 days without any treatment to determine the regeneration of lung tissues. Favipiravir was administered to rats by oral gavage at a daily dose of 200 mg/kg for 5 and 10 days, as in previous studies. At the end of the experiment, the histopathological and biochemical effects of favipiravir in the lung tissue were investigated. The data obtained from the study showed that favipiravir increased oxidative stress parameters, expression of apoptotic markers, and pro-inflammatory markers in lung tissue. Since malondialdehydes is an oxidant parameter, it increased in favipiravir-administered groups; It was determined that the antioxidant parameters glutathione, superoxide dismutase, glutathione peroxidase, and catalase decreased. Other markers used in the analysis are Bcl-2, Bax, NF-κB, interleukin (IL)-6, Muc1, iNOS, P2X7R, IL-6 and caspase-3. The levels of Bax, caspase-3, NF-κB, IL-6, Muc1, and P2X7R were increased in the Fav-treated groups compared with the control. However, the levels of Bcl-2 decreased in the Fav-treated groups. The present study proves that favipiravir, widely used today, causes side effects in lung tissue.

Enhanced favipiravir drug degradation using the synergy of PbO2-based anodic oxidation and Fe-MOF-based cathodic electro-Fenton.

Favipiravir (FAV) is a widely utilized antiviral drug effective against various viruses, including SARS-CoV-2, influenza, and RNA viruses. This article aims to introduce a novel approach, known as Linear-Paired Electrocatalytic Degradation (LPED), as an efficient technique for the electrocatalytic degradation of emerging pollutants. LPED involves simultaneously utilizing a carbon-Felt/Co-PbO2 anode and a carbon-felt/Co/Fe-MOF-74 cathode, working together to degrade and mineralize FAV. The prepared anode and cathode characteristics were analyzed using XPS, SEM, EDX mapping, XRD, LSV, and CV analyses. A rotatable central composite design-based quadratic model was employed to optimize FAV degradation, yielding statistically desirable results. Under optimized conditions (pH = 5, current density = 4.2 mA/cm2, FAV concentration = 0.4 mM), individual processes of cathodic electro-Fenton and anodic oxidation with a CF/Co-PbO2 anode achieved degradation rates of 58.9% and 89.5% after 120 min, respectively. In contrast, using the LPED strategy resulted in a remarkable degradation efficiency of 98.4%. Furthermore, a cyclic voltammetric study of FAV on a glassy carbon electrode was conducted to gather additional electrochemical insights and rectify previously published data regarding redox behavior, pH-dependent properties, and adsorption activities. The research also offers a new understanding of the LPED mechanism of FAV at the surfaces of both CF/Co-PbO2 and CF/Co/Fe-MOF-74 electrodes, utilizing data from cyclic voltammetry and LC-MS techniques. The conceptual strategy of LPED is generalizable in order to the synergism of anodic oxidation and cathodic electro-Fenton for the degradation of other toxic and resistant pollutants.

Luminescence of favipiravir in skin appendages and sclera. A controlled study and literature review.

BACKGROUND/OBJECTIVES: Favipiravir is an antiviral agent, recently used for COVID-19 infections. Several reports associate favipiravir intake with Wood's lamp fluorescence of hair, nails, and sclera. The present study was designed to elucidate the positivity rates, and sites of favipiravir-related fluorescence and to unravel the site-specific changes in fluorescence positivity rates by a function of time past exposure. METHODS: The study population comprised 50 patients and 50 control individuals. All patients in the patient group had received a full dose of favipiravir for COVID-19 infection. Fifty volunteers served as the control group. Wood's lamp examination was performed in a completely darkened room, and the positivity rate, extent, pattern, and distribution of fluorescence were recorded. RESULTS: Wood's light revealed fluorescence of the fingernails, toenails, sclera, and hair in 35 (70%), 35 (70%), 22 (44%), and 8 (16%) patients, respectively. No control individual tested positive by Wood's lamp. Statistical analysis revealed significant differences between patient and control groups in terms of Wood's light luminescence in the fingernails (p = .000), toenails (p = .000), sclera (p = .000) and hair (p = .003). Although fingernail, toenail, and hair fluorescence positivity rates declined or ceased at or after 91 days of favipiravir exposure, ocular fluorescence positivity rates were prolonged up to 188 days. CONCLUSIONS: These findings confirm that favipiravir may produce fluorescence of nails, sclera, and hair, detectable by Wood's light starting from the initial month and peaking at second- and third months following exposure to the medication. Although nail and hair fluorescence tend to abate after 3 months, ocular fluorescence may persist even longer than 6 months after cessation of the medication.

Findings from a discontinued clinical trial of favipiravir in high-risk patients with early-onset COVID-19.

INTRODUCTION: Favipiravir terminates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Accordingly, early administration of favipiravir to SARS-CoV-2-infected coronavirus disease 2019 (COVID-19) patients may be expected to suppress disease progression. METHODS: A randomized double-blind placebo-controlled trial was conducted to demonstrate efficacy of favipiravir in reducing disease progression in patients with mild COVID-19. The participants were unvaccinated patients with comorbidities and at risk of progression to severe disease. Patients were enrolled within 72 h of disease onset and randomized to receive either favipiravir (1800 mg/dose on Day 1 followed by 800 mg/dose) or matching placebo twice daily for 10 days. The primary endpoint was the proportion of patients requiring oxygen therapy within 28 days of randomization. RESULTS: The trial was discontinued after enrolling 84 patients due to slower than anticipated enrollment caused by rapid uptake of SARS-CoV-2-vaccines and the emergence of the Omicron variant. Results from the 84 patients demonstrated no significant difference in all clinical outcomes. In post-hoc analyses, favipiravir treatment showed higher efficacy in patients within 48 h of onset. No deaths or severe adverse events were documented in the favipiravir group. Plasma concentrations of favipiravir from Day 2 onward were maintained above 40 µg/mL. CONCLUSIONS: Conducting clinical trials for pathogens like SARS-CoV-2 that rapidly accumulate mutations leading to altered disease characteristics carries significant risks unless it can be done in a short period. Therefore, it would be important to prepare the comprehensive clinical trial platform that can appropriately and promptly evaluate drugs even under a pandemic.