Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase

Abstract

The ongoing Corona Virus Disease 2019 pandemic, caused by severe 20 acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp 25 complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of 30 COVID-19.2 Coronaviruses (CoV) are large genome, positive-strand RNA viruses of the order 35 Nidovirales that have recently attracted global attention due to the ongoing COVID-19 pandemic. Despite significant efforts to control its spread, SARS-CoV-2 has caused substantial health and economic burden, emphasizing the immediate need for antiviral treatments. As with all positive strand RNA viruses, an RdRp lies at the core of the viral replication machinery and for CoVs this is the nsp12 protein. The pivotal role of nsp12 in the viral life-cycle, lack of host 40 homologues and high level of sequence and structural conservation makes it an optimal target for therapeutics. However, there has been remarkably little biochemical characterization of nsp12 and a lack of fundamental data to guide the design of antiviral therapeutics and study their mechanism of action (MoA). A promising class of RdRp inhibitors are nucleoside analogues (NAs), small molecule drugs that are metabolized intracellularly into their active ribonucleoside 45 5'-triphosphate (RTP) forms and incorporated into the nascent viral RNA by error-prone viral RdRps. This can disrupt RNA synthesis directly via chain termination, or can lead to the accumulation of deleterious mutations in the viral genome. For CoVs, the situation is complicated by the post-replicative repair capacity provided by the nsp14 exonuclease (ExoN) that is essential for maintaining the integrity of their large ~30 kb genomes 1-3 . Nsp14 has been 50 shown to remove certain NAs after insertion by the RdRp into the nascent RNA, thus reducing their antiviral effects 4-6 . Despite this, several NAs currently being used for the treatment of other viral infections have been identified as potential anti-CoV candidates 7-9 . Among these is the purine base analogue T-705 (Favipiravir, Avigan, Extended data Fig. 1a,b) that has broadspectrum activity against a number of RNA viruses and is currently licensed in Japan for use in 55

Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model

“…Using VeroE6 cells and an antiviral assay based on reduction of cytopathic effect (CPE), we recorded EC50 and EC90 of 32 and 52.5 µg/mL using a multiplicity of infection (MOI) of 0.001, 70.0 and >78.5µg/mL with an MOI of 0.01 ( Figure S1) in accordance with previous studies [18][19][20] . Infectious titer reductions (fold change in comparison with untreated cells) were ≥2 with 19.6µg/mL of favipiravir and ranged between 11 and 342 with 78.5µg/mL.…”

“…This inhibits RNA strand extension and induces abnormal levels of mutation accumulation into the viral genome 16,17 . Recently, it was shown in vitro that favipiravir has a similar mechanism of action with SARS-CoV-2 through a combination of chain termination, reduced RNA synthesis and lethal mutagenesis 20 . Our genomic analysis confirmed the mutagenic effect of favipiravir in vivo.…”

“…Favipiravir is a prodrug that is metabolized intracellularly into its active ribonucleoside 5'-triphosphate form that acts as a nucleotide analogue to selectively inhibit RNA-dependent RNA polymerase and induce lethal mutagenesis 16,17 . Recently, several studies reported in vitro inhibitory activity of favipiravir against SARS-CoV-2 with 50% effective concentrations (EC50) ranging from 62 to >500µM (10 to >78µg/mL) [18][19][20] . Based on these results, more than 20 clinical trials on the management of COVID-19 by favipiravir are ongoing (https://clinicaltrials.gov/).…”

Detailed Molecular Interactions of Favipiravir with SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza Virus Polymerases In Silico

“…The agent could bind to RdRp proteins and could inhibit genome replication. A previous report showed that the agent inhibits SARS-CoV-2 in vitro [ 28 ]. This study suggests that inhibition of genome replication is termination [ 28 ].…”

“…A previous report showed that the agent inhibits SARS-CoV-2 in vitro [ 28 ]. This study suggests that inhibition of genome replication is termination [ 28 ]. However, detailed molecular interactions between the agent and the viral replication systems may not currently be known.…”