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

Abstract

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.

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“…Favipiravir is a prodrug that is metabolized to its active form, favipiravir-ribofuranosyl-5′-triphosphate (favipiravir-RTP), mainly by the enzyme human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in order to stop the replication process of the viral RNA genome (i.e., of the virus) (Smee et al 2009 ). However, limitations have restricted the use of favipiravir as an efficient anti-COVID-19 agent till now, e.g., reliable data regarding in vitro SARS-CoV-2 inhibition are still not available (Shiraki and Daikoku 2020 ; Dong et al 2020 ); broad data regarding in vivo SARS-CoV-2 inhibition and efficacy in preclinical animal studies are still not available (Dong et al 2020 ; Cai et al 2020 ); the few available animal experiments of favipiravir show the potential for teratogenic effects (Shiraki and Daikoku 2020 ); favipiravir has not been shown to be effective in primary human airway cells (Yoon et al 2018 ); lack of additional virus-toxic functional groups in favipiravir structure to augment its antiviral mechanism of action against the lethal and resistant SARS-CoV-2 (Dong et al 2020 ; Abdelnabi et al 2017 ); lipophilic/hydrophilic properties of favipiravir are not adequately balanced to achieve maximal bioavailability and distribution (especially to the lungs) in humans (Du and Chen 2020 ); expected binding affinities of active favipiravir-RTP molecule (as a viral RdRp inhibitor) with SARS-CoV-2 RdRp enzyme protein are not that great (as concluded mainly from the studies of active favipiravir-RTP binding affinities with the viral polymerase, e.g., Abdelnabi et al 2017 ); favipiravir/favipiravir-RTP structure is not an ideal hydrogen bond acceptor (Naydenova et al 2021 ); data concerning its clinical use in humans are not clear (Shiraki and Daikoku 2020 ; Cai et al 2020 ; Du and Chen 2020 ); favipiravir as an anti-COVID-19 drug is used primarily off-label (in Japan) as its use as anti-COVID-19 has not been approved (Shiraki and Daikoku 2020 ; Du and Chen 2020 ); and many favipiravir published articles and papers have irreproducible data (Dong et al 2020 ; Cai et al 2020 ).

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Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

“…These base pairs mimic natural C–G and U–A base pairs, respectively, and do not impair RdRp progression. This antiviral mechanism is conceptually similar to the recently suggested mutagenesis mode of action of favipiravir 47,48 , but is entirely distinct from that of remdesivir, which impairs RdRp progression 28 . However, like remdesivir, molnupiravir escapes viral RNA proofreading because M incorporation and M-directed misincorporation are apparently not recognized by the viral exonuclease 23,24 .…”

SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples

“…Additionally, in order to serve as a marker of replicating virus, sgRNA is expected to show a rapid decline after transcriptional inhibition due to ribonuclease degradation, in contrast to gRNA, which may be protected from degradation by viral capsids and therefore persist more durably (16). Therefore, we hypothesized that, upon treatment with SARS-CoV-2 RNA-dependent RNA polymerase inhibitors (17, 18, 19) in cell lines infected with SARS-CoV-2, we should observe a rapid decline of sgRNA after viral death when compared with gRNA.…”