Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

Abstract

Molnupiravir is an orally available antiviral drug candidate that is in phase III trials for the treatment of COVID-19 patients. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in patients. Here we establish the molecular mechanisms that underlie molnupiravir-induced RNA mutagenesis by the RNA-dependent RNA polymerase (RdRp) of the coronavirus SARS-CoV-2. Biochemical assays show that the RdRp readily uses the active form of molnupiravir, β-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. Incorporation of NHC monophosphate into nascent RNA does not impair further RdRp progression. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes containing mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

Broad spectrum antiviral nucleosides—Our best hope for the future

“…Lethal mutagenesis is a type of antiviral therapy that essentially increases the viral mutation rate by incorporating nucleosides that act as mutagens, to the point of destroying the viability of the virus. 10,77 One approved nucleoside that has been shown to work by this mechanism is ribavirin, although ribavirin has also been shown to work by other mechanisms and in general, its mechanism of action is not well understood. 6 Ribavirin (Fig.…”

“…13) and is reported to increase mutations of G to A and C to U in replicating coronaviruses. 77 So far it is showing great promise and is currently in Phase II/III clinical trials. Interestingly, the U.S. Department of Health and Human Services has committed to purchasing $1.2B worth of molnupiravir should it ultimately be approved by the FDA.…”

Profile of Patrick Cramer

“…Given Cramer's passion for polymerases, it is not surprising that his group also became interested in solving the structure of the polymerase from the novel coronavirus SARS-CoV-2. His group successfully solved this structure, which provides a basis for understanding how antivirals, such as remdesivir or molnupiravir, work against this novel coronavirus to interfere with its RNA synthesis (41)(42)(43). "We have now teamed up with chemists to search for new antiviral compounds," Cramer says.…”

Pentosan Polysulfate Inhibits Attachment and Infection by SARS-CoV-2 In Vitro: Insights into Structural Requirements for Binding

“…Additional treatments are therefore required and, indeed, during the writing of this article, the first orally available antiviral drug against COVID, molnupiravir, was approved for use in the United Kingdom. 4 5…”