Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

Abstract

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. 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 that contain 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 probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

Molnupiravir: coding for catastrophe

“…These structures thus provide a valuable direct visualization of how the NHC base of molnupiravir in the template strand base pairs with G and A to introduce G-to-A transition mutations. Kabinger et al 3 and Gordon et al 2 therefore arrive at similar conclusions about the mechanism of lethal mutagenesis by molnupiravir, with subtle differences. Gordon et al 2 stress that the C-to-U transition mutations occur when MNP is incorporated in the template strand because the two tautomers of NHC exist more equally than its substrate triphosphate form.…”

“…Molnupiravir is a prodrug derivatized from the ribonucleoside analog β-d-N 4 -hydroxycytidine (NHC) that is converted to its active form molnupiravir triphosphate (MTP) in the cell 6 . Both Gordon et al 2 and Kabinager et al 3 investigated the selective incorporation of MTP versus the natural nucleotides (ATP, GTP, UTP and CTP). They determined that MTP competes most effectively with CTP for incorporation into the product RNA.…”

“…Kabinger et al 3 used solid-phase synthesis to generate a template RNA strand with an internal MNP base to specifically asses the role of an NHC as the templating base. By contrast, Gorden et al 2 enzymatically synthesized a template RNA containing MNP.…”

“…In addition to the biochemical studies, Kabinger et al 3 acid scaffold. Although the cryo-EM maps cannot distinguish the two tautomeric forms, they reasonably assume that the amino form of molnupiravir base pairs with G, forming three hydrogen bonds.…”

Discovery, Development, and Patent Trends on Molnupiravir: A Prospective Oral Treatment for COVID-19

“…These features of molnupiravir also helped its rapid development as a treatment for COVID-19. The viricidal effect of molnupiravir is due to its mutagenic effects in the virus, wherein RdRp utilizes the active triphosphate form of molnupiravir (EIDD-2061, Figure 1C) as a substrate in place of cytidine/uridine triphosphate and generates mutated RNA copies [56]. A report states that active ribonucleosides, including molnupiravir, may also be mutagenic to the host [57].…”