How does this drug function to prevent SARS-CoV-2 replication?

Molnupiravir is a nucleoside analog, that can replace the nucleoside C or U. When the bioavailable form incorporated into viral genome during replication, the accumulated “error catastrophe” would lead to lethal mutagenesis of SARS-CoV-2. The key is that the drug-induced error rate of replication surpasses the error threshold that can sustain a virus population whereas the host genome or transcription remained stable owning to effective proofreading activity. For instance, the paper examined host interferon-stimulated gene 15 (ISG15) mRNA, a highly up-regulated innate immune-related gene after MERS-CoV infection. ISG15 mRNA error rates remained at the base line across all groups, including vehicle control and oral administration of molnupiravir at −2, +12, +24, or +48 hour post infection. It is important to take the considerations that Coronavirus (CoVs) are positive-sense RNA viruses that replicate through a negative-sense RNA intermediate. Therefore, the incorporation of molnupiravir (or EIDD-201) as a C or a U can occur in both polarities of RNA.

RNA-dependent RNA polymerase (RdRp), also known as nsp12, is a key component for the replication and transcription of viral RNA genome (RNA) (1). RdRp is consider the primary target of nucleoside analog (NA) inhibitor. Molnupiravir targets the RdRp of SARS-CoV-2. The biochemical mechanism of action of molnupiravir is entirely different from that of remdesivir or chain-terminating nucleoside analogs (2). Here, Sheahan et al. (2020) first showed that potent antiviral activities of molnupiravir against MERS-CoV and SARS-CoV-2 in the human lung epithelial cell line Calu-3 and in primary human airway epithelial cells (3). The sequences and structure motifs of RdRp or nsp12 proteins are highly conserved across the Coronavirus family including the newly emerged SARS-CoV-2. For example, the RdRp of SARS-CoV-2 has 99.1% similarity and 96% amino acid identity to that of SARS-CoV. Remdesivir was the first FDA-approved drug for the treatment of patients with COVID-19, but CoV resistance to remdesivir was reported with 5-fold increases in IC50. RdRp point mutations such as F480L and V557L confer the resistance of CoV to remdesivir in a model CoV mouse hepatitis virus (MHV) and in SARS-CoV. However, the remdesivir-resistant mutation strain is still susceptible to molnupiravir, suggestion that the two broad-spectrum drugs may select for exclusive and mutually sensitizing resistance pathways.

What are the most important findings in this paper that support the clinical development of the drug?

• Remdesivir is the first FDA-approved, broad-spectrum nucleoside analog drug for treatment of COVID-19. However, its efficiency is disputed because of CoVs resistance to remdesivir has been identified. Importantly, this study demonstrated that molnupiravir is effective against remdesivir-resistant virus and multiple distinct zoonotic CoVs.
• Second, it proved the drug has broad-spectrum and potent antiviral activity to phylogenic distantly CoVs, although many of which have up to 20% variation in the RdRp.
• Mechanistically, the paper provides insights that molnupiravir causes excessively high error rate of viral RNA production. For example, they conducted Primer ID next generation sequencing and showed MERS-CoVviral RNA transition mutation frequency is increased during NHC treatment. With 10 μM molnupiravir, a six-fold increase in error rate resulted in a 26,000-fold decrease in virus titer.
• Additionally, molnupiravir has both prophylactic and therapeutic effects on CoVs, demonstrating by infecting the mouse model with SARS-CoV and MERS-CoV.
• Considering the safety of molnupiravir, it should not cause mutation of host genome. In order words, the molnupiravir-induced error rate should not surpass the proofreading capacity of host. To examine this, the authors extracted total lung RNA, and they targeted sequencing viral nsp10 transcript and host ISG15 transcript, which is highly expressed in response to viral infection. Genome mutation rates during replication were found excessively high in viral nsp10 gene, reaching to over 40 transitions per 10k bases. In contrast, the host ISG15 remain largely unchanged.

Describe the main benefits and concerns surrounding the use of Molnupiravir in SARS-CoV-2 infected patients.

Benefits:

• Like other broad-spectrum, nucleoside analog antiviral drugs, molnupiravir targets viral polymerases but does not terminate RNA chain elongation. Therefore, molnupiravir can circumvent exonucleolytic proofreading that can remove misincorporated nucleotides from the nascent RNA 3’ end (2).
• Different from FDA-proved remdesivir, molnupiravir incorporation does not cause RdRp to stall.
• It is an orally bioavailable prodrug.
• The effectiveness is robust to many CoV RdRp proteins with variations.

Concerns:

• The study only tested the drug efficiency against SARS-CoV-2 in vitro in human lung epithelial cell or human airway epithelial cells. MERS-CoV in vivo infection experiments were conducted with genetically modified mice encoding a murine DPP4 receptor encoding two human residues at positions 288 and 330 (humanized DPP4 (hDPP4) 288/330 mice). However, testing SARS-CoV-2 in mouse model is still challenging because mouse ACE2 receptor is not compatible.
• The onsets of symptom are different between the mouse model and human. Therefore, the window in when to treat COVID-19 patients before peak virus replication requires more clinic experience.
• SARS-CoV, MERS-CoV, and SARS-CoV-2 disease severity increases with increasing age. This study has limitations in lacking aged mouse models that can recapitulate the age-specific pathogenesis as observed in aging human.

List of references

1. Zhao Y, He G, Huang W. 2021. A novel model of molnupiravir against SARS-CoV-2 replication: accumulated RNA mutations to induce error catastrophe. 1. Sig Transduct Target Ther 6:1–3.
2. Kabinger F, Stiller C, Schmitzová J, Dienemann C, Kokic G, Hillen HS, Höbartner C, Cramer P. 2021. Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. 9. Nat Struct Mol Biol 28:740–746.
3. Sheahan TP, Sims AC, Zhou S, Graham RL, Pruijssers AJ, Agostini ML, Leist SR, Schäfer A, Dinnon KH, Stevens LJ, Chappell JD, Lu X, Hughes TM, George AS, Hill CS, Montgomery SA, Brown AJ, Bluemling GR, Natchus MG, Saindane M, Kolykhalov AA, Painter G, Harcourt J, Tamin A, Thornburg NJ, Swanstrom R, Denison MR, Baric RS. 2020. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Science Translational Medicine 12:eabb5883.