Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template

Abstract

The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 is an important target in current drug development efforts for the treatment of coronavirus disease 2019. Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5’-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate in the RNA primer strand. When embedded in the template strand, NHC-monophosphate supported the formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand. The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 is an important target in current drug development efforts for the treatment of coronavirus disease 2019. Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5’-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate in the RNA primer strand. When embedded in the template strand, NHC-monophosphate supported the formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand. Decoding molnupiravir-induced mutagenesis in SARS-CoV-2Journal of Biological ChemistryVol. 297Issue 1PreviewMolnupiravir, a prodrug of the nucleoside derivative β-D-N4-hydroxycytidine (NHC), is currently in clinical trials for COVID-19 therapy. However, the biochemical mechanisms involved in molnupiravir-induced mutagenesis had not been explored. In a recent study, Gordon et al. demonstrated that NHC can be incorporated into viral RNA and subsequently extended and used as template for RNA-dependent RNA synthesis, proposing a mutagenesis model consistent with available virological evidence. Their study uncovers molecular mechanisms by which molnupiravir drives SARS-CoV-2 into error catastrophe. Full-Text PDF Open Access The discovery and development of potent antiviral drugs for the treatment of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains challenging. The virus can cause severe forms of coronavirus disease 2019 (COVID-19) that require hospitalization. Remdesivir (RDV) targets the viral RNA-dependent RNA polymerase (RdRp) and is currently the only antiviral agent approved by the US Food and Drug Administration (1U.S. Food and Drug AdministrationFact Sheet for Health Care Providers Emergency Use Authorization (EUA) of Remdesivir (GS-5734TM). U.S. Food and Drug Administration, Silver Spring, MD2020Google Scholar, 2Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Gotte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295: 6785-6797Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). Antibody therapies were granted emergency use authorization for the treatment of outpatients who are at high risk for progressing to severe disease and/or hospitalization (3U.S. Food and Drug AdministrationFact Sheet for Health Care Providers Emergency Use Authorization (EUA) of Casirivimab and Imdevimab. U.S. Food and Drug Administration, Silver Spring, MD2020Google Scholar) run up. Both RDV and antibody therapies are intravenously administered, which limits their utility especially for outpatient use. Oral drugs that can be used much earlier in the disease are under investigation, and molnupiravir (MK-4482/EIDD-2801) is perhaps the most advanced candidate compound in this category (4Vasudevan N. Ahlqvist G.P. McGeough C.P. Paymode D.J. Cardoso F.S.P. Lucas T. Dietz J.P. Opatz T. Jamison T.F. Gupton F.B. Snead D.R. A concise route to MK-4482 (EIDD-2801) from cytidine.Chem. Commun. (Camb.). 2020; 56: 13363-13364Crossref PubMed Google Scholar). 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Andres E.L. Bluemling G.R. Lockwood M.A. Sheahan T.P. Sims A.C. Natchus M.G. Saindane M. Kolykhalov A.A. Painter G.R. Baric R.S. et al.Small-molecule antiviral beta-d-N (4)-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance.J. Virol. 2019; 93e01348-19Crossref PubMed Scopus (202) Google Scholar, M. Yoon J.J. Cox R.M. Hart M. Sticher Z.M. Makhsous N. Plesker R. Barrena A.H. Reddy P.G. Mitchell D.G. Shean R.C. Bluemling G.R. Kolykhalov A.A. Greninger A.L. Natchus M.G. et al.Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and 2019; PubMed Scopus Google Scholar, T.P. Sims A.C. S. Pruijssers A.J. M.L. A. L.J. Chappell J.D. Lu X. et orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in and in 2020; PubMed Scopus Google Scholar). to NHC increases G to A and C to U transition mutations in and SARS-CoV-2 M.L. Pruijssers A.J. Chappell J.D. Gribble J. Lu X. Andres E.L. Bluemling G.R. Lockwood M.A. Sheahan T.P. Sims A.C. Natchus M.G. Saindane M. Kolykhalov A.A. Painter G.R. Baric R.S. et al.Small-molecule antiviral beta-d-N (4)-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance.J. Virol. 2019; 93e01348-19Crossref PubMed Scopus (202) Google Scholar, T.P. Sims A.C. S. Pruijssers A.J. M.L. A. L.J. Chappell J.D. Lu X. et orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in and in 2020; PubMed Scopus Google Scholar). in mutation frequencies which the antiviral broad-spectrum antiviral as or have been as nucleoside the of these is with in the higher X. X. J. J. M. T. C.K. S. J.P. Shi A SARS-CoV-2 for and of drugs for Commun. 2020; PubMed Scopus Google Scholar, A. B. J. F. V. F. E. B. O. B. incorporation of by the and viral RNA polymerase in SARS-CoV-2 Commun. 2020; PubMed Scopus Google Scholar, S. D. J.J. J.Y. R. The broad-spectrum antiviral ribonucleoside is an RNA virus PubMed Scopus Google Scholar). 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However, is currently not the the drug and drug and the of the observed mutations. we a biochemical to study the mechanism of action of and purified the with and and studied the efficiency of incorporation of NHC-TP in to natural NHC-TP is incorporated as a When the monophosphate is embedded in the base pairs with GTP or The incorporation of GTP inhibition of RNA synthesis, extension with the incorporated ATP is not and to transition mutations. The data in this study a model that the antiviral effects of molnupiravir and data on NHC-TP as a substrate for RNA polymerases are Z.M. Lu Mitchell D.G. J. L. Bluemling G.R. Guthrie D.B. Natchus M.G. Painter G.R. Kolykhalov A.A. of the for (4)-hydroxycytidine to replication and Agents Chemother. 2020; PubMed Scopus Google Scholar). It been that the respiratory syncytial virus NHC-TP as a substrate for incorporation template The incorporated not as a in this J.J. Toots M. Lee S. Lee M.E. Ludeke B. Luczo J.M. Ganti K. Cox R.M. Sticher Z.M. Edpuganti V. Mitchell D.G. Lockwood M.A. Kolykhalov A.A. Greninger A.L. Moore M.L. et al.Orally efficacious broad-spectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses.Antimicrob. Agents Chemother. 2018; 62e00766-18Crossref PubMed Scopus (123) Google Scholar). RNA polymerase can use NHC-TP as or Z.M. Lu Mitchell D.G. J. L. Bluemling G.R. Guthrie D.B. Natchus M.G. Painter G.R. Kolykhalov A.A. of the for (4)-hydroxycytidine to replication and Agents Chemother. 2020; PubMed Scopus Google Scholar). effects incorporation have not been reported. Here we for incorporation of NHC-TP by SARS-CoV-2 have the biochemical for and which C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Gotte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. 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The efficiency of incorporation of the natural nucleotide over the efficiency of incorporation of the analogue a SARS-CoV-2 shows a for CTP over NHC-TP the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), which shows that are incorporated the The data that with CTP is most RNA synthesis incorporation of template G at The concentrations of CTP and NHC-TP incorporation at most of the primer is to the product with formation of of CTP to formation and is under these both a product and a product are which to a of base both are in the of ATP and UTP the product NHC-TP as a in the In the of concentrations of ATP and UTP that product the primer is extended of or was incorporated CTP and NHC-TP shows that the incorporated is concentrations of ATP and UTP and similar of CTP and NHC-TP similar of activity are at high concentrations of ATP and UTP of or are extended from a product J. R. L.J. N. J. Identification and of a coronavirus RNA Virol. 2019; PubMed Scopus Google Scholar). In the of significant RNA synthesis the of the viral is in and embedded efficiency and of RNA synthesis be at a this is as a this we have an RNA template with a RNA polymerase we model with a or a at an incorporated as a in the template we RNA synthesis in the of concentrations of GTP of GTP is at concentrations as as In contrast, the incorporation of GTP is and concentrations of GTP are to the product to RNA synthesis the product template is not the are with GTP incorporation at the product formation on ATP which is at in order to is that concentrations of GTP in on both product formation template is with template that RNA synthesis be The of product to a of the of nucleotide we RNA synthesis in the of the template the concentrations of GTP and ATP an product that is into product at concentrations as as and The product is not with template which shows inhibition by in the The product is not the of ATP in the of GTP which that incorporation of GTP is the cause for both GTP and ATP can be incorporated with similar only GTP a that can be overcome by have that CTP UTP is incorporated embedded in the template shows base with GTP and the level of NHC-TP shows a for template The broad-spectrum antiviral agent a prodrug of is currently in a clinical for the treatment of SARS-CoV-2 F. N. M.J. L.J. Painter G.R. and of a novel broad-spectrum antiviral agent with activity against Agents Chemother. Scopus Google Scholar). The drug is orally bioavailable and can be to outpatients in the disease with the to data in a increase in G to A and C to U transition mutations that with increases in antiviral effects against M.L. Pruijssers A.J. Chappell J.D. Gribble J. Lu X. Andres E.L. Bluemling G.R. Lockwood M.A. Sheahan T.P. Sims A.C. Natchus M.G. Saindane M. Kolykhalov A.A. Painter G.R. Baric R.S. et al.Small-molecule antiviral beta-d-N (4)-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance.J. Virol. 2019; 93e01348-19Crossref PubMed Scopus (202) Google Scholar, T.P. Sims A.C. S. Pruijssers A.J. M.L. A. L.J. Chappell J.D. Lu X. et orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in and in 2020; PubMed Scopus Google Scholar). Molnupiravir is as a nucleotide Here we studied the biochemical mechanisms with the purified of on we a model that effects on both efficiency and of RNA synthesis demonstrated that NHC-TP predominantly as a and is incorporated template G as efficiency of incorporation of CTP over efficiency of incorporation of is with a of the of is that is incorporated its natural ATP C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Gotte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295: 6785-6797Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Gotte M. The antiviral compound inhibits RNA-dependent RNA polymerase from respiratory syndrome Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar). However, earlier studies have that in to is to the activity M.L. Pruijssers A.J. Chappell J.D. Gribble J. Lu X. Andres E.L. Bluemling G.R. Lockwood M.A. Sheahan T.P. Sims A.C. Natchus M.G. Saindane M. Kolykhalov A.A. Painter G.R. Baric R.S. et al.Small-molecule antiviral beta-d-N (4)-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance.J. Virol. 2019; 93e01348-19Crossref PubMed Scopus (202) Google Scholar). hepatitis C virus the of the 5’-triphosphate of is but in the of a is an inhibitor of hepatitis C virus replication A. N. L. J. of incorporation and the inhibition of nucleotide against hepatitis C virus Agents Chemother. PubMed Scopus Google Scholar). the for incorporation of NHC-TP have an on efficiency and of viral When is in the base is and both the GTP and ATP are with significant NHC-TP in forms that base of in the of of the antiviral drugs used against Chem. Chem. 2020; PubMed Google Scholar). The form C and base with the form U and base with A data that the NHC-TP substrate predominantly in its form and however, as in the both forms to and CTP or incorporation of GTP and of GTP inhibits incorporation of the concentrations to can overcome this and on RNA synthesis is not observed with ATP and incorporation of ATP a G to A transition mutation via and base or C to U are the viral RNA a C via the for the is and to the higher frequencies of G to A and C to U transition mutations in the of molnupiravir or The mechanism to influenza virus that shows the transition mutations in the of the but not to respiratory syncytial virus that shows a J.J. Toots M. Lee S. Lee M.E. Ludeke B. Luczo J.M. Ganti K. Cox R.M. Sticher Z.M. Edpuganti V. Mitchell D.G. Lockwood M.A. Kolykhalov A.A. Greninger A.L. Moore M.L. et al.Orally efficacious broad-spectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses.Antimicrob. Agents Chemother. 2018; 62e00766-18Crossref PubMed Scopus (123) Google Scholar, M. Yoon J.J. Cox R.M. Hart M. Sticher Z.M. Makhsous N. Plesker R. Barrena A.H. Reddy P.G. Mitchell D.G. Shean R.C. Bluemling G.R. Kolykhalov A.A. Greninger A.L. Natchus M.G. et al.Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and 2019; PubMed Scopus Google Scholar). However, biochemical data are and be important to study viral and polymerases that NHC-TP as In the study, we have that the incorporated can RNA synthesis in or as a in The to be that concentrations can overcome inhibition at concentrations of In that concentrations of ATP are higher that of GTP concentrations of and PubMed Scopus Google the be in a RNA and RNA with embedded used in this study were and from NHC-TP were from Schinazi and from were from The SARS-CoV-2 was by and as a by a and the on as C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Gotte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295: 6785-6797Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). incorporation by SARS-CoV-2 and data and were as by C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Gotte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295: 6785-6797Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Gotte M. The antiviral compound inhibits RNA-dependent RNA polymerase from respiratory syndrome Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar, E.P. Gordon C.J. Woolner E. D. Perry J.K. Feng J.Y. Porter D.P. Gotte M. inhibition of coronavirus RNA-dependent RNA polymerase by a mechanism of Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar). was or for and nucleotide incorporation RNA synthesis was from nucleotide incorporation were used to the for the natural nucleotide over The is as a of the incorporation of the natural nucleotide over the nucleotide The efficiency of nucleotide incorporation is by the of over The substrate for nucleotide incorporation is a primer by incorporation of into a of the primer is at a however, its is the product in the is by the to the primer product and to the in the primer and the product The product is by the substrate in order to the of the which is not as the of is as product over The is as is the of with the RNA with embedded were as by E.P. Gordon C.J. Woolner E. D. Perry J.K. Feng J.Y. Porter D.P. Gotte M. inhibition of coronavirus RNA-dependent RNA polymerase by a mechanism of Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar). are in data are the E.P. Gordon C.J. Woolner E. D. Perry J.K. Feng J.Y. Porter D.P. Gotte M. inhibition of coronavirus RNA-dependent RNA polymerase by a mechanism of Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar). The that have of with the of this Woolner and for M. J. and E. T. J. E. and R. F. S. E. T. M. J. and E. T. M. J. and E. T. J. and E. T. M. and R. F. S. M. J. and E. T. data M. and E. T. M. J. E. T. and R. F. S. and M. J. and E. T. M. M. M. study was supported by to M. from the of Health and from the of and by the for the Health R. F. S. is supported by and The is the of the and not the of the of

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