Small-molecule Inhibition of SARS-CoV-2 NSP14 RNA Cap Methyltransferase

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid development of highly effective vaccines against SARS-CoV-2 has altered the trajectory of the pandemic, and antiviral therapeutics have further reduced the number of COVID-19 hospitalizations and deaths. Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that encode various structural and non-structural proteins, including those critical for viral RNA replication and evasion from innate immunity. Here we report the discovery and development of a first-in-class non-covalent small-molecule inhibitor of the viral guanine-N7 methyltransferase (MTase) NSP14. High-throughput screening identified RU-0415529, which inhibited SARS-CoV-2 NSP14 by forming a unique ternary S-adenosylhomocysteine (SAH)-bound complex. Hit-to-lead optimization of RU-0415529 resulted in TDI-015051 with a dissociation constant (K) of 61 pM and a half-maximal effective concentration (EC) of 11 nM, inhibiting virus infection in a cell-based system. TDI-015051 also inhibited viral replication in primary small airway epithelial cells and in a transgenic mouse model of SARS CoV-2 infection with an efficacy comparable with the FDA-approved reversible covalent protease inhibitor nirmatrelvir. The inhibition of viral cap methylases as an antiviral strategy is also adaptable to other pandemic viruses.

Citing Articles

Strategies for the Viral Exploitation of Nuclear Pore Transport Pathways.

Zhang X, Lim K, Qiu Y, Hazawa M, Wong R Viruses. 2025; 17(2).

PMID: 40006906 PMC: 11860923. DOI: 10.3390/v17020151.

References

Phillips N . The coronavirus is here to stay - here's what that means. Nature. 2021; 590(7846):382-384. DOI: 10.1038/d41586-021-00396-2. View

Dolgin E . Pan-coronavirus vaccine pipeline takes form. Nat Rev Drug Discov. 2022; 21(5):324-326. DOI: 10.1038/d41573-022-00074-6. View

Chaudhary N, Weissman D, Whitehead K . mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov. 2021; 20(11):817-838. PMC: 8386155. DOI: 10.1038/s41573-021-00283-5. View

Li G, Hilgenfeld R, Whitley R, De Clercq E . Therapeutic strategies for COVID-19: progress and lessons learned. Nat Rev Drug Discov. 2023; 22(6):449-475. PMC: 10113999. DOI: 10.1038/s41573-023-00672-y. View

Malone B, Urakova N, Snijder E, Campbell E . Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat Rev Mol Cell Biol. 2021; 23(1):21-39. PMC: 8613731. DOI: 10.1038/s41580-021-00432-z. View

Owen D, Allerton C, Anderson A, Aschenbrenner L, Avery M, Berritt S . An oral SARS-CoV-2 M inhibitor clinical candidate for the treatment of COVID-19. Science. 2021; 374(6575):1586-1593. DOI: 10.1126/science.abl4784. View

Nencka R, Silhan J, Klima M, Otava T, Kocek H, Krafcikova P . Coronaviral RNA-methyltransferases: function, structure and inhibition. Nucleic Acids Res. 2022; 50(2):635-650. PMC: 8789044. DOI: 10.1093/nar/gkab1279. View

Decroly E, Ferron F, Lescar J, Canard B . Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol. 2011; 10(1):51-65. PMC: 7097100. DOI: 10.1038/nrmicro2675. View

Both G, Furuichi Y, Muthukrishnan S, Shatkin A . Ribosome binding to reovirus mRNA in protein synthesis requires 5' terminal 7-methylguanosine. Cell. 1975; 6(2):185-95. DOI: 10.1016/0092-8674(75)90009-4. View

10.

Muthukrishnan S, Both G, Furuichi Y, Shatkin A . 5'-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation. Nature. 1975; 255(5503):33-7. DOI: 10.1038/255033a0. View

11.

Both G, Banerjee A, Shatkin A . Methylation-dependent translation of viral messenger RNAs in vitro. Proc Natl Acad Sci U S A. 1975; 72(3):1189-93. PMC: 432492. DOI: 10.1073/pnas.72.3.1189. View

12.

Ferron F, Decroly E, Selisko B, Canard B . The viral RNA capping machinery as a target for antiviral drugs. Antiviral Res. 2012; 96(1):21-31. PMC: 7114304. DOI: 10.1016/j.antiviral.2012.07.007. View

13.

Zhang J, Zheng Y . SAM/SAH Analogs as Versatile Tools for SAM-Dependent Methyltransferases. ACS Chem Biol. 2015; 11(3):583-97. PMC: 5772741. DOI: 10.1021/acschembio.5b00812. View

14.

Czarna A, Plewka J, Kresik L, Matsuda A, Karim A, Robinson C . Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity. Structure. 2022; 30(8):1050-1054.e2. PMC: 9125827. DOI: 10.1016/j.str.2022.04.014. View

15.

Ma Y, Wu L, Shaw N, Gao Y, Wang J, Sun Y . Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex. Proc Natl Acad Sci U S A. 2015; 112(30):9436-41. PMC: 4522806. DOI: 10.1073/pnas.1508686112. View

16.

Ferron F, Subissi L, Silveira De Morais A, Le N, Sevajol M, Gluais L . Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA. Proc Natl Acad Sci U S A. 2017; 115(2):E162-E171. PMC: 5777078. DOI: 10.1073/pnas.1718806115. View

17.

Kottur J, Rechkoblit O, Quintana-Feliciano R, Sciaky D, Aggarwal A . High-resolution structures of the SARS-CoV-2 N7-methyltransferase inform therapeutic development. Nat Struct Mol Biol. 2022; 29(9):850-853. PMC: 10388636. DOI: 10.1038/s41594-022-00828-1. View

18.

Liu C, Shi W, Becker S, Schatz D, Liu B, Yang Y . Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme. Science. 2021; 373(6559):1142-1146. PMC: 9836006. DOI: 10.1126/science.abi9310. View

19.

Bootsma A, Wheeler S . Tuning Stacking Interactions between Asp-Arg Salt Bridges and Heterocyclic Drug Fragments. J Chem Inf Model. 2018; 59(1):149-158. DOI: 10.1021/acs.jcim.8b00563. View

20.

Craft M, Waldrop G . Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate. J Enzyme Inhib Med Chem. 2021; 37(1):100-108. PMC: 8667948. DOI: 10.1080/14756366.2021.1994558. View