The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Department of Organic Chemistry Faculty of Science Charles University Prague 128 00 Czech Republic

Department of Pharmacology and Toxicology University of Toronto Toronto Ontario M5S 1A8 Canada

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Náměstí 2 Prague 6 166 10 Czech Republic

QBI COVID 19 Research Group San Francisco California 94158 United States

Zobrazit více v PubMed

Zhu N.; Zhang D.; Wang W.; Li X.; Yang B.; Song J.; Zhao X.; Huang B.; Shi W.; Lu R.; et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 2020, 382, 727–733. 10.1056/nejmoa2001017. PubMed DOI PMC

Brant A. C.; Tian W.; Majerciak V.; Yang W.; Zheng Z. M. SARS-CoV-2: from its discovery to genome structure, transcription, and replication. Cell Biosci. 2021, 11, 136.10.1186/s13578-021-00643-z. PubMed DOI PMC

Park G. J.; Osinski A.; Hernandez G.; Eitson J. L.; Majumdar A.; Tonelli M.; Henzler-Wildman K.; Pawlowski K.; Chen Z.; Li Y.; et al. The mechanism of RNA capping by SARS-CoV-2. Nature 2022, 609, 793–800. 10.1038/s41586-022-05185-z. PubMed DOI PMC

V’Kovski P.; Kratzel A.; Steiner S.; Stalder H.; Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat. Rev. Microbiol. 2021, 19, 155–170. 10.1038/s41579-020-00468-6. PubMed DOI PMC

Cross S. T.; Michalski D.; Miller M. R.; Wilusz J. RNA regulatory processes in RNA virus biology. Wiley Interdiscip. Rev.: RNA 2019, 10, e153610.1002/wrna.1536. PubMed DOI PMC

Dong H.; Fink K.; Zust R.; Lim S. P.; Qin C. F.; Shi P. Y. Flavivirus RNA methylation. J. Gen. Virol. 2014, 95, 763–778. 10.1099/vir.0.062208-0. PubMed DOI

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

Slanina H.; Madhugiri R.; Bylapudi G.; Schultheiß K.; Karl N.; Gulyaeva A.; Gorbalenya A. E.; Linne U.; Ziebuhr J. Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit. Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e202231011810.1073/pnas.2022310118. PubMed DOI PMC

Chen Y.; Cai H.; Pan J.; Xiang N.; Tien P.; Ahola T.; Guo D. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3484–3489. 10.1073/pnas.0808790106. PubMed DOI PMC

Decroly E.; Imbert I.; Coutard B.; Bouvet M.; Selisko B.; Alvarez K.; Gorbalenya A. E.; Snijder E. J.; Canard B. Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2’O)-methyltransferase activity. J. Virol. 2008, 82, 8071–8084. 10.1128/jvi.00407-08. PubMed DOI PMC

Tahir M. Coronavirus genomic nsp14-ExoN, structure, role, mechanism, and potential application as a drug target. J. Med. Virol. 2021, 93, 4258–4264. 10.1002/jmv.27009. PubMed DOI PMC

Krafcikova P.; Silhan J.; Nencka R.; Boura E. Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin. Nat. Commun. 2020, 11, 3717.10.1038/s41467-020-17495-9. PubMed DOI PMC

Rona G.; Zeke A.; Miwatani-Minter B.; de Vries M.; Kaur R.; Schinlever A.; Garcia S. F.; Goldberg H. V.; Wang H.; Hinds T. R.; et al. The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target. Cell Death Differ. 2022, 29, 285–292. 10.1038/s41418-021-00900-1. PubMed DOI PMC

Case J. B.; Ashbrook A. W.; Dermody T. S.; Denison M. R. Mutagenesis of S-Adenosyl-L-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity. J. Virol. 2016, 90, 7248–7256. 10.1128/jvi.00542-16. PubMed DOI PMC

Pan R.; Kindler E.; Cao L.; Zhou Y.; Zhang Z.; Liu Q.; Ebert N.; Zust R.; Sun Y.; Gorbalenya A. E.; et al. N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition. mBio 2022, 13, e036622110.1128/mbio.03662-21. PubMed DOI PMC

Kasprzyk R.; Jemielity J. Enzymatic Assays to Explore Viral mRNA Capping Machinery. ChemBioChem 2021, 22, 3236–3253. 10.1002/cbic.202100291. PubMed DOI PMC

Jung E.; Soto-Acosta R.; Xie J.; Wilson D. J.; Dreis C. D.; Majima R.; Edwards T. C.; Geraghty R. J.; Chen L. Bisubstrate Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 Nsp14 Methyltransferase. ACS Med. Chem. Lett. 2022, 13, 1477–1484. 10.1021/acsmedchemlett.2c00265. PubMed DOI PMC

Ahmed-Belkacem R.; Hausdorff M.; Delpal A.; Sutto-Ortiz P.; Colmant A. M. G.; Touret F.; Ogando N. S.; Snijder E. J.; Canard B.; Coutard B.; et al. Potent Inhibition of SARS-CoV-2 nsp14 N7-Methyltransferase by Sulfonamide-Based Bisubstrate Analogues. J. Med. Chem. 2022, 65, 6231–6249. 10.1021/acs.jmedchem.2c00120. PubMed DOI

Ahmed-Belkacem R.; Sutto-Ortiz P.; Guiraud M.; Canard B.; Vasseur J. J.; Decroly E.; Debart F. Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase. Eur. J. Med. Chem. 2020, 201, 112557.10.1016/j.ejmech.2020.112557. PubMed DOI PMC

Devkota K.; Schapira M.; Perveen S.; Khalili Yazdi A.; Li F.; Chau I.; Ghiabi P.; Hajian T.; Loppnau P.; Bolotokova A.; et al. Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors. SLAS Discovery 2021, 26, 1200–1211. 10.1177/24725552211026261. PubMed DOI PMC

Bobileva O.; Bobrovs R.; Kanepe I.; Patetko L.; Kalnins G.; Sisovs M.; Bula A. L.; Gri̅nberga S.; Boroduskis M. R.; Ramata-Stunda A.; et al. Potent SARS-CoV-2 mRNA Cap Methyltransferase Inhibitors by Bioisosteric Replacement of Methionine in SAM Cosubstrate. ACS Med. Chem. Lett. 2021, 12, 1102–1107. 10.1021/acsmedchemlett.1c00140. PubMed DOI PMC

Otava T.; Sala M.; Li F.; Fanfrlik J.; Devkota K.; Perveen S.; Chau I.; Pakarian P.; Hobza P.; Vedadi M.; et al. The Structure-Based Design of SARS-CoV-2 nsp14 Methyltransferase Ligands Yields Nanomolar Inhibitors. ACS Infect. Dis. 2021, 7, 2214–2220. 10.1021/acsinfecdis.1c00131. PubMed DOI

Bobileva O.; Bobrovs R.; Sirma E. E.; Kanepe I.; Bula A. L.; Patetko L.; Ramata-Stunda A.; Grinberga S.; Jirgensons A.; Jaudzems K. 3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors. Molecules 2023, 28, 768.10.3390/molecules28020768. PubMed DOI PMC

Imprachim N.; Yosaatmadja Y.; Newman J. A. Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development. Nucleic Acids Res. 2023, 51, 475–487. 10.1093/nar/gkac1207. PubMed DOI PMC

Singh I.; Li F.; Fink E.; Chau I.; Li A.; Rodriguez-Hernández A.; Glenn I.; Zapatero-Belinchón F. J.; Rodriguez M.; Devkota K.; et al. Structure-based discovery of inhibitors of the SARS-CoV-2 Nsp14 N7-methyltransferase. J. Med. Chem. 2023, 66, 7785–7803. 10.1021/acs.jmedchem.2c02120. PubMed DOI PMC

Kasprzyk R.; Spiewla T. J.; Smietanski M.; Golojuch S.; Vangeel L.; De Jonghe S.; Jochmans D.; Neyts J.; Kowalska J.; Jemielity J. Identification and evaluation of potential SARS-CoV-2 antiviral agents targeting mRNA cap guanine N7-Methyltransferase. Antiviral Res. 2021, 193, 105142.10.1016/j.antiviral.2021.105142. PubMed DOI PMC

Pearson L. A.; Green C. J.; Lin D.; Petit A. P.; Gray D. W.; Cowling V. H.; Fordyce E. A. F. Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry. SLAS Discovery 2021, 26, 749–756. 10.1177/24725552211000652. PubMed DOI PMC

Liu Q.; Cai X.; Yang D.; Chen Y.; Wang Y.; Shao L.; Wang M. W. Cycloalkane analogues of sinefungin as EHMT1/2 inhibitors. Bioorg. Med. Chem. 2017, 25, 4579–4594. 10.1016/j.bmc.2017.06.032. PubMed DOI

Knapp D. C.; Serva S.; D’Onofrio J.; Keller A.; Lubys A.; Kurg A.; Remm M.; Engels J. W. Fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension. Chem.—Eur. J. 2011, 17, 2903–2915. 10.1002/chem.201001952. PubMed DOI PMC

Kielkowski P.; Pohl R.; Hocek M. Synthesis of acetylene linked double-nucleobase nucleos(t)ide building blocks and polymerase construction of DNA containing cytosines in the major groove. J. Org. Chem. 2011, 76, 3457–3462. 10.1021/jo200436j. PubMed DOI

Kalcic F.; Zgarbova M.; Hodek J.; Chalupsky K.; Dracinsky M.; Dvorakova A.; Strmen T.; Sebestik J.; Baszczynski O.; Weber J.; et al. Discovery of Modified Amidate (ProTide) Prodrugs of Tenofovir with Enhanced Antiviral Properties. J. Med. Chem. 2021, 64, 16425–16449. 10.1021/acs.jmedchem.1c01444. PubMed DOI

Emeny J. M.; Morgan M. J. Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J. Gen. Virol. 1979, 43, 247–252. 10.1099/0022-1317-43-1-247. PubMed DOI

Czarna A.; Plewka J.; Kresik L.; Matsuda A.; Karim A.; Robinson C.; O’Byrne S.; Cunningham F.; Georgiou I.; Wilk P.; et al. Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity. Structure 2022, 30, 1050–1054.e2. 10.1016/j.str.2022.04.014. PubMed DOI PMC

Jones G.; Willett P.; Glen R. C.; Leach A. R.; Taylor R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 1997, 267, 727–748. 10.1006/jmbi.1996.0897. PubMed DOI

Trott O.; OlsonVina A. J. AutoDock improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455–461. 10.1002/jcc.21334. PubMed DOI PMC

Wang Z.; Sun H.; Yao X.; Li D.; Xu L.; Li Y.; Tian S.; Hou T. Comprehensive evaluation of ten docking programs on a diverse set of protein-ligand complexes: the prediction accuracy of sampling power and scoring power. Phys. Chem. Chem. Phys. 2016, 18, 12964–12975. 10.1039/c6cp01555g. PubMed DOI

Schrödinger L.; DeLano W.. PyMOL, 2020. Retrieved from http://www.pymol.org/pymol.

Structure of SARS-CoV-2 MTase nsp14 with the inhibitor STM957 reveals inhibition mechanism that is shared with a poxviral MTase VP39

Meeting report of the 37th International Conference on Antiviral Research in Gold Coast, Australia, May 20-24, 2024, organized by the International Society for Antiviral Research

Discovery of highly potent SARS-CoV-2 nsp14 methyltransferase inhibitors based on adenosine 5'-carboxamides