Seven coronaviruses have infected humans (HCoVs) to-date. SARS-CoV-2 caused the current COVID-19 pandemic with the well-known high mortality and severe socioeconomic consequences. MERS-CoV and SARS-CoV caused epidemic of MERS and SARS, respectively, with severe respiratory symptoms and significant fatality. However, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause respiratory illnesses with less severe symptoms in most cases. All coronaviruses use RNA capping to evade the immune systems of humans. Two viral methyltransferases, nsp14 and nsp16, play key roles in RNA capping and are considered valuable targets for development of anti-coronavirus therapeutics. But little is known about the kinetics of nsp10-nsp16 methyltransferase activities of most HCoVs, and reliable assays for screening are not available. Here, we report the expression, purification, and kinetic characterization of nsp10-nsp16 complexes from six HCoVs in parallel with previously characterized SARS-CoV-2. Probing the active sites of all seven by SS148 and WZ16, the two recently reported dual nsp14 / nsp10-nsp16 inhibitors, revealed pan-inhibition. Overall, our study show feasibility of developing broad-spectrum dual nsp14 / nsp10-nsp16-inhibitor therapeutics.

Drug Discovery Program Ontario Institute for Cancer Research Toronto Ontario Canada; Department of Pharmacology and Toxicology University of Toronto Toronto Ontario M5S 1A8 Canada

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague 6 Czech Republic

Structural Genomics Consortium University of Toronto Toronto Ontario M5G 1L7 Canada

Structural Genomics Consortium University of Toronto Toronto Ontario M5G 1L7 Canada; Drug Discovery Program Ontario Institute for Cancer Research Toronto Ontario Canada

Structural Genomics Consortium University of Toronto Toronto Ontario M5G 1L7 Canada; Drug Discovery Program Ontario Institute for Cancer Research Toronto Ontario Canada; Department of Pharmacology and Toxicology University of Toronto Toronto Ontario M5S 1A8 Canada; QBI COVID 19 Research Group San Francisco CA USA

Zobrazit více v PubMed

Cui J., Li F., Shi Z.L. Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 2019;17(3):181–192. PubMed PMC

Donaldson E.F., Haskew A.N., Gates J.E., Huynh J., Moore C.J., Frieman M.B. Metagenomic analysis of the viromes of three north American bat species: viral diversity among different bat species that share a common habitat. J. Virol. 2010;84(24):13004–13018. PubMed PMC

Huynh J., Li S., Yount B., Smith A., Sturges L., Olsen J.C., et al. Evidence supporting a zoonotic origin of human coronavirus strain NL63. J. Virol. 2012;86(23):12816–12825. PubMed PMC

Pfefferle S., Oppong S., Drexler J.F., Gloza-Rausch F., Ipsen A., Seebens A., et al. Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana. Emerg. Infect. Dis. 2009;15(9):1377–1384. PubMed PMC

Kesheh M.M., Hosseini P., Soltani S., Zandi M. An overview on the seven pathogenic human coronaviruses. Rev. Med. Virol. 2022;32(2) PubMed

Drosten C., Gunther S., Preiser W., van der Werf S., Brodt H.R., Becker S., et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 2003;348(20):1967–1976. PubMed

Fouchier R.A., Kuiken T., Schutten M., van Amerongen G., van Doornum G.J., van den Hoogen B.G., et al. Aetiology: Koch’s postulates fulfilled for SARS virus. Nature. 2003;423(6937):240. PubMed PMC

Ksiazek T.G., Erdman D., Goldsmith C.S., Zaki S.R., Peret T., Emery S., et al. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 2003;348(20):1953–1966. PubMed

Zhong N.S., Zheng B.J., Li Y.M., Poon Xie Z.H., Chan K.H., et al. Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People’s Republic of China, in February, 2003. Lancet. 2003;362(9393):1353–1358. PubMed PMC

Zaki A.M., van Boheemen S., Bestebroer T.M., Osterhaus A.D., Fouchier R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012;367(19):1814–1820. PubMed

Cheng V.C., Lau S.K., Woo P.C., Yuen K.Y. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin. Microbiol. Rev. 2007;20(4):660–694. PubMed PMC

Corman V.M., Muth D., Niemeyer D., Drosten C. Hosts and sources of endemic human coronaviruses. Adv. Virus Res. 2018;100:163–188. PubMed PMC

Andersen K.G., Rambaut A., Lipkin W.I., Holmes E.C., Garry R.F. The proximal origin of SARS-CoV-2. Nat. Med. 2020;26(4):450–452. PubMed PMC

Furuichi Y., Shatkin A.J. Viral and cellular mRNA capping: past and prospects. Adv. Virus Res. 2000;55:135–184. PubMed PMC

Menachery V.D., Debbink K., Baric R.S. Coronavirus non-structural protein 16: evasion, attenuation, and possible treatments. Virus Res. 2014;194:191–199. PubMed PMC

Snijder E.J., Bredenbeek P.J., Dobbe J.C., Thiel V., Ziebuhr J., Poon L.L., et al. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 2003;331(5):991–1004. PubMed PMC

Chen Y., Cai H., Pan J., Xiang N., Tien P., Ahola T., et al. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc. Natl. Acad. Sci. U. S. A. 2009;106(9):3484–3489. PubMed PMC

Chen Y., Su C., Ke M., Jin X., Xu L., Zhang Z., et al. Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2’-O-methylation by nsp16/nsp10 protein complex. PLoS Pathog. 2011;7(10) PubMed PMC

Decroly E., Debarnot C., Ferron F., Bouvet M., Coutard B., Imbert I., et al. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2’-O-methyltransferase nsp10/nsp16 complex. PLoS Pathog. 2011;7(5) PubMed PMC

Khalili Yazdi A., Li F., Devkota K., Perveen S., Ghiabi P., Hajian T., et al. A high-throughput radioactivity-based assay for screening SARS-CoV-2 nsp10-nsp16 complex. SLAS Discov. 2021;26(6):757–765. PubMed PMC

Devkota K., Schapira M., Perveen S., Khalili Yazdi A., Li F., Chau I., et al. Probing the SAM binding site of SARS-CoV-2 Nsp14 in vitro using SAM competitive inhibitors guides developing selective Bisubstrate inhibitors. SLAS Discov. 2021;26(9):1200–1211. PubMed PMC

Otava T., Sala M., Li F., Fanfrlik J., Devkota K., Perveen S., et al. The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields Nanomolar inhibitors. ACS Infect. Dis. 2021;7(8):2214–2220. PubMed

Martin Klima, Aliakbar Khalili Yazdi, Li Fengling, Chau Irene, Taraneh Hajian, Albina Bolotokova H., Ümit Kaniskan, Han3 Yulin, Wang Ke, Li Deyao, Luo Minkui, Jin Jian, Evzen Boura, Masoud Vedadi. Crystal structure of SARS-CoV-2 nsp10-nsp16 in complex with small molecule inhibitors, SS148 and WZ16. Protein Sci. 2022;31(9):e4395. PubMed PMC

Minasov G., Rosas-Lemus M., Shuvalova L., Inniss N.L., Brunzelle J.S., Daczkowski C.M., et al. Mn(2+) coordinates Cap-0-RNA to align substrates for efficient 2’-O-methyl transfer by SARS-CoV-2 nsp16. Sci. Signal. 2021;14(689) PubMed PMC

Romano M., Ruggiero A., Squeglia F., Maga G., Berisio R. A structural view of SARS-CoV-2 RNA replication machinery: RNA synthesis, proofreading and final capping. Cells. 2020;9(5) PubMed PMC

Bouvet M., Debarnot C., Imbert I., Selisko B., Snijder E.J., Canard B., et al. In vitro reconstitution of SARS-coronavirus mRNA cap methylation. PLoS Pathog. 2010;6(4) PubMed PMC

Devarkar S.C., Wang C., Miller M.T., Ramanathan A., Jiang F., Khan A.G., et al. Structural basis for m7G recognition and 2’-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I. Proc. Natl. Acad. Sci. U. S. A. 2016;113(3):596–601. PubMed PMC

Dostalik P., Krafcikova P., Silhan J., Kozic J., Chalupska D., Chalupsky K., et al. Structural analysis of the OC43 coronavirus 2’-O-RNA methyltransferase. J. Virol. 2021;95(15) PubMed PMC

Copeland R.A. John Wiley & Sons, Inc.; 2005. Evaluation of Enzyme Inhibitors in Drug Discovery. PubMed

Abdool Karim S.S., de Oliveira T. New SARS-CoV-2 variants - clinical, public health, and vaccine implications. N. Engl. J. Med. 2021;384(19):1866–1868. PubMed PMC

Lamb Y.N. Nirmatrelvir plus ritonavir: first approval. Drugs. 2022;82(5):585–591. doi: 10.1007/s40265-022-01692-5. PubMed DOI PMC

Malden D.E., Hong V., Lewin B.J., Ackerson B.K., Lipsitch M., Lewnard J.A., et al. Hospitalization and emergency department encounters for COVID-19 after Paxlovid treatment - California, December 2021-may 2022. MMWR Morb. Mortal. Wkly Rep. 2022;71(25):830–833. PubMed

Daffis S., Szretter K.J., Schriewer J., Li J., Youn S., Errett J., et al. 2’-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature. 2010;468(7322):452–456. PubMed PMC

Almazan F., Dediego M.L., Galan C., Escors D., Alvarez E., Ortego J., et al. Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J. Virol. 2006;80(21):10900–10906. PubMed PMC

Menachery V.D., Yount B.L., Jr., Josset L., Gralinski L.E., Scobey T., Agnihothram S., et al. Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-o-methyltransferase activity. J. Virol. 2014;88(8):4251–4264. PubMed PMC

Zust R., Cervantes-Barragan L., Habjan M., Maier R., Neuman B.W., Ziebuhr J., et al. Ribose 2’-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat. Immunol. 2011;12(2):137–143. PubMed PMC

Scheer S., Ackloo S., Medina T.S., Schapira M., Li F., Ward J.A., et al. A chemical biology toolbox to study protein methyltransferases and epigenetic signaling. Nat. Commun. 2019;10(1):19. PubMed PMC

Li A.S.M., Li F., Eram M.S., Bolotokova A., Dela Sena C.C., Vedadi M. Chemical probes for protein arginine methyltransferases. Methods. 2020;175:30–43. PubMed

Dhankhar P., Dalal V., Kotra D.G., Kumar P. In-silico approach to identify novel potent inhibitors against GraR of S. aureus. Front. Biosci. (Landmark Ed) 2020;25(7):1337–1360. PubMed

Gupta D.N., Dalal V., Savita B.K., Dhankhar P., Ghosh D.K., Kumar P., et al. In-silico screening and identification of potential inhibitors against 2Cys peroxiredoxin of Candidatus Liberibacter asiaticus. J. Biomol. Struct. Dyn. 2022;40(19):8725–8739. PubMed

Waterhouse A., Bertoni M., Bienert S., Studer G., Tauriello G., Gumienny R., et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(W1):W296–W303. PubMed PMC

Guex N., Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997;18(15):2714–2723. PubMed

Morris G.M., Huey R., Lindstrom W., Sanner M.F., Belew R.K., Goodsell D.S., et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 2009;30(16):2785–2791. PubMed PMC

The zymogenic form of SARS-CoV-2 main protease: A discrete target for drug discovery

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

Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors

Structure of monkeypox virus poxin: implications for drug design

Discovery and structural characterization of monkeypox virus methyltransferase VP39 inhibitors reveal similarities to SARS-CoV-2 nsp14 methyltransferase