A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

Department of Microbiology Immunology and Center for Structural Biology of Infectious Diseases Northwestern University Feinberg School of Medicine Chicago Illinois 60611 United States

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

Department of Pharmacology Northwestern University Feinberg School of Medicine Chicago Illinois 60611 United States

Drug Discovery Program Ontario Institute for Cancer Research Toronto Ontario M5G 0A3 Canada

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague 6 160 00 Czech Republic

Northwestern Synchrotron Research Center Life Sciences Collaborative Access Team Northwestern University Argonne Illinois 60439 United States

Structural Chemistry Gilead Pharmaceuticals San Mateo California 94404 United States

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

Takeda Development Center Americas Inc San Diego California 92121 United States

WuXi AppTec Co Ltd China Pilot Free Trade Zone Shanghai 201308 China

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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