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.

• Klíčová slova
• antiviral, coronavirus, covalent inhibitors, nsp16 methyltransferase, structural biology,
• MeSH
• COVID-19 * MeSH
• krysa rodu Rattus MeSH
• methyltransferasy MeSH
• myši MeSH
• S-adenosylmethionin chemie   metabolismus   MeSH
• SARS-CoV-2 * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• methyltransferasy MeSH
• S-adenosylmethionin MeSH

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.

• MeSH
• aminokyseliny chemie   MeSH
• Coronavirus účinky léků   enzymologie   genetika   MeSH
• lidé MeSH
• methyltransferasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• metylace MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• objevování léků MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• methyltransferasy MeSH
• RNA virová MeSH

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.

• MeSH
• adenosin analogy a deriváty   metabolismus   farmakologie   MeSH
• Betacoronavirus enzymologie   MeSH
• chemické modely MeSH
• COVID-19 MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• katalytická doména MeSH
• koronavirové infekce virologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• methyltransferasy chemie   metabolismus   MeSH
• molekulární modely MeSH
• pandemie MeSH
• RNA čepičky MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 MeSH
• stabilita RNA MeSH
• virová pneumonie virologie   MeSH
• virové nestrukturální proteiny chemie   metabolismus   MeSH
• virové regulační a přídatné proteiny chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• inhibitory enzymů MeSH
• methyltransferasy MeSH
• NSP10 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP16 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA 2'-O-methyltransferase MeSH Prohlížeč
• RNA čepičky MeSH
• RNA virová MeSH
• sinefungin MeSH Prohlížeč
• virové nestrukturální proteiny MeSH
• virové regulační a přídatné proteiny MeSH