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.

• Keywords
• Coronavirus, Enzyme kinetics, Enzyme purification, RNA methyltransferase, RNA virus, Viral protein, nsp10-nsp16 complex,
• MeSH
• COVID-19 * MeSH
• Humans MeSH
• Methyltransferases chemistry   MeSH
• Pandemics MeSH
• RNA MeSH
• SARS-CoV-2 genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• NSP10 protein, SARS-CoV-2 MeSH Browser
• NSP16 protein, SARS-CoV-2 MeSH Browser
• RNA MeSH
• SS148 MeSH Browser
• WZ16 MeSH Browser

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.

• Keywords
• antiviral, coronavirus, covalent inhibitors, nsp16 methyltransferase, structural biology,
• MeSH
• COVID-19 * MeSH
• Rats MeSH
• Methyltransferases MeSH
• Mice MeSH
• S-Adenosylmethionine chemistry   metabolism   MeSH
• SARS-CoV-2 * metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Methyltransferases MeSH
• S-Adenosylmethionine MeSH

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.

• Keywords
• SARS-CoV-2, inhibitor, methylation, virus,
• MeSH
• Chromatography, Liquid MeSH
• COVID-19 virology   MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Methyltransferases genetics   metabolism   MeSH
• Methylation MeSH
• Gene Expression Regulation, Viral MeSH
• RNA Caps MeSH
• RNA, Viral genetics   MeSH
• SARS-CoV-2 enzymology   genetics   MeSH
• Substrate Specificity MeSH
• Viral Nonstructural Proteins genetics   metabolism   MeSH
• Viral Regulatory and Accessory Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• NSP10 protein, SARS-CoV-2 MeSH Browser
• NSP16 protein, SARS-CoV-2 MeSH Browser
• RNA Caps MeSH
• RNA, Viral MeSH
• Viral Nonstructural Proteins MeSH
• Viral Regulatory and Accessory Proteins MeSH

SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.

• Keywords
• COVID-19, SARS-CoV-2, SS148, WZ16, nsp10, nsp16,
• MeSH
• COVID-19 Drug Treatment * MeSH
• Humans MeSH
• Methyltransferases chemistry   MeSH
• RNA, Viral metabolism   MeSH
• SARS-CoV-2 * MeSH
• Viral Nonstructural Proteins chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• RNA, Viral MeSH
• Viral Nonstructural Proteins MeSH

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

• Keywords
• SARS-CoV-2, capping enzyme, coronavirus, methyltransferase, nsp10, nsp14, nsp16,
• MeSH
• COVID-19 virology   MeSH
• Humans MeSH
• Methyltransferases analysis   genetics   metabolism   MeSH
• Antibodies, Monoclonal analysis   MeSH
• RNA Caps genetics   metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• SARS-CoV-2 chemistry   enzymology   genetics   MeSH
• Protein Transport MeSH
• Viral Nonstructural Proteins analysis   genetics   metabolism   MeSH
• Viral Regulatory and Accessory Proteins analysis   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• Antibodies, Monoclonal MeSH
• NSP10 protein, SARS-CoV-2 MeSH Browser
• NSP16 protein, SARS-CoV-2 MeSH Browser
• RNA Caps MeSH
• RNA, Viral MeSH
• Viral Nonstructural Proteins MeSH
• Viral Regulatory and Accessory Proteins 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
• Adenosine analogs & derivatives   metabolism   pharmacology   MeSH
• Betacoronavirus enzymology   MeSH
• Models, Chemical MeSH
• COVID-19 MeSH
• Enzyme Inhibitors metabolism   pharmacology   MeSH
• Catalytic Domain MeSH
• Coronavirus Infections virology   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Methyltransferases chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Pandemics MeSH
• RNA Caps MeSH
• RNA, Viral metabolism   MeSH
• SARS-CoV-2 MeSH
• RNA Stability MeSH
• Pneumonia, Viral virology   MeSH
• Viral Nonstructural Proteins chemistry   metabolism   MeSH
• Viral Regulatory and Accessory Proteins chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine MeSH
• Enzyme Inhibitors MeSH
• Methyltransferases MeSH
• NSP10 protein, SARS-CoV-2 MeSH Browser
• NSP16 protein, SARS-CoV-2 MeSH Browser
• RNA 2'-O-methyltransferase MeSH Browser
• RNA Caps MeSH
• RNA, Viral MeSH
• sinefungin MeSH Browser
• Viral Nonstructural Proteins MeSH
• Viral Regulatory and Accessory Proteins MeSH

The OC43 coronavirus is a human pathogen that usually causes only the common cold. One of its key enzymes, similar to other coronaviruses, is the 2'-O-RNA methyltransferase (MTase), which is essential for viral RNA stability and expression. Here, we report the crystal structure of the 2'-O-RNA MTase in a complex with the pan-methyltransferase inhibitor sinefungin solved at 2.2-Å resolution. The structure reveals an overall fold consistent with the fold observed in other coronaviral MTases. The major differences are in the conformation of the C terminus of the nsp16 subunit and an additional helix in the N terminus of the nsp10 subunits. The structural analysis also revealed very high conservation of the S-adenosyl methionine (SAM) binding pocket, suggesting that the SAM pocket is a suitable spot for the design of antivirals effective against all human coronaviruses. IMPORTANCE Some coronaviruses are dangerous pathogens, while some cause only common colds. The reasons are not understood, although the spike proteins probably play an important role. However, to understand the coronaviral biology in sufficient detail, we need to compare the key enzymes from different coronaviruses. We solved the crystal structure of 2'-O-RNA methyltransferase of the OC43 coronavirus, a virus that usually causes mild colds. The structure revealed some differences in the overall fold but also revealed that the SAM binding site is conserved, suggesting that development of antivirals against multiple coronaviruses is feasible.

• Keywords
• OC43, coronavirus, crystal structure, methyltransferase,
• MeSH
• Betacoronavirus enzymology   genetics   MeSH
• Protein Conformation, alpha-Helical MeSH
• Crystallography, X-Ray MeSH
• Methyltransferases chemistry   genetics   MeSH
• Binding Sites MeSH
• Viral Proteins chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• RNA 2'-O-methyltransferase MeSH Browser
• Viral Proteins MeSH