AT-9010 (2'-methyl-2'-fluoro guanosine triphosphate) is a GTP analog whose prodrug, AT-752 is under consideration in human medicine as a potential antiviral drug against certain flaviviruses. It was previously believed to inhibit viral replication by acting primarily as a chain terminator. However, it was discovered recently that it also binds the GTP binding site of the methyltransferase (MTase) domain of the orthoflavivirus polymerase, thus interfering with RNA capping. Here, we investigated the binding of AT-9010 to Ntaya and Zika virus MTases. Structural analysis using X-ray crystallography revealed similar interactions between the base and sugar moieties of AT-9010 and key residues in both MTases, although differences in hydrogen bonding were observed. Our analysis also suggested that the triphosphate part of AT-9010 is flexible. Despite minor variations, the overall binding mode of AT-9010 was found to be the same for all of the flaviviral MTases examined, suggesting a structural basis for the efficacy of AT-9010 against multiple orthoflavivirus MTases.

• MeSH
• Antiviral Agents * chemistry   pharmacology   metabolism   MeSH
• Flaviviridae * enzymology   MeSH
• Guanosine Triphosphate * analogs & derivatives   metabolism   chemistry   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Methyltransferases * metabolism   chemistry   genetics   MeSH
• Models, Molecular MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Viral Proteins * metabolism   chemistry   MeSH
• Zika Virus * enzymology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents * MeSH
• Guanosine Triphosphate * MeSH
• Methyltransferases * MeSH
• Viral Proteins * MeSH

The emergence of SARS-CoV-2, the causative agent of COVID-19, has highlighted the need for advanced antiviral strategies. Targeting the coronaviral methyltransferase nsp14, which is essential for RNA capping, offers a promising approach for the development of small-molecule inhibitors. We designed and synthesized a series of adenosine 5'-carboxamide derivatives as potential nsp14 inhibitors and identified coumarin analogs to be particularly effective. Structural modifications revealed the importance of the 5'-carboxyl moiety for the inhibitory activity, showing superior efficacy compared to other modifications. Notably, compound 18l (HK370) demonstrated high selectivity and favorable in vitro pharmacokinetic properties and exhibited moderate antiviral activity in cell-based assays. These findings provide a robust foundation for developing targeted nsp14 inhibitors as a potential treatment for COVID-19 and related diseases.

• Publication type
• Journal Article MeSH

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 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.

• Publication type
• Journal Article MeSH

Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.

• MeSH
• COVID-19 * MeSH
• Zika Virus Infection * MeSH
• Humans MeSH
• Methyltransferases metabolism   MeSH
• RNA, Viral genetics   MeSH
• RNA MeSH
• SARS-CoV-2 genetics   MeSH
• Viral Nonstructural Proteins chemistry   MeSH
• Monkeypox virus genetics   metabolism   MeSH
• Zika Virus * genetics   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
• RNA MeSH
• Viral Nonstructural 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

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
• Amino Acids chemistry   MeSH
• Coronavirus drug effects   enzymology   genetics   MeSH
• Humans MeSH
• Methyltransferases antagonists & inhibitors   chemistry   metabolism   MeSH
• Methylation MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Drug Discovery MeSH
• RNA, Viral chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Methyltransferases MeSH
• RNA, Viral 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

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

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