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

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

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 recent pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) calls the whole world into a medical emergency. For tackling Coronavirus Disease 2019 (COVID-19), researchers from around the world are swiftly working on designing and identifying inhibitors against all possible viral key protein targets. One of the attractive drug targets is guanine-N7 methyltransferase which plays the main role in capping the 5'-ends of viral genomic RNA and sub genomic RNAs, to escape the host's innate immunity. We performed homology modeling and molecular dynamic (MD) simulation, in order to understand the molecular architecture of Guanosine-P3-Adenosine-5',5'-Triphosphate (G3A) binding with C-terminal N7-MTase domain of nsp14 from SARS-CoV-2. The residue Asn388 is highly conserved in present both in N7-MTase from SARS-CoV and SARS-CoV-2 and displays a unique function in G3A binding. For an in-depth understanding of these substrate specificities, we tried to screen and identify inhibitors from the Traditional Chinese Medicine (TCM) database. The combination of several computational approaches, including screening, MM/GBSA, MD simulations, and PCA calculations, provides the screened compounds that readily interact with the G3A binding site of homology modeled N7-MTase domain. Compounds from this screening will have strong potency towards inhibiting the substrate-binding and efficiently hinder the viral 5'-end RNA capping mechanism. We strongly believe the final compounds can become COVID-19 therapeutics, with huge international support.[Formula: see text]The focus of this study is to screen for antiviral inhibitors blocking guanine-N7 methyltransferase (N7-MTase), one of the key drug targets involved in the first methylation step of the SARS-CoV-2 RNA capping mechanism. Compounds binding the substrate-binding site can interfere with enzyme catalysis and impede 5'-end cap formation, which is crucial to mimic host RNA and evade host cellular immune responses. Therefore, our study proposes the top hit compounds from the Traditional Chinese Medicine (TCM) database using a combination of several computational approaches.Communicated by Ramaswamy H. Sarma.

• Keywords
• COVID-19, Methyltransferase, N7-MTase, RNA capping, SARS-CoV-2, TCM, ensemble sampling, molecular dynamics, natural products, nsp14,
• MeSH
• Antiviral Agents pharmacology   MeSH
• COVID-19 * MeSH
• Exoribonucleases metabolism   MeSH
• Guanine MeSH
• Humans MeSH
• Methyltransferases * metabolism   MeSH
• RNA, Viral MeSH
• SARS-CoV-2 MeSH
• Molecular Dynamics Simulation MeSH
• Viral Nonstructural Proteins MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antiviral Agents MeSH
• Exoribonucleases MeSH
• Guanine MeSH
• Methyltransferases * MeSH
• RNA, Viral MeSH
• Viral Nonstructural 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

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

• Keywords
• SARS-CoV-2, TBEV, discovery, drug, flavivirus, high-throughput screening, papain-like, protease, virus,
• MeSH
• Antiviral Agents pharmacology   MeSH
• COVID-19 Drug Treatment MeSH
• Fluorescent Dyes chemistry   MeSH
• Protease Inhibitors pharmacology   MeSH
• Coronavirus Papain-Like Proteases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• RNA Helicases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• RNA Viruses enzymology   MeSH
• High-Throughput Screening Assays methods   MeSH
• SARS-CoV-2 enzymology   MeSH
• Serine Endopeptidases chemistry   genetics   metabolism   MeSH
• Viral Nonstructural Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Encephalitis Viruses, Tick-Borne enzymology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents MeSH
• Fluorescent Dyes MeSH
• Protease Inhibitors MeSH
• Coronavirus Papain-Like Proteases MeSH
• NS3 protein, flavivirus MeSH Browser
• papain-like protease, SARS-CoV-2 MeSH Browser
• RNA Helicases MeSH
• Serine Endopeptidases MeSH
• Viral Nonstructural 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

Stimulator of interferon genes (STING) binds cyclic dinucleotides (CDNs), which induce a large conformational change of the protein. The structural basis of activation of STING by CDNs is rather well understood. Unliganded STING forms an open dimer that undergoes a large conformational change (∼10 Å) to a closed conformation upon the binding of a CDN molecule. This event activates downstream effectors of STING and subsequently leads to activation of the type 1 interferon response. However, a previously solved structure of STING with 3',3'-c-di-GMP shows Mg atoms mediating the interaction of STING with this CDN. Here, it is shown that no Mg atoms are needed for this interaction; in fact, magnesium can in some cases obstruct the binding of a CDN to STING.

• Keywords
• 3′,3′-c-di-GMP, CDN, STING, cGAS, crystal structure,
• MeSH
• Cyclic GMP chemistry   metabolism   MeSH
• Magnesium metabolism   MeSH
• Crystallography, X-Ray MeSH
• Membrane Proteins chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cyclic GMP MeSH
• Magnesium MeSH
• Membrane Proteins MeSH
• STING1 protein, human MeSH Browser

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

• Keywords
• BCX4430, attenuation, drug resistance, galidesivir, mutation, tick-borne encephalitis virus,
• MeSH
• Adenine analogs & derivatives   chemistry   pharmacology   MeSH
• Adenosine analogs & derivatives   MeSH
• Alleles MeSH
• Drug Resistance, Microbial MeSH
• Antiviral Agents chemistry   pharmacology   MeSH
• Cell Line MeSH
• Genotype MeSH
• Encephalitis, Tick-Borne drug therapy   virology   MeSH
• Disease Models, Animal MeSH
• Mutation * MeSH
• Mice MeSH
• Pyrrolidines chemistry   pharmacology   MeSH
• Amino Acid Substitution * MeSH
• Drug Resistance, Viral * MeSH
• Viral Nonstructural Proteins genetics   MeSH
• Encephalitis Viruses, Tick-Borne drug effects   physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Adenosine MeSH
• Antiviral Agents MeSH
• galidesivir MeSH Browser
• Pyrrolidines MeSH
• Viral Nonstructural Proteins MeSH

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2'-C-methyl- or 4'-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2'-methylated nucleosides, 7-deaza-2'-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2'-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4'-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro However, the strong anti-WNV effect of 4'-azidocytidine and 4'-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2'-C-methylated or 4'-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

• Keywords
• West Nile virus, antiviral agents, flavivirus, nucleoside analogs,
• MeSH
• Antiviral Agents therapeutic use   MeSH
• Cell Line MeSH
• Chlorocebus aethiops MeSH
• Disease Models, Animal MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Swine MeSH
• Disease Progression MeSH
• Virus Replication drug effects   MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   MeSH
• Tubercidin analogs & derivatives   therapeutic use   MeSH
• Vero Cells MeSH
• Viremia drug therapy   MeSH
• West Nile virus drug effects   genetics   MeSH
• West Nile Fever drug therapy   pathology   virology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 7-deaza-2'-C-methyladenosine MeSH Browser
• Antiviral Agents MeSH
• RNA-Dependent RNA Polymerase MeSH
• Tubercidin MeSH