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
• antivirové látky * chemie   farmakologie   metabolismus   MeSH
• Flaviviridae * enzymologie   MeSH
• guanosintrifosfát * analogy a deriváty   metabolismus   chemie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• methyltransferasy * metabolismus   chemie   genetika   MeSH
• molekulární modely MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virové proteiny * metabolismus   chemie   MeSH
• virus zika * enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• guanosintrifosfát * MeSH
• methyltransferasy * MeSH
• virové proteiny * 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
• infekce virem zika * MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• RNA virová genetika   MeSH
• RNA MeSH
• SARS-CoV-2 genetika   MeSH
• virové nestrukturální proteiny chemie   MeSH
• virus opičích neštovic genetika   metabolismus   MeSH
• virus zika * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA virová MeSH
• RNA MeSH
• virové nestrukturální proteiny 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

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.

• Klíčová slova
• COVID-19, Methyltransferase, N7-MTase, RNA capping, SARS-CoV-2, TCM, ensemble sampling, molecular dynamics, natural products, nsp14,
• MeSH
• antivirové látky farmakologie   MeSH
• COVID-19 * MeSH
• exoribonukleasy metabolismus   MeSH
• guanin MeSH
• lidé MeSH
• methyltransferasy * metabolismus   MeSH
• RNA virová MeSH
• SARS-CoV-2 MeSH
• simulace molekulární dynamiky MeSH
• virové nestrukturální proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky MeSH
• exoribonukleasy MeSH
• guanin MeSH
• methyltransferasy * MeSH
• RNA virová MeSH
• virové nestrukturální proteiny 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.

• Klíčová slova
• OC43, coronavirus, crystal structure, methyltransferase,
• MeSH
• Betacoronavirus enzymologie   genetika   MeSH
• konformace proteinů, alfa-helix MeSH
• krystalografie rentgenová MeSH
• methyltransferasy chemie   genetika   MeSH
• vazebná místa MeSH
• virové proteiny chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA 2'-O-methyltransferase MeSH Prohlížeč
• virové proteiny 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.

• Klíčová slova
• SARS-CoV-2, TBEV, discovery, drug, flavivirus, high-throughput screening, papain-like, protease, virus,
• MeSH
• antivirové látky farmakologie   MeSH
• farmakoterapie COVID-19 MeSH
• fluorescenční barviva chemie   MeSH
• inhibitory proteas farmakologie   MeSH
• koronavirové proteasy podobné papainu antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• lidé MeSH
• preklinické hodnocení léčiv MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• RNA-helikasy antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• RNA-viry enzymologie   MeSH
• rychlé screeningové testy metody   MeSH
• SARS-CoV-2 enzymologie   MeSH
• serinové endopeptidasy chemie   genetika   metabolismus   MeSH
• virové nestrukturální proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• fluorescenční barviva MeSH
• inhibitory proteas MeSH
• koronavirové proteasy podobné papainu MeSH
• NS3 protein, flavivirus MeSH Prohlížeč
• papain-like protease, SARS-CoV-2 MeSH Prohlížeč
• RNA-helikasy MeSH
• serinové endopeptidasy MeSH
• virové nestrukturální proteiny 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

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.

• Klíčová slova
• 3′,3′-c-di-GMP, CDN, STING, cGAS, crystal structure,
• MeSH
• guanosinmonofosfát cyklický chemie   metabolismus   MeSH
• hořčík metabolismus   MeSH
• krystalografie rentgenová MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• guanosinmonofosfát cyklický MeSH
• hořčík MeSH
• membránové proteiny MeSH
• STING1 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• BCX4430, attenuation, drug resistance, galidesivir, mutation, tick-borne encephalitis virus,
• MeSH
• adenin analogy a deriváty   chemie   farmakologie   MeSH
• adenosin analogy a deriváty   MeSH
• alely MeSH
• antibiotická rezistence MeSH
• antivirové látky chemie   farmakologie   MeSH
• buněčné linie MeSH
• genotyp MeSH
• klíšťová encefalitida farmakoterapie   virologie   MeSH
• modely nemocí na zvířatech MeSH
• mutace * MeSH
• myši MeSH
• pyrrolidiny chemie   farmakologie   MeSH
• substituce aminokyselin * MeSH
• virová léková rezistence * MeSH
• virové nestrukturální proteiny genetika   MeSH
• viry klíšťové encefalitidy účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenin MeSH
• adenosin MeSH
• antivirové látky MeSH
• galidesivir MeSH Prohlížeč
• pyrrolidiny MeSH
• virové nestrukturální proteiny 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.

• Klíčová slova
• West Nile virus, antiviral agents, flavivirus, nucleoside analogs,
• MeSH
• antivirové látky terapeutické užití   MeSH
• buněčné linie MeSH
• Cercopithecus aethiops MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• prasata MeSH
• progrese nemoci MeSH
• replikace viru účinky léků   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   MeSH
• tubercidin analogy a deriváty   terapeutické užití   MeSH
• Vero buňky MeSH
• viremie farmakoterapie   MeSH
• virus západního Nilu účinky léků   genetika   MeSH
• západonilská horečka farmakoterapie   patologie   virologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 7-deaza-2'-C-methyladenosine MeSH Prohlížeč
• antivirové látky MeSH
• RNA-dependentní RNA-polymerasa MeSH
• tubercidin MeSH