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.

• Klíčová slova
• COVID-19, SARS-CoV-2, SS148, WZ16, nsp10, nsp16,
• MeSH
• farmakoterapie COVID-19 * MeSH
• lidé MeSH
• methyltransferasy chemie   MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 * MeSH
• virové nestrukturální proteiny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA virová MeSH
• virové nestrukturální proteiny 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.

• Klíčová slova
• SARS-CoV-2, inhibitor, methylation, virus,
• MeSH
• chromatografie kapalinová MeSH
• COVID-19 virologie   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• metylace MeSH
• regulace exprese virových genů MeSH
• RNA čepičky MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 enzymologie   genetika   MeSH
• substrátová specifita MeSH
• virové nestrukturální proteiny genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• NSP10 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP16 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA čepičky MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH
• virové regulační a přídatné proteiny 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

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