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A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex.

ABSTRACT: Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.

SUBMITTER: Khalili Yazdi A

PROVIDER: S-EPMC8216315 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19.

Project description:SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.

| S-EPMC7803792 | biostudies-literature

Substrate Specificity of SARS-CoV-2 Nsp10-Nsp16 Methyltransferase.

Project description: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.

| S-EPMC8472550 | biostudies-literature

Identification of novel mutations in the methyltransferase complex (Nsp10-Nsp16) of SARS-CoV-2.

Project description:A recent outburst of the pandemic caused by a member of the coronaviridae family identified as SARS-CoV-2. The highly contagious nature of the virus allows it to spread rapidly worldwide and caused severe healthcare and economic distress. So far, no proper line of treatment or vaccines has been available against SARS-CoV-2. Since, the infected people rapidly increased, causing the saturation of healthcare systems with coronavirus disease (COVID-19) patients. As the virus spread to new locations it also acquired various mutations. Here, in this study, we focused on identifying mutations in one of the crucial complex of SARS-CoV-2, the Nsp10-Nsp16 2'-O-methyltransferase complex. This complex plays indispensable role in the post-transcriptional modifications of viral RNA by its capping. We analysed 208 sequences of Nsp10-Nsp16 reported from India and compared with first reported sequence from Wuhan, China. Our analysis revealed a single mutation in Nsp10 and five mutations in Nsp16 protein. We also show that these mutations are leading to alteration in the secondary structure of Nsp10-Nsp16. Further, the protein modelling studies revealed that the mutation of both Nsp10-Nsp16 impacts the protein dynamicity and stability. Altogether, this study provides novel insights into the variations observed in the proteins of SARS-CoV-2 that might have functional consequences.

| S-EPMC7547569 | biostudies-literature

Discovery of SARS-CoV-2 Nsp14 and Nsp16 Methyltransferase Inhibitors by High-Throughput Virtual Screening.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput virtual screening against these two enzymes. The docking of seven million commercially available drug-like compounds and S-adenosylmethionine (SAM) co-substrate analogues against both MTases resulted in 80 virtual screening hits (39 against nsp14 and 41 against nsp16), which were purchased and tested using an enzymatic homogeneous time-resolved fluorescent energy transfer (HTRF) assay. Nine compounds showed micromolar inhibition activity (IC50 < 200 μM). The selectivity of the identified inhibitors was evaluated by cross-checking their activity against human glycine N-methyltransferase. The majority of the compounds showed poor selectivity for a specific MTase, no cytotoxic effects, and rather poor cell permeability. Nevertheless, the identified compounds represent good starting points that have the potential to be developed into efficient viral MTase inhibitors.

| S-EPMC8705538 | biostudies-literature

The crystal structure of nsp10-nsp16 heterodimer from SARS-CoV-2 in complex with S-adenosylmethionine.

Project description:SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2'-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 Å. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

| S-EPMC7263505 | biostudies-literature

Computational Analysis of SAM Analogs as Methyltransferase Inhibitors of nsp16/nsp10 Complex from SARS-CoV-2.

Project description:Methyltransferases (MTases) enzymes, responsible for RNA capping into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are emerging important targets for the design of new anti-SARS-CoV-2 agents. Here, analogs of S-adenosylmethionine (SAM), obtained from the bioisosteric substitution of the sulfonium and amino acid groups, were evaluated by rigorous computational modeling techniques such as molecular dynamics (MD) simulations followed by relative binding free analysis against nsp16/nsp10 complex from SARS-CoV-2. The most potent inhibitor (2a) shows the lowest binding free energy (-58.75 Kcal/mol) and more potency than Sinefungin (SFG) (-39.8 Kcal/mol), a pan-MTase inhibitor, which agrees with experimental observations. Besides, our results suggest that the total binding free energy of each evaluated SAM analog is driven by van der Waals interactions which can explain their poor cell permeability, as observed in experimental essays. Overall, we provide a structural and energetic analysis for the inhibition of the nsp16/nsp10 complex involving the evaluated SAM analogs as potential inhibitors.

| S-EPMC9697258 | biostudies-literature

Molecular Docking Studies to Identify Promising Natural Inhibitors Targeting SARS-CoV-2 Nsp10-Nsp16 Protein Complex

Project description:ObjectivesUnavailability of potential drugs/vaccines in the outbreak of the pandemic severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) have devastated the human population globally. Several druggable targets have been analyzed against different viral proteins such as the spike protein. The study aims to explore the potential of natural compounds as an effective drug against a novel nsp10-nsp16 complex of SARS-CoV-2 using in silico approaches.Materials and methodsIn silico screening (Docking analysis) was performed for 10 shortlisted natural compounds viz. allicin, ajoene, carvacrol, coumarin, curcumin, menthol, eugenol, theaflavin, ursolic acid, and catechin against a novel target of SARS-CoV-2, that has been anticipated to provide valuable lead molecules and potentially druggable compounds for the treatment of SARS-CoV-2.ResultsTheaflavin and catechin, the natural components of black tea and green tea, out of 10 shortlisted compounds have shown excellent performance in our docking studies with the minimum binding energy of -11.8 kcal/mol and -9.2 kcal/mol respectively, against a novel nsp10-nsp16 complex of SARS-CoV-2 that indicates their potential for inhibitory molecular interactions against the virus to assist rapid drug designing from natural products.ConclusionEither consumption of black tea and green tea or repurposing them as drug candidates may help individuals to fight against SARS-CoV-2, subject to their in vivo and in vitro further experimental validations.

| S-EPMC8892556 | biostudies-literature

Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex.

Project description:To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9?Å resolution and crystal structure determination is in progress.

| S-EPMC3053173 | biostudies-literature

Crystal structure of SARS-CoV-2 nsp10-nsp16 in complex with small molecule inhibitors, SS148 and WZ16.

Project description: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.

| S-EPMC9375521 | biostudies-literature

In-Silico Drug Designing of Spike Receptor with Its ACE2 Receptor and Nsp10/Nsp16 MTase Complex Against SARS-CoV-2.

Project description:The realm Riboviria constitutes Coronaviruses, which led to the emergence of the pandemic COVID 19 in the twenty-first century affected millions of lives. At present, the management of COVID 19 largely depends on antiviral therapeutics along with the anti-inflammatory drug. The vaccine is under the final clinical phase, and emergency use is available. We aim at ACE2 and Nsp10/Nsp16 MTase as potential drug candidate in COVID 19 management in the present work. For drug designing, various computational simulation strategies have been employed like Swiss-Model, Hawk Dock, HDOCK, py Dock, and PockDrug for homology modeling, binding energies of the molecule with a target, simulate the conformation and binding poses, statistics of protein lock with target key and drug ability, respectively. The current in-silico screening depicts that the spike protein receptor is complementary to the target when bound to each other and forms a stable complex. The MMGBSA free energy binding property of receptor and ligand is critical. The intermolecular Statistics with the target Nsp10/Nsp16 MTase complex are plausible. We have also observed a high-affinity pocket binding site with the target. Therefore, the favorable intermolecular interactions and Physico-chemical properties emanate as a drug candidate treating COVID-19. This study has approached computational tools to analyze the conformation, binding affinity, and drug ability of receptor-ligand. Thus, the spike receptor with its ACE2 receptor with Nsp10/Nsp16 MTase complex would be a potent drug against SARS CoV-2 and can cure the infection as per consensus scoring.

| S-EPMC7966892 | biostudies-literature

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