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.
Keywords:
COVID-19; SARS-CoV-2; methyltransferase; nsp10-nsp16 complex; nsp16.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine / analogs & derivatives*
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Adenosine / chemistry
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Adenosine / pharmacology
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COVID-19 / virology
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Cloning, Molecular
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Enzyme Assays
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / pharmacology
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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High-Throughput Screening Assays*
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Humans
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Kinetics
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Methylation
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Methyltransferases
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Models, Molecular
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RNA Caps / antagonists & inhibitors*
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RNA Caps / genetics
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RNA Caps / metabolism
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RNA, Viral / antagonists & inhibitors*
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RNA, Viral / genetics
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RNA, Viral / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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SARS-CoV-2 / drug effects
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SARS-CoV-2 / enzymology
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SARS-CoV-2 / genetics
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Tritium
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Viral Nonstructural Proteins / antagonists & inhibitors*
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Viral Nonstructural Proteins / chemistry
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / metabolism
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Viral Regulatory and Accessory Proteins / antagonists & inhibitors*
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Viral Regulatory and Accessory Proteins / chemistry
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Viral Regulatory and Accessory Proteins / genetics
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Viral Regulatory and Accessory Proteins / metabolism
Substances
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Enzyme Inhibitors
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NSP10 protein, SARS-CoV-2
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NSP16 protein, SARS-CoV-2
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RNA Caps
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RNA, Viral
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Recombinant Proteins
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Viral Nonstructural Proteins
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Viral Regulatory and Accessory Proteins
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Tritium
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Methyltransferases
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Adenosine
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sinefungin