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SLAS Discov ; 26(6): 757-765, 2021 07.
Article in English | MEDLINE | ID: covidwho-1194439


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.

Adenosine/analogs & derivatives , High-Throughput Screening Assays , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/chemistry , Adenosine/pharmacology , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Methylation , Methyltransferases , Models, Molecular , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Tritium , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism