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

SLAS Discov. 2021 Jul;26(6):757-765. doi: 10.1177/24725552211008863. Epub 2021 Apr 19.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • 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
  • High-Throughput Screening Assays*
  • Humans
  • Kinetics
  • Methylation
  • Methyltransferases
  • Models, Molecular
  • RNA Caps / antagonists & inhibitors*
  • RNA Caps / genetics
  • RNA Caps / metabolism
  • RNA, Viral / antagonists & inhibitors*
  • 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 / antagonists & inhibitors*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism
  • Viral Regulatory and Accessory Proteins / antagonists & inhibitors*
  • Viral Regulatory and Accessory Proteins / chemistry
  • Viral Regulatory and Accessory Proteins / genetics
  • Viral Regulatory and Accessory Proteins / metabolism

Substances

  • Enzyme Inhibitors
  • NSP10 protein, SARS-CoV-2
  • NSP16 protein, SARS-CoV-2
  • RNA Caps
  • RNA, Viral
  • Recombinant Proteins
  • Viral Nonstructural Proteins
  • Viral Regulatory and Accessory Proteins
  • Tritium
  • Methyltransferases
  • Adenosine
  • sinefungin