Fluorogenic in vitro activity assay for the main protease Mpro from SARS-CoV-2 and its adaptation to the identification of inhibitors

STAR Protoc. 2021 Sep 17;2(3):100793. doi: 10.1016/j.xpro.2021.100793. Epub 2021 Aug 17.

Abstract

This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Studies to identify potential inhibitors of Mpro mainly rely on in silico molecular dynamics simulations or on FRET (Fluorescence Resonance Energy Transfer) substrates. The protocol is based on an aminomethyl coumarin substrate. High sensitivity, specificity, and an easily detectable fluorescent read-out are the advantages offered by this rapid assay, which allows high throughput screening of new Mpro inhibitors.

Keywords: Biotechnology and bioengineering; Chemistry; Clinical Protocol; Health Sciences; High Throughput Screening; Molecular/Chemical Probes; Protein Biochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / pharmacology*
  • COVID-19 / metabolism
  • COVID-19 / virology
  • COVID-19 Drug Treatment*
  • Coronavirus 3C Proteases / antagonists & inhibitors*
  • Coronavirus 3C Proteases / metabolism
  • Coumarins / metabolism
  • Drug Discovery
  • Fluorescence Resonance Energy Transfer / methods*
  • High-Throughput Screening Assays
  • Humans
  • In Vitro Techniques
  • Protease Inhibitors / pharmacology*
  • SARS-CoV-2 / enzymology*
  • Viral Proteins / antagonists & inhibitors*

Substances

  • Antiviral Agents
  • Coumarins
  • Protease Inhibitors
  • Viral Proteins
  • coumarin
  • 3C-like proteinase, SARS-CoV-2
  • Coronavirus 3C Proteases