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

This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARSCoV-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.

Automated summary

This protocol outlines an in vitro fluorogenic assay for measuring the proteolytic activity of Mpro and identifying inhibitors, with the advantages of high sensitivity, specificity, and easy detectable fluorescent read-out for high throughput screening of new Mpro inhibitors.