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.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Coronavirus 3C Proteases/antagonists & inhibitors , Fluorescence Resonance Energy Transfer/methods , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , COVID-19/metabolism , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Coumarins/metabolism , Drug Discovery , High-Throughput Screening Assays , Humans , In Vitro TechniquesABSTRACT
Because of the essential roles of SARS-CoV-2 papain-like protease (PLpro) in the viral polyprotein processing and suppression of host immune responses, it is a crucial target for drug discovery against COVID-19. To develop robust biochemical methodologies for inhibitor screening against PLpro, extensive characterization of recombinant protein is important. Here we report cloning, expression, and purification of the recombinant SARS-CoV-2 PLpro, and explore various parameters affecting its stability and the catalytic activity. We also report the optimum conditions which should be used for high-throughput inhibitor screening using a fluorogenic tetrapeptide substrate.