Fluorogenic in vitro activity assay for the main protease Mpro from SARS-CoV-2 and its adaptation to the identification of inhibitors
Julien Ihssen, Greta Faccio, Chunyan Yao, Teja Širec, Urs Spitz
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.
Topics & Concepts
ProteaseFörster resonance energy transferIn silicoIn vitroChemistryHigh-throughput screeningSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2)Computational biologyBiochemistryEnzymeCoronavirus disease 2019 (COVID-19)FluorescenceBiologyMedicineGeneInfectious disease (medical specialty)PhysicsDiseaseQuantum mechanicsPathologyComputational Drug Discovery MethodsSARS-CoV-2 and COVID-19 ResearchMosquito-borne diseases and control