Assay Method Information

Assay Name:  Fluorescence MPro inhibition assay
Description:  Compounds were seeded into assay-ready plates (Greiner 384 low volume, cat 784900) using an Echo 555 acoustic dispenser, and DMSO was back-filled for a uniform concentration in assay plates (DMSO concentration maximum 1%) Screening assays were performed in duplicate at 20uM and 50uM. Hits of greater than 50% inhibition at 50uM were confirmed by dose response assays. Dose response assays were performed in 12 point dilutions of 2-fold, typically beginning at 100uM. Highly active compounds were repeated in a similar fashion at lower concentrations beginning at 10uM or 1uM. Reagents for Mpro assay were dispensed into the assay plate in 10ul volumes for a final volume of 20uL. Final reaction concentrations were 20mM HEPES pH7.3, 1.0mM TCEP, 50mM NaCl, 0.01% Tween-20, 10% glycerol, 5nM Mpro, 375nM fluorogenic peptide substrate ([5-FAM]-AVLQSGFR-[Lys(Dabcyl)]-K-amide). Mpro was pre-incubated for 15 minutes at room temperature with compound before addition of substrate and a further 30 minute incubation. Protease reaction was measured in a BMG Pherastar FS with a 480/520 ex/em filter set. Raw data was mapped and normalized to high (Protease with DMSO) and low (No Protease) controls using Genedata Screener software. Normalized data was then uploaded to CDD Vault (Collaborative Drug Discovery). Dose response curves were generated for IC50 using nonlinear regression with the Levenberg Marquardt algorithm with minimum inhibition = 0% and maximum inhibition = 100%.
Affinity data for this assay

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