Assay Method Information

Assay Name:  Fluorescence Mpro inhibition assay (S1)
Description:  Compounds were seeded into assay-ready plates (Greiner 384 low volume, cat. no. 784900) using an Echo 555 acoustic dispenser, and dimethyl sulfoxide (DMSO) was back-filled for a uniform concentration in assay plates (DMSO concentration maximum 1%) Screening assays were performed in duplicate at 20 μM and 50 μM. Hits of greater than 50% inhibition at 50 μM were confirmed by dose response assays. Dose response assays were performed in 12-point dilutions of twofold, typically beginning at 100 μM. Highly active compounds were repeated in a similar fashion at lower concentrations beginning at 10 μM or 1 μM. Reagents for Mpro assay were dispensed into the assay plate in 10 μl volumes for a final volume of 20 μl.Final reaction concentrations were 20 mM HEPES pH 7.3, 1.0 mM TCEP, 50 mM NaCl, 0.01% Tween-20, 10% glycerol, 5 nM Mpro, 375 nM fluorogenic peptide substrate ([5-FAM]-AVLQSGFR-[Lys(Dabcyl)]-K-amide). Mpro was pre-incubated for 15 min at room temperature with compound before addition of substrate and a further 30-min incubation. Protease reaction was measured in a BMG Pherastar FS with a 480/520 excitation/emission filter set. Raw data were mapped and normalized to high (Protease with DMSO) and low (No Protease) controls using Genedata Screener software. Normalized data were 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%.The assay was calibrated at different enzyme concentrations to confirm linearity and response of protease activity, as well as optimization of buffer components for most stable and reproducible assay conditions. Substrate concentration was chosen after titration to minimize saturation of signal in the plate reader while obtaining a satisfactory and robust dynamic range of typically five- to sixfold over control without enzyme. We used low substrate concentrations of the bright FRET peptide to avoid inner filter effect (60) and to bias toward detection of competitive inhibitors (61). As positive control, under our assay condition, nirmatrelvir has IC50 of 2.6 nM.
Affinity data for this assay

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