| Assay Method Information | |
| | Mpro Enzyme Activity Assay |
| Description: | Representative Compounds of the Disclosure, PF-00835231, and PF-07321332 were tested for their capability to inhibit the SARS-CoV-2 main protease Mpro by using a biochemical FRET-based Mpro enzyme activity assay. Representative compounds of Formula I′ and II′ were also tested. See PCT/US2021/046311. Briefly, recombinant Mpro protein (Ser1-Gln306; with proven proteolytic activity) was purchased from Biosynth Carbosynth (Staad, Switzerland). An EDANS- and Dabcyl-labeled peptide was purchased from Life Technologies GmbH (Darmstadt, Germany), and served as substrate peptide for Mpro proteolytic cleavage allowing fluorescence resonance energy transfer (FRET) read-out. Due to the Mpro-mediated cleavage of the substrate peptide, the EDANS fluorescence (λexc.=336 nm; λem.=490 nm) becomes dequenched (from disappearing Dabcyl) and increases with increasing Mpro activity. The assay buffer was 20 mM Tris buffer supplemented with 100 mM NaCl, and 1 mM EDTA, adjusted to pH 7.3 with 1N HCl. The test compounds were diluted from 20 mM stocks in DMSO; the stock of the substrate peptide was 250 μM in aqua bidest. The catalytic activity of the recombinant Mpro enzyme was 20 U/mg. It was checked in advance that neither the assay buffer nor the Mpro protein by itself emit fluorescence at 490 nm under 336 nm excitation. The basal emission of the uncleaved substrate peptide was subtracted from all results by baseline correction. The enzyme assay was carried out in black U-form half-area 96-wells. Each assay sample was finally composed of 0.4 μL substrate peptide stock (3× ad 20 μL assay buffer to yield finally 2 μM; 100 pmol), 0.1 μL Mpro enzyme (20 mU in assay buffer ad 20 μL) and 20 μL of 3× (in assay buffer) test compound dilution, resulting in a final sample volume of 60 μL. The final test compound concentrations were: 10 μM for compound fast-screening, and 0-200 μM for IC50 determinations. Initially, Mpro enzyme and test compound was added and mixed in 96-well and pre-incubated for 30 min in the dark with 200 rpm swiveling at room temperature. Subsequently, the reaction was started by addition of the substrate peptide, and followed by a fluorescence kinetic (λexc.=336 nm/λem.=500 nm/CutOff=435 nm; 30 min with 2 min increment by using a SpectraMax M5 multiwell plate reader (Molecular Devices, San Jose, CA, USA). |
| Affinity data for this assay | |
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