| Assay Method Information | |
| | Rapid-Fire MS Biochemical Assay measuring Mpro activity (5nM Mpro) |
| Description: | Assay buffer preparation: 50 mM HEPES pH 7.3, 150 mM NaCl, 1 mM EDTA, 0.01% pluronic f-127(Catalog # 59005, Biotium) was prepared using UltraPure Distilled Water (Catalog # 10977015, Thermo Scientific). The Mpro enzyme stock consisted of a 10 nM solution of Mpro made in buffer. The substrate solution stock consisted of a 10 µM solution of Mpro peptide (AVLSGFRKK (SEQ ID NO:2); Purchased from Vivitide) made in buffer. The quench solution consisted of a 2% acetic acid solution spiked with 200 nM of the internal standard peptide (13C AVLQ; Purchased from Vivitide).The Rapidfire Mass Spectrometry setup consisted of a Rapidfire autosampler platform (Agilent) coupled with a Sciex 6500 QQQ Mass Spectrometer (Sciex). A C18 Rapidfire cartridge type C (Catalog # G9205A) was used in the analysis, the load solvent consisted of 0.1% formic acid flowed at 1.5 mL/min and the elute solvent consisted of 75% acetonitrile, 5% isopropal alcohol, 20% ultrapure distilled water with 0.1% formic acid. MS transitions were created for the substrate peptide AVLQSGFRKK (SEQ ID NO:3) (378.6 Da 482.6 Da), product peptide AVLQ (430.3 Da- 260.4Da) and the internal standard peptide 13C AVLQ (434.3 Da- 288.3 Da) Compound plates were prepared in 384 Echo plate (cat# LPL0200, Labcyte) and the starting concentration of compound was 10 mM, then 1 to 3 serial dilution in 100% DMSO, 8 µl/ well). To generate assay ready plates, 10 nl of compound were transferred to a 384-well clear assay plate using an Echo 555 Liquid Handler (Labcyte).5 µL of Mpro enzyme solution was dispensed in each well throughout entire plate (Col 1-24) using buffer distributor (Multidrop Combi from Thermo Scientific) with 5 µL of assay buffer only dispensed to no enzyme control in Col 24. Enzyme allowed to preincubate with compounds for 15 minutes at room temperature (25^C) prior to starting reaction by addition of 5 µL of substrate peptide solution to all wells in the plate (Col 1-24). The plates were incubated for 120 minutes at room temperature with a final volume of 10 µl/ well buffer, such that the compound concentration was diluted 1000-fold to final concentration. Final assay concentrations were 5 nM Mpro and 5 µM substrate peptide. After incubation, reaction was quenched by adding 40 µL of quench solution and plates were analyzed by the Rapidfire/Sciex MS platform.Mass spectral data was analyzed as ratiometric measurements between the integrated areas of the peak for the product peptide and the labeled internal standard peptide for each sample (Multiquant, Sciex). Percent activity measurements were calculated comparing each ratio of product/Internal standard from compound treatments to the ratios calculated for the DMSO only wells with and without enzyme (Neutral and active controls respectively). Mpro Inhibitor qualified absolute IC50 values (concentration of inhibitor that inhibits Mpro activity at least 50%), were determined with 8 inhibitor concentrations measured in duplicate for each inhibitor. The qualified absolute IC50 was calculated by non-linear regression analysis to sigmoidal-logistic curves by HELIOS (PROD 2) system. |
| Affinity data for this assay | |
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