| Assay Method Information | |
| | RapidFire MPro inhibition assay |
| Description: | The assay was performed according to the published procedure. Briefly, compounds were seeded into assay-ready plates (Greiner 384PP, cat# 781280) using an ECHO 650T dispenser and DMSO was back-filled for a uniform concentration in assay plates (DMSO concentration < 1%, final volume = 500 nL.). A 15 uM enzyme stock solution is prepared in 20 mM HEPES, pH 7.5 and 300 mM NaCl, and subsequently diluted to a working solution of 300 nM Mpro in assay buffer (20 mM HEPES, pH 7.5 and 50 mM NaCl) before the addition of 25 uL to each well using a Multidrop Combi (Thermo Scientific). After a quick centrifugation step (1000 rpm, 15 s) the plate is incubated for 15 min at room temperature. The reaction is initiated with the addition of 25 uL of 4 uM 11-mer (TSAVLQSGFRK-NH2, initially custom synthesized by the Schofield group, GLBiochem, used until March 2021), or 10 uM 37-mer (ALNDFSNSGSDVLYQPPQTSITSAVLQSGFRKMAFPS-NH2, GLBiochem, used after March 2021), dissolved in assay buffer. After centrifugation (1000 rpm, 14 s) the reaction is incubated for 10 min (11-mer) or 5 min (37-mer) at room temperature before quenching with 10 % formic acid. The reactions are analysed with MS using RapidFire (RF) 365 high-throughput sampling robot (Agilent) connected to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight (Q-TOF) mass spectrometer using electrospray. All compounds are triaged by testing the % inhibition at 5 and 50 uM final concentration. Dose response curves uses an 11-point range of 100--0.0017 uM inhibitor concentrations. RapidFire integrator software (Agilent) was used to extract the charged states from the total ion chromatogram data followed by peak integration. For the 11-mer peptide the m/z (+1) charge states of both the substrate (1191.67 Da) and cleaved N-terminal product TSAVLQ (617.34 Da) were used and the 37-mer peptide the m/z (+2) charge states of the substrate (3960.94 Da) and m/z (+1) of the cleaved C-terminal product SGFRKMAFPS (1125.57 Da). Percentage conversion (product peak integral / (product peak integral + substrate peak integral))*100) and percentage inhibitions were calculated and normalised against DMSO control with deduction of any background signal in Microsoft Excel. IC50s were calculated using Levenberg Marquardt algorithm used to fit a restrained Hill equation to the dose-response data with both GraphPad PRISM and CDD. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |