| Assay Method Information | |
| | fluorescence resonance energy transfer (FRET)-based CoV-2 3CLpro inhibition assay |
| Description: | SARS-CoV-2 3CLpro expression and purification is based on a published procedure and our modified protocol is found in the supplementary file. A highly sensitive FRET based protease assay was developed to identify inhibitors of 3CL proteases based on a published protocol. The peptide substrate (Dabcyl)KTSAVLQSGFRKM(Glu)(EDANS) was synthesized by Genscript (USA). The test compounds were 3-fold serially diluted in 100% DMSO to 15 concentrations, starting at 3.33 mM. 1.5 μl of the serially diluted compounds were transferred to a black 384 well assay plate (Cat. 781900, Greiner). 23.5 μl of 2.13X concentration of SARS-CoV-2 Chis-3CLpro enzyme prepared in assay buffer was added to the compounds and incubated for 30 mins at 25 ◦C. 25 μl of 2X concentration of peptide substrate was added to the assay plate and incubated at 37 ◦C for 1.5 h. The final assay contained 12.5 nM of enzyme, 6 μM substrate and 3% DMSO in assay buffer containing 50 mM HEPES at pH 7.5, 100 mM NaCl, and 0.01% Triton X-100 and 1 mM DTT. The starting test compound concentration started at 100 μM. The FRET signal was measured using an excitation wavelength of 340 nm (UV[TRF] 340/60 nm, Barcode 101), emission wavelength of 490 nm (DSPPsion 486/10 filter, Barcode 220) and Lance/DELFIA D400 single mirror (Barcode 412) on Envision plate reader (2104 EnVision Multilabel Plate Readers, Perkin Elmer). The dose response curves were fitted with a variable slope using GraphPad Prism software (GraphPad, USA) to determine a compound s IC50. |
| Affinity data for this assay | |
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