Assay Method Information

Assay Name:  Inhibition of proteolytic activity
Description:  Inhibition of proteolytic activity was tested using recombinant SARS-CoV-2Mpro, which was expressed and purified as previously described.8,12 For the kinetic assays, 100 nM Mpro in reaction buffer (20 mM Tris, 100 mM NaCl, 1 mM DTT, pH 7.3) was incubated with or without compound in DMSO at varying concentrations to a final DMSO concentration of 6% for 15 min with shaking at room temperature. The reaction was initiated by addition of substrate (Dabcyl-KTSAVLQSGFRKM-E(Edans-NH2); GL Biochem) in reaction buffer, which is cleaved by Mpro, generating a product containing a free Edans group. Fluorescence was monitored at an excitation wavelength of 360 nm and emission wavelength of 460 nm. Baseline subtraction controlled for intrinsic fluorescence of each compound as well as intrinsic fluorescence of the uncleaved FRET substrate. All tested compounds had purity of at least 95% based on HPLC, and all measurements were performed in triplicate and averaged.
Affinity data for this assay
 

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