| Assay Method Information | |
| | Mpro Enzymatic Assay |
| Description: | Recombinant Mpro was obtained and Mpro enzymatic assays were performed as previously described (Tietjen et al., 2021). Briefly, 5 μL of 25 nM recombinant Mpro protein was diluted in 25 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM DTT, and 0.005% Tween was dispensed into black 384-well plates. Test compounds were serially diluted into 100% DMSO, and 100 nL was added to Mpro dilutions using a Janus MDT Nanohead (PerkinElmer). Wells were then treated with 5 μL of 5 μM fluorogenic substrate ({DABCYL}-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-(EDANS)-NH2; Bachem, Vista, CA, USA) and monitored for fluorescence at 355 nm excitation and 460 nm emission every 5 minutes for up to 120 minutes using an Envision plate reader (PerkinElmer). Rate of substrate cleavage was determined using linear regression of the raw data values obtained during the time course. Slopes of these progress curves were then normalized to percent inhibition, where 100% equaled the rate in the absence of Mpro and 0% equaled rate of cleavage in the presence of Mpro and 0.1% DMSO. |
| Affinity data for this assay | |
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