| Assay Method Information | |
| | Cov Mpro Biochemical IC50 Assay |
| Description: | This biochemical assay measured small molecule inhibitor IC50 values against recombinant SARS-CoV-2 Main protease (Mpro). Compounds from 100 μM to 5 nM in 3 fold dilutions were prepared and pre-incubated for 20 minutes while mixing at room temperature with 2 Mpro. The final Mpro concentration was 7.5 nM with the fluorogenic peptide substrate (DABCYL-KTSAVLQ/SGFRKME-EDANS) at its Km value of 40 μM in a reaction volume of 40 μM, Product formation was quantitated in kinetic mode every 2.5 minutes for 8 cycles by monitoring fluorescence at EX/EM wavelength of 340/460. Rates of reaction (slope with background subtracted) from inhibitor wells were normalized to DMSO wells and then curve fitted for IC50 using the PRISM algorithm: log(inhibitor) vs. normalized response Variable slope The IC50 value for each compound was defined as the concentration reducing enzyme activity by 50%. |
| Affinity data for this assay | |
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