| Assay Method Information | |
| | Mpro Enzyme Activity Assay |
| Description: | The recombinant SARS-CoV-2 Mpro (with a final concentration of 750 nM) was mixed with a series of dilutions for each compound in 25 μL assay buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT) and incubated for 10 min. The reaction was initiated by adding 25 μL of fluorescent substrate (MCA-AVLQ↓SGFR-Lys (Dnp)-Lys-NH2), with a final concentration of 20 μM, and the fluorescence signal at 320 nm (excitation)/405 nm (emission) was measured using the microplate reader (BMG). The Vmax of the reaction with the addition of compounds at different concentrations was calculated, together with the Vmax of the reaction with the addition of DMSO, that were used to generate an IC50 curve. For each compound, the IC50 value of anti-SARS-CoV-2 Mpro was measured using 9 concentrations, with 3 independent repeated experiments. |
| Affinity data for this assay | |
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