| Assay Method Information | |
| | Enzyme inhibition assay and Ki values determination |
| Description: | To evaluate the potency of synthesized compounds against Mpro, the proteolytic activity of 50 nM Mpro -His and Mpro was first measured in the presence and absence of 25 µM compound using the fluorescent peptide Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH2 (SEQ ID NO: 5) (GenScript Biotech NJ, USA) as the reporter substrate at a concentration of 15 µM. Compounds were incubated with Mpro for 20 min at room temperature in reaction buffer composed of 20 mM Tris-HCL, pH 7.3, 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.02% Tween-20. Hydrolysis of the fluorescent peptide was monitored at an emission wavelength of 460 nm with excitation wavelength at 360 nm, using a TECAN M200 plate reader (TECAN, M nnedorf, Switzerland). Compounds that inhibited Mpro activity by less than 50% were considered inactive (Table 1).To determine the Ki values of active compounds, 25 nM Mpro was mixed with increasing concentrations of compounds (from 40 nM to 4000 nM with two-fold dilutions) and hydrolysis of 15 µM fluorescent peptide was monitored. Initial hydrolysis rates of fluorescent peptide were plotted as a function of compound concentrations and Ki values were obtained by fitting the data into the Morrison equation with standard error from triplicates. |
| Affinity data for this assay | |
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