| Assay Method Information | |
| | 3CLpro Inhibitory Activity Test |
| Description: | The inhibitory activity of the compounds against SARS-CoV-2 3CLpro was determined using fluorescence resonance energy transfer.10 μL of the compound solutions prepared at different concentrations (final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95 nM, in DMSO) and 40 L of SARS-CoV-2 3CLpro (Shanghai Biyuntian Biotechnology Co., Ltd., final concentration: 0.5 μM, diluted with Tris-HCl buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.4)) were mixed, added to a black 96-well plate, incubate at 37° C. for 10 min. A reaction was initiated by adding 50 μL of the fluorescent substrate Dabcyl-KTSAVLQSGFRKME-Edans (Shanghai Biyuntian Biotechnology Co., Ltd., the final concentration: 20 μM), incubated for 10 min, and measured by a multifunctional microplate reader (Thermo Fisher Scientific Co., Ltd., Varioskan Flash) for fluorescence detection, the excitation wavelength: 340 nm, the emission wavelength: 490 nm. The fluorescence value was recorded to calculate the inhibition percentage of the sample. DMSO without compound was used as the enzyme activity control, and the Tris-HCl buffer without SARS-CoV-2 3CLpro was used as the blank control, and the treatment methods were the same. The IC50 values of the samples (compounds 1-18) were calculated by nonlinear regression analysis using GraphPad Prism software.Inhibition Rate (%)=(RFU enzyme activity control −RFU sample)/(RFU enzyme activity control −RFU blank control)×100%. |
| Affinity data for this assay | |
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