| Assay Method Information | |
| | Inhibitory Activity against 3CLPpro |
| Description: | The inhibitory activity of the compounds against SARS-CoV-2 3CLpro was determined by fluorescence resonance energy transfer technique. A suitable amount of the above compounds was respectively weighed, and formulated in DMSO to give solutions over suitable concentration gradients. 5 μL of the formulated solution and 91 μL of Assay Reagent (Assay Buffer: 2019-nCoV Mpro/3CLpro=90:1, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) were added to a 96-well black plate, mixed uniformly, and incubated for 10 min at 37° C. in the dark. 4 μL of Substrate (100 μM Dabcyl-KTSAVLQSGFRKME-Edans, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was rapidly added to each well, and mixed uniformly. After incubation for 5 min at 37° C. in the dark, the signal gradually became stable. The fluorescence in 5-30 min was detected on a multifunctional plate reader (Thermo Fisher Technology Co., LTD., Varioskan Flash) and the inhibition percentage of the sample was calculated. The excitation wavelength was 340 nm and the emission wavelength was 490 nm. The Assay Reagent free of the compound was used as a control with 100% enzyme activity, the Assay Buffer free of SARS-CoV-2 Mpro/3CLpro was used as a blank control, and S-216722 (Shandong Xuanshuo Medical Technology Co., Ltd.) and PF-07321332 (Jinan Jianfeng Chemical Co., Ltd.) were used as positive controls. The rest of the treatment method was the same. |
| Affinity data for this assay | |
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