2.2. Enzymatic inhibition assay of SARS-CoV-2 3CLpro or SARS-CoV 3CLpro by FRET

Dabcyl-KNSTLQSGLRKE-Edans (Sangon Biotech, Shanghai, China) was synthesized as a substrate to measure the protease activity of SARS-CoV-2 3CL^pro. For the inhibition assay of SARS-CoV-2 3CL^pro, 4 μg/mL protease was incubated with the indicated concentrations of tested compounds in reaction buffer (0.1 M PBS, 1 mM EDTA, pH 7.4) for 30 min at 37 °C. The fluorogenic substrate at a final concentration of 20 μM was added to initiate the reaction. The fluorescence intensity change was measured immediately every 2 min for 20 min at 340 nm (excitation) / 490 nm (emission) using Spectramax® ID3 (Molecular Devices, California, USA) plate reader. The inhibition ratios of the protease with compounds added at various concentrations were calculated compared to the reaction including the solvent control. An FDA-Approved Drug Library containing an array of 1,018 compounds was obtained from Selleck Chemicals (# L1300) and used for screening the inhibitors by a FRET assay against SARS-CoV-2 3CL^pro. Vitamin K3 analogues were purchased from MCE. The IC₅₀ values of Vitamin K3 and its analogues were calculated by fitting the curve of normalized inhibition ratio with the concentration of the test compounds. Similarly, the inhibitory activities of SARS-CoV 3CL^pro by Vitamin K3 and its analogues were also determined according to the previously published method [^32,33].