A fluorescence resonance energy transfer (FRET) protease assay was applied to measure the inhibitory activity of compounds against SARS-CoV-2 3CLpro and SARS-CoV 3CLpro. The fluorogenic substrate MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 was synthesized by GenScript (Nanjing, China). The FRET-based protease assay was performed as follows. The recombinant SARS-CoV-2 3CLpro (30 nM final concentration) or SARS-CoV 3CLpro (100 nM final concentration) was mixed with serial dilutions of each compound, oral liquid or the dissolved lyophilized powder in 80 µL assay buffer (50 mM Tris–HCl, pH 7.3, 1 mM EDTA) and incubated for 10 min. The reaction was initiated by adding 40 µL fluorogenic substrate at a final concentration of 20 µM. After that, the fluorescence signal at 320 nm (excitation)/405 nm (emission) measured immediately every 35 s for 3.5 min with a Bio-Tek Synergy4 plate reader. The initial velocities of reactions with compounds added at various concentrations compared to the reaction added with DMSO were calculated and used to generate inhibition profiles.
The inhibition of SARS-CoV-2 PLpro by compounds as well as Shuanghuanglian preparations was measured similar to that described previously [23 (link)] with a fluorogenic peptide (RLRGG-AMC) synthesized by GenScript (Nanjing, China). The reactions were performed in a total volume of 120 μL. First, 50 nM PLpro was incubated with the indicated concentrations of tested compounds or Shuanghuanglian preparations in the condition of 50 mM HEPES, pH 7.5, 0.1 mg/mL BSA, 5 mM DTT for 10 min. The reactions were initiated by the addition of 10 µM fluorogenic peptide. After that, the fluorescence signal at 360 nm (excitation)/460 nm (emission) was measured immediately every 1 min for 5 min with a Bio-Tek Synergy4 plate reader. The initial velocities of reactions with compounds or Shuanghuanglian added at various concentrations compared to the reaction added with DMSO were calculated and used to generate inhibition profiles.
For each compound or Shuanghuanglian preparation, at least three independent experiments were performed for the determination of IC50 values. The IC50 values were expressed as the mean ± SD and determined via nonlinear regression analysis using GraphPad Prism software 8.0 (GraphPad Software, Inc., San Diego, CA, USA).
The inhibition of SARS-CoV-2 PLpro by compounds as well as Shuanghuanglian preparations was measured similar to that described previously [23 (link)] with a fluorogenic peptide (RLRGG-AMC) synthesized by GenScript (Nanjing, China). The reactions were performed in a total volume of 120 μL. First, 50 nM PLpro was incubated with the indicated concentrations of tested compounds or Shuanghuanglian preparations in the condition of 50 mM HEPES, pH 7.5, 0.1 mg/mL BSA, 5 mM DTT for 10 min. The reactions were initiated by the addition of 10 µM fluorogenic peptide. After that, the fluorescence signal at 360 nm (excitation)/460 nm (emission) was measured immediately every 1 min for 5 min with a Bio-Tek Synergy4 plate reader. The initial velocities of reactions with compounds or Shuanghuanglian added at various concentrations compared to the reaction added with DMSO were calculated and used to generate inhibition profiles.
For each compound or Shuanghuanglian preparation, at least three independent experiments were performed for the determination of IC50 values. The IC50 values were expressed as the mean ± SD and determined via nonlinear regression analysis using GraphPad Prism software 8.0 (GraphPad Software, Inc., San Diego, CA, USA).
