Skip to main content
. 2021 Aug 23;13(8):1669. doi: 10.3390/v13081669

Figure 1.

Figure 1

Development of SARS-CoV-2 3CLpro inhibitor screening assay using AlphaScreen. (A) Schematic representation of AlphaScreen-based assay for SARS-CoV-2 3CLpro enzymatic activity. A high signal is detected when the substrate is not cleaved by 3CLpro, and the signal becomes weaker when the substrate is cleaved. (B) Results of an enzymatic assay using wild-type SARS-CoV-2 3CLpro and its inactive mutant, C145A. 3CLpro decreases the signal when the substrate is cleaved. Substrate cleavage was also detected by immunoblots. Error bars obtained from duplicate testing. p-value < 0.005 (***). WT-Wild type. (C) Optimization of the enzyme assay. A concentration gradient of 3CLpro was reacted with 100 nM of the recombinant substrate and the signal was detected. Error bars obtained from duplicate testing. (D) The inhibitory effect of 3CLpro using the known SARS-CoV-2 3CLpro inhibitor GC376. Error bars obtained from duplicate testing. (E) Screening of 91 compounds using the 3CLpro enzyme assay. The substrate and enzyme concentration was 100 nM in this experiment. GC376 was used as a positive control. ATRA showed in the red bar. Error bars obtained from duplicate testing.