2.5. Fluorescence Resonance Energy Transfer (FRET) Assay

The enzyme activity of SARS-CoV-2 M^pro was determined by FRET assay, as previously detailed [41]. For the activity assay of TMPRSS2, 100 μL reaction volume containing 25 μM TMPRSS2 protein in the presence or absence of SB was pre-incubated at room temperature for 30 min. The reaction was initiated by addition of the fluorescent protein substrate and monitored continuously for 2 h at 536 nm after excitation at 506 nm using a Synergy H1 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The concentrations of SB used in the FRET assays of SARS-CoV-2 M^pro were from 0.5 mg/mL to 4 mg/mL with two-fold series dilution. The IC₅₀ value of SB on inhibition of SARS-CoV-2 M^pro was determined by using 7 different concentrations of SB and calculated by Prism 9 software. The final concentration of SB used in the FRET assays of TMPRSS2 was 4 mg/mL.