4.4. Enzymatic Inhibition Assay

The protease enzymatic activities of SARS-CoV-2 Mpro and PLpro were measured using a FRET-based peptide substrate: MCA-AVLQSGFR-K(DNP)-K-NH2 and fluorogenic substrate: Z-RLRGG-AMC, respectively. The protease enzymatic activities of human thrombin, human TMPRSS2, and human cathepsin L were performed using the fluorogenic substrates Boc-VPR-AMC, Boc-QAR-AMC, and Z-FR-AMC, respectively.

The SARS-CoV-2 Mpro (50 nM final concentration) enzymatic reaction was carried out in reaction buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.01% Tween 20 using 10 µM of MCA-AVLQSGFR-K(DNP)-K-NH2 FRET-based peptide as a substrate. SARS-CoV-2 PLpro (24.46 nM final concentration) enzymatic assays were performed in reaction buffer containing 50 mM HEPES pH 6.5, 150 mM NaCl, and 0.01% Tween 20 using 50 µM of Z-RLRGG-AMC fluorogenic substrate. The positive control for these assays was 10 µM Ebselen. Human thrombin (678.3 nM final concentration) enzymatic assays were carried out in 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5 using 100 µM Boc-VPR-AMC as the substrate and 100 µM of dabigatran as a positive control. Human TMPRSS2 (at a 30 nM final concentration) was assayed in 50 mM Tris pH 8, 150 mM NaCl, and 0.01% Tween 20 buffer using 10 µM Boc-QAR-AMC substrate and 10 µM Nafamostat as a positive control. A human cathepsin L (at 1 nM) activity assay was performed in 50 mM MES, 5 mM DTT, 1 mM EDTA, and 0.005% (w/v) Brij-35, pH 6.0 using 35 µM Z-FR-AMC as the substrate and 10 µM of E64 as a positive control. The negative controls for all assays were 0.2% DMSO. All experiments were assayed in a black 384-well microplate (BD Falcon, Dhaka, Bangladesh) in tahe total volume of 50 µM at 37 °C. For the Mpro FRET-based peptide substrate, the fluorescence signals were monitored at wavelengths of 320 and 400 nm for excitation and emission, respectively. The fluorescence signals of the PLpro fluorogenic substrate were monitored at wavelengths of 360 and 460 nm for excitation and emission, respectively. All fluorescence signals were detected using a Synergy HTX Multi-Mode Microplate Reader (BioTek) and data were visualized using Gen5 Software (BioTek, Hong Kong). The dose–response curves of each compound against all selected proteases were performed on 10 concentrations in triplicate ranging from 50 mM to 100 nM and the IC₅₀ values of each compound were calculated accordingly using the SciPy and Matplotlib Python packages.