Compounds in Table 1 were diluted in 25 mM Tris buffer (pH = 8.0) to a final concentration of 100 μM for screen. DMSO was used as a solvent control. In all, 10 μL compound solution was add to black 96-well plate (Greiner). In total, 30 μL of 2 μM GMpro was added to the plate and incubated with the compounds at 37 °C incubator for 30 min. In total, 330 μL of 25 mM Tris buffer was also added as blank control. Then 10 μL of 20 μM peptide substrate (Dabcyl-TSAVLQ↓SGFRKMK-Edans) solution (Genscript) in DMSO was added. The RFU value was measured with an excitation wavelength of 340 nm and emission wavelength of 490 nm at 37 °C for 1 h by using a microplate reader (TECAN Infinite 200 Pro, Switzerland). The RFU change curves vs time with or without inhibitors were plotted by GraphPad Prism 8.0.
Because GC376 and Boceprevir are time-dependent covalent inhibitors, we evaluated the enzyme inhibitory activity without any preincubation. In all, 20 mM GC376 and Boceprevir in DMSO were diluted to 60 μM to 0.015 μM and 120 μM to 0.03 μM by 25 mM Tris buffer (pH = 8.0) respectively. In total, 30 μL inhibitor solution with a series of concentration in 25 mM Tris buffer (pH = 8.0) was mixed with 10 μL of 100 μM peptide substrate firstly. In total, 30 μL Tris buffer was also mixed with 10 μL of 100 μM peptide substrate as negative control. Then, 10 μL of 200 nM final concentration of Mpro was added to the plate. The RFU value was measured with an excitation wavelength of 360 nm and emission wavelength of 490 nm at 37 °C for 1 h by using SpectraMax Paradigm Muti-Mode Detection Platform (Molecular Devices, USA)39 (link). Experiments were performed in triplicate. First 1200 s change of fluorescence value was used to calculate the reaction rate v0 by SoftMax Pro 7.1. The reaction rate of different inhibitor concentration is divided by the reaction rate of the negative control to calculate the inhibition rate with Microsoft Excel 2016. Inhibition curve was plotted by GraphPad Prism 8.0.
Because GC376 and Boceprevir are time-dependent covalent inhibitors, we evaluated the enzyme inhibitory activity without any preincubation. In all, 20 mM GC376 and Boceprevir in DMSO were diluted to 60 μM to 0.015 μM and 120 μM to 0.03 μM by 25 mM Tris buffer (pH = 8.0) respectively. In total, 30 μL inhibitor solution with a series of concentration in 25 mM Tris buffer (pH = 8.0) was mixed with 10 μL of 100 μM peptide substrate firstly. In total, 30 μL Tris buffer was also mixed with 10 μL of 100 μM peptide substrate as negative control. Then, 10 μL of 200 nM final concentration of Mpro was added to the plate. The RFU value was measured with an excitation wavelength of 360 nm and emission wavelength of 490 nm at 37 °C for 1 h by using SpectraMax Paradigm Muti-Mode Detection Platform (Molecular Devices, USA)39 (link). Experiments were performed in triplicate. First 1200 s change of fluorescence value was used to calculate the reaction rate v0 by SoftMax Pro 7.1. The reaction rate of different inhibitor concentration is divided by the reaction rate of the negative control to calculate the inhibition rate with Microsoft Excel 2016. Inhibition curve was plotted by GraphPad Prism 8.0.
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