A fluorescence resonance energy transfer (FRET) protease assay was applied to measure the inhibitory activity of compounds against the SARS-CoV-2 3CLpro. The fluorogenic substrate (Dabcyl-TSAVLQ↓SGFRKMK-Edans) was synthesized by GenScript. The FRET-based protease assay was performed as follows. The recombinant SARS-CoV-2-3CLpro 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. The reaction was initiated by adding 40 μL fluorogenic substrate with a final concentration of 20 μM. After that, the fluorescence signal at 360 nm (excitation)/490 nm (emission) was immediately measured every 30 s for 10 min with a BioTek Synergy4 plate reader. The Vmax of reactions added with compounds at various concentrations compared to the reaction added with DMSO were calculated and used to generate IC50 curves. For each compound, at least three independent experiments were performed for the determination of IC50 values.
Dabcyl tsavlq sgfrkmk edans
Manufactured by GenScript
Dabcyl-TSAVLQ↓SGFRKMK-Edans is a fluorogenic peptide substrate designed for the detection and measurement of protease activity. The substrate consists of a quencher (Dabcyl) and a fluorophore (Edans) separated by a protease cleavage sequence (TSAVLQ↓SGFRKMK). Upon cleavage by the target protease, the quencher and fluorophore are separated, resulting in an increase in fluorescence emission that can be monitored.
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Lab products found in correlation
Synergy 4, by Agilent Technologies (1 mentions)
Prism 8, by GraphPad (1 mentions)
Black 96 well plate, by Greiner (1 mentions)
Spectramax paradigm muti mode detection platform, by Molecular Devices (1 mentions)
Spectramax paradigm multi mode detection platform, by Molecular Devices (1 mentions)
Leupeptin hemisulfate, by Targetmol (1 mentions)
3 protocols using dabcyl tsavlq sgfrkmk edans
1
FRET-based Inhibition Assay for SARS-CoV-2 3CLpro
2
Fluorescence-Based Protease Activity Assay
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In all, 10 μL of 100 μM substrate solution (Dabcyl-TSAVLQ↓SGFRKMK-Edans) (Genscript) was added to black 96-well plate (Greiner) with 40 μL final concentration of 200 nM GMpro or native Mpro in 25 mM Tris buffer (pH = 8.0). The relative fluorescence units (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)31 (link). Experiments were performed in triplicate. Then the progress curve of peptide hydrolysis was plotted by GraphPad Prism 8.0. First 1000 s change of fluorescence value was used to calculate the initial rate v0 by SoftMax Pro 7.1.
3
SARS-CoV-2 Main Protease Inhibition Assay
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A total of 20 mM leupeptin hemisulfate (catalog number T6564; TargetMol) in deionized water was diluted to 2 mM to 31.25 μM with 25 mM Tris buffer (pH 8.0). A 30-μl inhibitor solution with a series of concentrations in 25 mM Tris buffer (pH 8.0) was first mixed with 10 μl 100 μM peptide substrate (Dabcyl-TSAVLQ↓SGFRKMK -Edans; GenScript). Next, 10 μl of a final concentration of 200 nM Mpro was added to the plate. The relative fluorescence unit (RFU) value was measured with an excitation wavelength of 360 nm and an emission wavelength of 490 nm at 37°C for 1 h by using a SpectraMax Paradigm multimode detection platform (Molecular Devices, USA). Experiments were performed in triplicate. The enzyme activity reaction rate and inhibition rate were calculated by using MS Excel. The inhibition curve was plotted by using GraphPad Prism 8.0.
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