License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
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The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol details the fluorescence assay for Enterovirus coxsackievirus A16 (CVA16) 2A protease activity measurement. This method is intended to measure the activity of viral proteases by using a specific labelled-peptide that allows the detection of the cleaved product. The substrate contains the cleavage-sequence specific to the tested protease and is labeled in C-terminal by the fluorophore Edans (ex 336 nm; em: 455 nm) and in N-ternimal by the quencher Dabcyl (ex 472 nm). In the case of a non-cleaved substrate, the proximity of Dabcyl to Edans prevents the emission and the detection of the fluorescence at 455 nm. The cleavage of the peptide by the protease allows Edans’ fluorescence emission and detection.
EV-D68 3C: Protein stocks were stored at -80 °C and used as 2x solution (1 micromolar (µM), 0.5 micromolar (µM) final assay concentration) in assay buffer.
Substrate: Dabcyl-KEALFQGPPQFE-Edans (LifeTein, USA) prepared as a stock solution at 5 millimolar (mM) in DMSO and used at 2x solution (40 micromolar (µM), 20 micromolar (µM) final concentration assay concentration) in assay buffer.
Positive control: GC376 (Pubchem CID 71481119), 50 micromolar (µM) top final assay concentration.
Plate reader: Pherastar FS, BMG Labtech (Germany), 350-460 FI optic module, the plate is read every 30 s for 2 hours and shacked during 5 s before the first reading.
CVA16 2A protease Assay
CVA16 2A protease Assay
3h
This method is intended to measure the activity of viral proteases by using a specific labelled-peptide that allows the detection of the cleaved product. The substrate contains the cleavage-sequence specific to the tested protease and is labeled in C-terminal by the fluorophore Edans (ex 336 nm; em: 455 nm) and in N-ternimal by the quencher Dabcyl (ex 472 nm). In the case of a non-cleaved substrate, the proximity of Dabcyl to Edans prevents the emission and the detection of the fluorescence at 455 nm. The cleavage of the peptide by the protease allows Edans’ fluorescence emission and detection.
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Reagents and equipment
Reagents and equipment
Assay buffer:50 millimolar (mM)Tris pH 7.0, 150 millimolar (mM) NaCl, 10 % (v/v) glycerol and 1 millimolar (mM) TCEP (optional).
Incubation:01:00:00 at room temperature.
CVA16 2A protease: protein stocks were stored at -80C and used as 2 Mass Percentsolution (5 micromolar (µM), 2.5 micromolar (µM)final assay concentration) in assay buffer.
Positive control: Telaprevir (Pubchem CID 3010818), 50 micromolar (µM) top final assay concentration.
Substrate: Dabcyl-TAITTLGKFGQE-Edans (LifeTein, USA) prepared as a stock solution at 10 mM in DMSO and used at 2x solution (20 micromolar (µM), 2.5 micromolar (µM) final concentration assay concentration) in assay buffer.
Liquid handler: Echo‱ acoustic liquid handler (Beckman Coulter, USA).
Plate reader: Pherastar FS, BMG Labtech (Germany), 350-460 FI optic module, the plate is read every 00:00:30 for 02:00:00 and shacked 00:00:05 before each reading.
3h 0m 35s
CVA16 2A protease IC50 Measurement
CVA16 2A protease IC50 Measurement
3h
Add 50 µLof 2x protein 0.5 micromolar (µM) solution to each well containing the compounds to be tested previously dispensed onto the plate.
Incubate the mix for 01:00:00 at Room temperature and initiate the enzymatic reaction by the addition of 50 µL of 2x (10 micromolar (µM)) substrate solution using the plate reader injector.
1h
Read the fluorescence intensity at 350/460 nm every 00:00:30 for 02:00:00 in kinetic mode, which includes a shaking step of the plate before each measurement.
2h 0m 30s
Calculate the IC50 by plotting the initial velocity against various concentrations of tested inhibitor by using a four-parameter dose−response curve in Prism (v8.0) software.
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Calculate the mean (μ) and the standard deviation (σ) of fluorescence intensity and then calculate the signal-to-background ratio and the Z' or Z-factor. (s: signal; c: control).
