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 D68 (EV-D68) 3C 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.
EV-D68 3C Pro Assay
EV-D68 3C Pro 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.
Incubation:01:00:00 at Room temperature.
EV-D68 3C: protein stocks were stored at -80C 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 10 millimolar (mM)in DMSO and used at 22 Mass Percent solution (40 micromolar (µM), 20 micromolar (µM) final concentration assay concentration) in assay buffer.
Plates: Non-binding, black 384-plate, small volume Greiner for the assay. Non-binding, 96-well polypropylene plates for the serial dilutions of the protease and substrate.
Plate reader: Pherastar FS, BMG Labtech (Germany), 350-460 FI optic module, the plate is read every 30 seconds for 2 hours and shacked 5 s before each reading.
1h
EV-D68 3C Pro assay development and optimization
EV-D68 3C Pro assay development and optimization
3h
This step aims to minimize the amount of costly reagents used for the assay and to find the best protease/substrate ratio allowing a high robustness determined by Z' calculation. A serial dilution of the protease is added to a substrate serial dilution and monitored for fluorescence intensity.
Prepare a serial dilution of the protease in assay buffer starting from 1 micromolar (µM)2 Mass Percent, at least 1000 µL, and perform a cascade dilution by transferring 500 µL of the first 2x solution to the next well which contains 500 µL of buffer. Transfer 10uL of 2x solution onto 384 well plate by repeat-pipetting, from the left to the right to cover several columns of the plate.
Prepare a serial dilution of the substrate starting from a solution of 40 micromolar (µM)2 Mass Percent, 1000 µL, by transferring in cascade 500 µL to the next well containing the same amount of buffer. Transfer 10uL of the 2x solution onto the protease solution by repeat-pipetting from the bottom to the top of the plate.
Read the fluorescence intensity at 350/460 nm every00:00:30 for 02:00:00 in kinetic mode a with shaking step of the plate before each measurement.
2h 0m 30s
Plot the fluorescent intensity as a function of time for each substrate concentration and determine a reaction time for the analysis which shows a complete (or almost complete) reaction. In the example below, the reaction time for the analysis is set to 120 min.
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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 Zfactor. (s: signal; c: control).
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Choose the substrate and protease concentrations that allow the best signal-to-background and the highest Z', in this example: 5µM substrate and 0.25µM protease.
EV-D68 3C Pro IC50 Measurement
EV-D68 3C Pro IC50 Measurement
3h
Add 10 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 10 µL of 2x (10 micromolar (µM)) substrate solution using the plate reader injector.
1h
Read the fluorescence intensity at 350/460 nm every 30 seconds for 02:00:00 in kinetic mode, which includes a shaking step of the plate before each measurement.
2h
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.
