TITLE: Fluorescence assay for Enterovirus A71 3C protease activity measurement
AUTHORS: Charline Giroud, oleg.fedorov

[DESCRIPTION]
This protocol details the fluorescence assay for Enterovirus A71 (EV-A71) 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.

[STEPS]
SECTION: EV-A71 3C Pro IC50 Measurement
2. Assay buffer: 50 millimolar (mM)Tris pH 7.0, 150 millimolar (mM) NaCl, 10 % (v/v) glycerol and 1 millimolar (mM) TCEP (optional).
Incubation: 60 min at Room temperature.
EV-A71 3C: protein stocks were stored at -80C and used as 2x solution ( 5 micromolar (µM), 22.5 micromolar (µM) final assay concentration) in assay buffer.
Positive control: GC376 (Pubchem CID 71481119), 50 µM top final assay concentration.
Substrate: Dabcyl-KEALFQGPPQFE-Edans (LifeTein, USA) prepared as a stock solution at 10 mM in DMSO and used at 2x solution20 micromolar (µM)(10 micromolar (µM) final concentration assay concentration) in assay buffer.
Plates: Non-binding, black 384-plate, Greiner for the assay.
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 30 s for 120 min and shacked 5 s before each reading.

SECTION: EV-A71 3C Pro IC50 Measurement
4. Incubate the mix for 60 min at Room temperature and initiate the enzymatic reaction by adding of 50 µL of 2x (10 micromolar (µM)) substrate solution using the plate reader injector.

SECTION: EV-A71 3C Pro IC50 Measurement
5. Read the fluorescence intensity at 350/460 nm every 30 seconds for 120 min in kinetic mode, which includes a shaking step of the plate before each measurement.

SECTION: EV-A71 3C Pro IC50 Measurement
6. Calculate the IC50 by plotting the initial velocity against various concentrations of tested inhibitors by using a four-parameter dose−response curve in Prism (v8.0) software.
  

SECTION: EV-A71 3C Pro Assay
1. 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.
  

SECTION: EV-A71 3C Pro IC50 Measurement
3. 50 µL of 2x protein solution were added to each well containing the compounds to
be tested previously dispensed onto the plate.