Jun 20, 2025
Version 2
Fluorescence assay for MERS-CoV Mpro protease activity measurement for screening against antiviral molecules V.2
- Charline Giroud1,2,3,
- Oleg Fedorov1,2,3
- 1Center for Medicines Discovery;
- 2University of Oxford;
- 3ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Charline Giroud, Oleg Fedorov 2025. Fluorescence assay for MERS-CoV Mpro protease activity measurement for screening against antiviral molecules. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rzm4g1b/v2Version created by Mary-Ann Xavier
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
Protocol status: Working
We use this protocol and it's working
Created: February 10, 2025
Last Modified: June 20, 2025
Protocol Integer ID: 119854
Keywords: FRET assay, enzymatic assay, viral proteases, Coronavirus, MERS-CoV , MPro, IC50, activity of viral protease, viral protease, cov mpro protease activity measurement, protease activity measurement, sequence specific to the tested protease, protocol for protein expresssion, antiviral molecules this protocol detail, cleavage of the peptide, tested protease, protein production protocol, detection of the fluorescence, protein expresssion, antiviral molecule, protease, fluorescence, protein production, fluorescence emission, labelled peptide, peptide, assay, proximity of dabcyl, purification, protein
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
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 MERS-CoV Mpro 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. In this current version we added the protein production protocol and the plasmid used for this assay. In this new version, we added the protocol for protein expresssion and purification and the Addgene id.
Attachments
nyiwb5id7.docx
203KB
Materials
Reagents
- Assay buffer: 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 10 millimolar (mM) glycerol,7 millimolar (mM) DTT, 5 millimolar (mM) EDTA, 1 millimolar (mM) TCEP (optional) and 0.05 % volume Tween-20 .
- Incubation: 01:00:00 at Room temperature .
- MERS-CoV Mpro: Protein stocks were stored at -80 °C and used as 2x solution (10 micromolar (µM) , 5 micromolar (µM) final assay concentration) in assay buffer.
- Substrate: Edans-GVLQSGLV-LysDabcyl-K (LifeTein, USA) prepared as a stock solution at 10 millimolar (mM) in DMSO and used at 2x solution (20 micromolar (µM) , 10 micromolar (µM) final concentration) in assay buffer.
- Positive control: GC376 (Pubchem CID 71481119), 50 micromolar (µM) top final assay concentration.
- Plates: Greiner, 384-well plate, black, small volume, non-binding #7840900
- 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 2 hours and shacked during 5 s before the first reading.
Protocol materials
MERS-CoV MproaddgeneCatalog #228646
Troubleshooting
Protein production
2d
The protein used in this assay was expressed and purified following the protocol below with plasmid MERS-CoV MproaddgeneCatalog #228646
MERS-CoV Mpro 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
3h 0m 35s
Assay buffer: 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 10 millimolar (mM) glycerol,7 millimolar (mM) DTT, 5 millimolar (mM) EDTA, 1 millimolar (mM) TCEP (optional) and 0.05 % volume Tween-20 .
Incubation: 01:00:00 at room temperature.
MERS-MPro: protein stocks were stored at -80C and used as 2 x solution (10 micromolar (µM) , 5 micromolar (µM) final assay concentration) in assay buffer.
Positive control: GC376 (Pubchem CID 71481119), 25 micromolar (µM) top final assay concentration.
Substrate: Edans-GVLQSGLV-LysDabcyl-K (LifeTein, USA) prepared as a stock solution at 10 mM in DMSO and used at 2x solution (20 micromolar (µM) , 10 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
MERS-MPro IC50 Measurement
3h 0m 30s
Add 10 µL of 2x protein 10 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 to initiate the enzymatic reaction by the addition of 10 µL of 2x (20 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 inhibitors by using a four-parameter dose−response curve in Prism (v8.0) software.
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).
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