Jun 08, 2025
Version 5
MERS-CoV Mpro fluorescence dose response for antiviral testing V.5
- Haim Barr1,2,
- Noa Lahav1,2
- 1Weizmann Institute of Science;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Haim Barr, Noa Lahav 2025. MERS-CoV Mpro fluorescence dose response for antiviral testing . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7r1rlx9/v5Version created by Noa Lahav
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: May 30, 2023
Last Modified: June 08, 2025
Protocol Integer ID: 82635
Keywords: Coronaviridae, Fluorescence assay, Protease assay, TR-FRET, Screening, Assay, Inhibitor, MERS-CoV, Mpro, response for antiviral testing, antiviral testing, cov mpro fluorescence, treatments for viral infectious disease, biochemical assay, direct enzyme activity measurement method, viral infectious disease, final experiment concentration, fluorescence intensity, cov, dose response, fluorescence, final assay, assay
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 is a functional, biochemical assay used to identify treatments for viral infectious disease in MERS-CoV 3C-like protease.
Utilizing a direct enzyme activity measurement method, the experiment was performed in a 384-well plate reading the fluorescence intensity. This assay tested the mode of action of inhibition.
This assay can run as dose response or single point as described in the guidelines tab.
Final Experiment Concentrations
| A | B | C | |
| Reagent | Concentration | Units | |
| MERS Mpro | 50 | nM | |
| MERS Substrate peptide | 550 | nM | |
| HEPES pH=7.3 | 20 | mM | |
| NaCl | 50 | mM | |
| BSA | 0.1 | mg/ml | |
| Triton X-100 | 0.01 | % (v/v) | |
| TCEP | 1 | mM |
For more information, please see the Materials section.
Guidelines
Compound Plate Design for Dose Response:
Total assay volume: 20 µL
Compounds top assay concentration: 100 µM
Dilution factor: 2
Dose response points: 12
Number of replicates: 2
Backfill with DMSO: yes
Compounds Plate Design for 2-Point Assay:
Total Assay Volume: 20 µL
Compounds Assay Concentration: 100 µM and 50 µM
Dilution Factor: 2
Dose Response Points: 2
Number of Replicates: 2
Backfill with DMSO: Yes
Materials
Assay Buffer Reagents (Concentration listed are the stock concentrations)
- 40 millimolar (mM) HEPES Buffer (pH 7.3)Fisher ScientificCatalog #BP299-1 (or similar)
- 100 millimolar (mM) Sodium ChlorideFisher ScientificCatalog #S271 (or similar)
- 10 mg/mL BSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S (or similar)
- 10 % volume Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML (or similar)
- 1000 millimolar (mM) TCEP HClP212121Catalog #SV-TCEP (or similar)
(all components are added fresh to the assay buffer before each experiment)
-------------------------------------------------------
Additional Reagents
- 507000 nanomolar (nM) MERS Mpro Enzyme*
*Note: Stock solutions were diluted with fresh assay buffer to create a 100 nanomolar (nM) solution before each experiment
- 750000 nanomolar (nM) MERS Substrate*
*Note: MERS Substrate (5-FAM)-GVLQSGLV-K(Dabcyl)-K-NH2 Stock was purchased from Peptide 2.0, dissolved in DMSO to 750000 nanomolar (nM) , aliquoted and stored at -80 °C . Before each experiment, stock was diluted with assay buffer to yield a concentration of 1100 nanomolar (nM) .
Protocol materials
Sodium ChlorideFisher ScientificCatalog #S271
BSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S
TCEP HClP212121Catalog #SV-TCEP
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
HEPES Buffer (pH 7.3)Fisher ScientificCatalog #BP299-1
Middle East respiratory syndrome coronavirus (MERS-CoV) Mpro strain HCoV-EMC 3CL protease domain addgeneCatalog #228646
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -20 C until use.
Protein expression and purification
We used the following plasmid in the protein expression and purification protocol.Middle East respiratory syndrome coronavirus (MERS-CoV) Mpro strain HCoV-EMC 3CL protease domain addgeneCatalog #228646
Prepare Reagents
PREPARE all of the reagents/buffers required for this experiment.
| A | B | C | D | E | |
| Reagent | Stock Concentration | Concentration Loaded into dispenser | Final Concentration in plate | Units | |
| MERS Mpro Enzyme stock | 507000 | 100 | 50 | nM | |
| MERS Substrate | 750000 | 1100 | 550 | nM | |
| Assay Buffer | |||||
| HEPES (pH 7.3) | 40 | 20 | 20 | mM | |
| Sodium Chloride | 100 | 50 | 50 | mM | |
| BSA | 10 | 0.1 | 0.1 | mg/mL | |
| Triton X-100 | 10 | 0.01 | 0.01 | % by volume | |
| TCEP | 1000 | 1 | 1 | mM | |
For more information, please see the Materials sec
Prepare 384 Well Plate
15m
PRIME the dispenser with Assay Buffer by selecting the PRIME button until the tubes are filled completely.
DISPENSE 10 µL Assay Buffer to Columns 1 and 23 of assay plate
Note: These will represent the inhibitor control columns (Contain: Substrate, Assay Buffer, DMSO, no experimental compounds)
EMPTY the dispenser tubes.
- Discard Assay Buffer discharged from the dispenser tubes.
PRIME the dispenser with 100 nanomolar (nM) MERS MPro Enzyme by selecting the PRIME button until the tubes are filled completely.
DISPENSE 10 µL 100 nanomolar (nM) MERS MPro Enzyme to Columns 2 through 22 and Column 24
Note:
- Be sure to cycle dispensing several times on an empty plate to detect and avoid dispensing issues.
- 100 nanomolar (nM) MERS MPro Enzyme is two times the final concentration for the assay. It is diluted in the plate to a final concentration of 50 nanomolar (nM) MERS MPro Enzyme .
- Column 2 and 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO, no compounds)
EMPTY the dispenser tubes .
- Discard the 100 nanomolar (nM) MERS MPro enzyme discharged from the tubes.
CENTRIFUGE plate 1500 rpm, Room temperature, 00:01:00 to remove bubbles
1m
INCUBATE plate for 00:15:00 at Room temperature
During Incubation: wash and prepare the dispenser to the next step
15m
PRIME the dispenser with 1100 nanomolar (nM) MERS Substrate by selecting the PRIME button until the tubes are filled completely.
- Note: Be sure to cycle dispensing on an empty plate to detect and avoid dispensing issues.
DISPENSE 10 µL 1100nM MERS Substrate into Columns 1 - 24 (the full plate)
Note:
- 1100 nanomolar (nM) MERS Substrate is two times the final concentration for the assay. It is diluted in the plate to a final concentration of 550 nanomolar (nM) MERS Substrate
CENTRIFUGE plate 1500 rpm, Room temperature, 00:01:00 in plate centrifuge to remove bubbles
1m
INCUBATE plate for 01:00:00 at Room temperature
⚠ Make sure the plate is protected from light!
Recommended: Clean the dispenser during this incubation step
1h
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) via the "MERS Protocol" on the PHERAstar FS Control Software.
- Software is a standard Fluorescence Assay set for Optimal excitation wavelength 485 nm, emission wavelength 528 nm, and a Gain of 300.
Equipment
PHERAstar FS
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
0471B0001A
SKU
LINK
Expected result
Gain 300 should yield ~10,000 RFU in full reaction and ~6,000 RFU in Buffer Control
Experimental Design
Figure 1: Graphical depiction of assay principal and its use in screening campaign