TITLE: MERS-CoV Mpro fluorescence dose response for antiviral testing 
AUTHORS: Haim Barr, Noa Lahav

[DESCRIPTION]
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
   Final Assay (in plate)  Reagent ConcentrationUnitsMERS Mpro50nMSubstrate550nMHEPES pH=7.320mMNaCl50mMBSA0.1mg/mlTriton X-1000.01%TCEP1mM


[BEFORE_START]
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -20 C until use.

[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


[STEPS]
SECTION: Prepare 384 Well Plate
3. PRIME the dispenser with Assay Buffer by selecting the PRIME button until the tubes are filled completely. 

SECTION: Prepare 384 Well Plate
3.1. DISPENSE 10 µL to Columns 1 and 23 of assay plate 
Note: These will represent the inhibitor control columns (Contain: Substrate, Assay Buffer, DMSO, no experimental compounds)

SECTION: Prepare 384 Well Plate
3.2. EMPTY the dispenser tubes. 
Discard Assay Buffer discharged from the dispenser tubes.
SECTION: Prepare 384 Well Plate
4. PRIME the dispenser with 100 nanomolar (nM) by selecting the PRIME button until the tubes are filled completely. 
 

SECTION: Prepare 384 Well Plate
4.1. DISPENSE 10 µL 100 nanomolar (nM) 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) is two times the final concentration for the assay. It is diluted in the plate to a final concentration of 50 nanomolar (nM).
Column 2 and 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO, no compounds)

SECTION: Prepare 384 Well Plate
4.2. EMPTY the dispenser tubes .
Discard the 100 nanomolar (nM) discharged from the tubes.
SECTION: Prepare 384 Well Plate
5. CENTRIFUGE plate 1500 rpm, 1 minto remove bubbles
SECTION: Prepare 384 Well Plate
6. INCUBATE plate for 15 min at Room temperature 

During Incubation: wash and prepare the dispenser to the next step
7. PRIME the dispenser with 1100 nanomolar (nM) 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.
8. DISPENSE 10 µL into Columns 1 - 24 (the full plate)

Note: 
1100 nanomolar (nM) is two times the final concentration for the assay. It is diluted in the plate to a final concentration of 550 nanomolar (nM)
9. CENTRIFUGE plate 1500 rpm, 1 min in plate centrifuge to remove bubbles
10. INCUBATE plate for 60 min at Room temperature 
⚠ Make sure the plate is protected from light!

Recommended: Clean the dispenser during this incubation step
 

SECTION: Read Plate Fluorescence
11. 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.
 

 

SECTION: Prepare Reagents
2. PREPARE all of the reagents/buffers required for this experiment.
 

SECTION: Experimental Design
12. Figure 1: Graphical depiction of assay principal and its use in screening campaign
SECTION: Protein expression and purification
1. We used the following plasmid in the protein expression and purification protocol.