TITLE: SARS-CoV-2 Main Protease (Mpro) Fluorescence Dose Response
AUTHORS: Haim Barr, Noa Lahav

[DESCRIPTION]
This is a functional, biochemical assay used to identify treatments for viral infectious disease that target SARS-COV-2 Main Protease (MPro). 

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. 

It was developed at the Weizmann Institute of Science, as a part of the ASAP Drug Discovery Consortium. 
-------------------------------------------------------
Experiment Assay Concentrations
 




[BEFORE_START]
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -200˚C until use.

[GUIDELINES]
Plate Information:
        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

[STEPS]
SECTION: Prepare 384 Well Plate
1. PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. 
SECTION: Prepare 384 Well Plate
1.1. 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
1.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
SECTION: Prepare Reagents
2. PRIME Multi-Drop Combi Tube Dispensing Cassette with 10 nanomolar (nM) by selecting the PRIME button on the Combi Dispenser until the tubes were filled completely. 
Note: Be sure to cycle dispensing several times on a a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).

SECTION: Prepare Reagents
2.1. DISPENSE 10 µL 10 nanomolar (nM)  to Columns 2 through 22 and also Column 24.

Note: 
10 nanomolar (nM) is two times the final concentration for the assay. It is diluted to be a final concentration of 5 nanomolar (nM) .
Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO; no experimental compounds)
SECTION: Prepare Reagents
2.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the 10 nanomolar (nM) discharged from the cassette.

SECTION: Prepare Reagents
3. CENTRIFUGE 15000 rpm, 1 min plate to remove bubbles
SECTION: Prepare Reagents
4. INCUBATE plate for 15 min at Room temperature
SECTION: Prepare Reagents
5. PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. Then, EMPTY the Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
SECTION: Prepare Reagents
6. PRIME Multi-Drop Combi Tube Dispensing Cassette with 750 nanomolar (nM) by selecting the PRIME button on the Combi Dispenser until the tubes were filled completely. 
Note: Be sure to cycle dispensing several times on a a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare Reagents
6.1. DISPENSE 10 µL 750 nanomolar (nM)  into Columns 1 through 24 (the full plate)

Note: 
 750 nanomolar (nM) is two times the final concentration for the assay. It is diluted to be a final concentration of 375 nanomolar (nM) 

SECTION: Prepare Reagents
7. CENTRIFUGE plate at 15000 rpm, 1 min  in plate centrifuge to remove bubbles

SECTION: Prepare Reagents
8. INCUBATE plate atRoom temperature for 30 min 
⚠ Make sure the plate is protected from light!

Recommended: Clean/Empty the Multi-Drop Combi Reagent Dispenser and Dispensing Cassette during this incubation step
SECTION: Read Plate Fluorescence
9. READ and RECORD the plate Relative fluorescence units (RFU) via the "SARS Endpoint protocol" on the PHERAstar FS Control Software.