Apr 28, 2024

Public workspaceSARS-CoV-2 Mpro fluorescence dose response V.4

DOI
dx.doi.org/10.17504/protocols.io.81wgbye9nvpk/v4
  • 1Weizmann Institute of Science;
  • 2ASAP Drug Discovery Consortium
  • Haim Barr: General acknowledgement: The Wohl Drug Discovery institute, The Nancy and Stephen Grand Israel National Center for Personalized Medicine.;
  • ASAP Discovery
Noa Lahav
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DOI: dx.doi.org/10.17504/protocols.io.81wgbye9nvpk/v4
Protocol Citation: Haim Barr, Noa Lahav 2024. SARS-CoV-2 Mpro fluorescence dose response. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbye9nvpk/v4Version 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: April 12, 2024
Last Modified: October 07, 2024
Protocol Integer ID: 98134
Keywords: SARS-CoV-2, Main Protease, dose response, Coronavirus
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 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.
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Experiment Assay Concentrations
ABC
ReagentFinal Assay ConcentrationUnits
SARS Mpro5nM
SARS Substrate375nM
HEPES (pH 7.3)20mM
NaCl50mM
Glycerol10% by volume
TWEEN 200.01% by volume
TCEP1mM
For more information, please check out the "Materials" Section



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
Materials
Assay Buffer Reagents (Concentration listed are Stock Solution Concentrations)
  1. Concentration40 millimolar (mM) ReagentHEPES 1M Solution pH 7.3Fisher ScientificCatalog #AAJ16924K2 (or similar)
  2. Concentration100 millimolar (mM) ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888-25G (or similar)
  3. Concentration50 % volume ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516 (or similar)
  4. Concentration10 % volume ReagentTWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9416 (or similar)
  5. Concentration1000 millimolar (mM) ReagentTris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259 (TCEP) (or similar)
*Note: all components are added fresh to the assay buffer before each experiment
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Additional Reagents:
Concentration710 micromolar (µM) SARS MPro Enzyme
  • The Enzyme original stock was originally Concentration750 micromolar (µM) and was diluted to create aliquots of Concentration20000 nanomolar (nM) using a storage buffer (50 mM Tris pH 7.5, 1 mM DTT, 50 mM NaCl, 1 mM EDTA, 50% Glycerol).
  • Before an experiment, the 20000 nM aliquots were diluted with Assay Buffer to create a Concentration10 nanomolar (nM) solution to be loaded into the Combi.

Concentration20000 micromolar (µM) SARS MPro Substrate
  • SARS MPro Substrate Stock ([5-FAM]-AVLQSGFR-[Lys(Dabcyl)-K-amide) was purchased and dissolved in DMSO and yielded a concentration of Concentration20000 micromolar (µM)
  • Before an experiment, the SARS MPro Substrate Stock had an intermediate dilution step with DMSO to yield a Concentration100 micromolar (µM) SARS MPro Substrate Solution. Then, before adding the SARS MPro Substrate to the Combi, it was diluted again with Assay Buffer to yield a concentration of Concentration750 nanomolar (nM) . The final concentration of SARS MPro Substrate for the assay was Concentration375 nanomolar (nM)
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 -200˚C until use.
Prepare 384 Well Plate
Prepare 384 Well Plate
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.
DISPENSE Amount10 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 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.
Prepare Reagents
Prepare Reagents
PRIME Multi-Drop Combi Tube Dispensing Cassette with Concentration10 nanomolar (nM) SARS MPro 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).

DISPENSE Amount10 µL Concentration10 nanomolar (nM) SARS MPro to Columns 2 through 22 and also Column 24.
Note:
  • Concentration10 nanomolar (nM) SARS Mpro is two times the final concentration for the assay. It is diluted to be a final concentration of Concentration5 nanomolar (nM) SARS MPro .
  • Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO; no experimental compounds)
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 Concentration10 nanomolar (nM) SARS MPro discharged from the cassette.

CENTRIFUGE Centrifigation15000 rpm, Room temperature, 00:01:00 plate to remove bubbles
1m
INCUBATE plate for Duration00:15:00 at TemperatureRoom temperature
15m
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.
PRIME Multi-Drop Combi Tube Dispensing Cassette with Concentration750 nanomolar (nM) SARS Substrate 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).
DISPENSE Amount10 µL Concentration750 nanomolar (nM) SARS Substrate into Columns 1 through 24 (the full plate)

Note:
  • Concentration750 nanomolar (nM) SARS Substrate is two times the final concentration for the assay. It is diluted to be a final concentration of Concentration375 nanomolar (nM) SARS Substrate

CENTRIFUGE plate at Centrifigation15000 rpm, Room temperature, 00:01:00 in plate centrifuge to remove bubbles

1m
INCUBATE plate atTemperatureRoom temperature for Duration00:30:00
⚠ Make sure the plate is protected from light!

Recommended: Clean/Empty the Multi-Drop Combi Reagent Dispenser and Dispensing Cassette during this incubation step
30m
Read Plate Fluorescence
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) via the "SARS Endpoint protocol" on the PHERAstar FS Control Software.

Expected result
Gain 300 should yield ~10,000 RFU in full reaction and ~6,000 RFU in Buffer Control