Apr 28, 2023
Version 2
Flaviviruses (West Nile, Zika, Dengue) NS2B/NS3 Fluorescence Dose Response V.2
This protocol is a draft, published without a DOI.
- Haim Barr1,2,
- Noa Lahav1,2
- 1Weizmann Institute of Science;
- 2ASAP Drug Discovery Consortium
Protocol Citation: Haim Barr, Noa Lahav 2023. Flaviviruses (West Nile, Zika, Dengue) NS2B/NS3 Fluorescence Dose Response. protocols.io https://protocols.io/view/flaviviruses-west-nile-zika-dengue-ns2b-ns3-fluore-cthtwj6nVersion created by ASAP Discovery
Manuscript citation:
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 28, 2023
Last Modified: April 09, 2024
Protocol Integer ID: 81171
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
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Abstract
This is a functional, biochemical assay used to identify treatments for viral infectious diseases related to viral Flaviviridae infection, (specifically West Nile, Zika, and Dengue) and targets the conserved NS2B/NS3 protein.
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 Concentrations (From Stock to Assay)
| A | B | C | D | E | |
| Reagent | Stock | Concentration Loaded into GNF | Final Concentration in Assay Plate | Units | |
| Substrate | 10000 | 10 | 5 | µM | |
| DENV NS2B/NS3 | 217000 | 200 | 100 | nM | |
| ZIKV NS2B/NS3 | 225000 | 200 | 100 | nM | |
| WNV NS2B/NS3 | 222000 | 200 | 100 | nM |
Assay Buffer
| A | B | C | D | E | |
| Reagent | Stock | Concentration Loaded into GNF | Final Concentration in Assay Plate | Units | |
| HEPES (pH 7.3) | 20 | 10 | 10 | mM | |
| NaCl | 100 | 50 | 50 | mM | |
| Glycerol | 50 | 5 | 5 | % | |
| Igepal | 10 | 0.05 | 0.05 | % | |
| TCEP | 1000 | 1 | 1 | mM |
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Compound Plate Design for Dose Response:
Total assay volume: 20 µL
Compounds top assay concentration: 60 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: 16 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 Stock Solution Concentrations)
- 20 millimolar (mM) HEPES 1M Solution pH 7.3Fisher ScientificCatalog #AAJ16924K2
- 100 millimolar (mM) Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
- 50 % volume Glycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
- 10 Mass Percent IGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
- 1000 millimolar (mM) Tris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259
*Note: all components are added fresh to the assay buffer before each experiment
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Additional Reagents:
West Nile Virus (WNV) Reagents:
222000 nanomolar (nM) WNV NS2B/NS3 Enzyme
- WNV NS2B/NS3was originally 222000 nanomolar (nM) and was diluted to 200 nanomolar (nM) with freshly made Assay Buffer before each experiment
10000 nanomolar (nM) WNV Enzyme Substrate
- Enzyme Substrate was Boc-Gly-Arg-Arg-AMC acetate saltBiosynthCatalog #FB110553
- Substrate stock was created by dissolving the substrate in DMSO to create 10 millimolar (mM) Substrate Stock Before each experiment, the Substrate Stock was diluted again to be 10 micromolar (µM) Substrate before every experiment with freshly made Assay Buffer
Zika (ZIKV) Virus Reagents:
225000 nanomolar (nM) ZIKV NS2B/NS3 Enzyme
- WNV NS2B/NS3was originally 225000 nanomolar (nM) and was diluted to 200 nanomolar (nM) with freshly made Assay Buffer before each experiment
10000 nanomolar (nM) ZIKV Enzyme Substrate
- Enzyme Substrate was Boc-Gly-Arg-Arg-AMC acetate saltBiosynthCatalog #FB110553
- Substrate stock was created by dissolving the substrate in DMSO to create 10 millimolar (mM) Substrate Stock Before each experiment, the Substrate Stock was diluted again to be 10 micromolar (µM) Substrate before every experiment with freshly made Assay Buffer
Dengue (DENV) Reagents:
217000 nanomolar (nM) DENV NS2B/NS3 Enzyme
- WNV NS2B/NS3was originally 217000 nanomolar (nM) and was diluted to 200 nanomolar (nM) with freshly made Assay Buffer before each experiment
10000 nanomolar (nM) WNV Enzyme Substrate
- Enzyme Substrate was Bz-Nle-KRR-AMC (hydrochloride)Cayman Chemical CompanyCatalog #27710
- Substrate Stock was created by dissolving the substrate in DMSO to create 10 millimolar (mM) Substrate Stock Before each experiment, the Substrate Stock was diluted again to be 10 micromolar (µM) Substrate before every experiment with freshly made Assay Buffer
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.
Determine which Flavivirus is needed and prepare solutions
Determine which Flavivirus is needed and prepare solutions
Determine which Flavivirus is needed and prepare solutions based on the materials section.
| A | B | C | D | E | |
| Reagent | Stock | Loaded into GNF | Final in assay plate | units | |
| Choice of NS2B/NS3 Enzyme Protein | |||||
| DENV NS2B/NS3 | 217000 | 200 | 100 | nM | |
| ZIKV NS2B/NS3 | 225000 | 200 | 100 | nM | |
| WNV NS2B/NS3 | 222000 | 200 | 100 | nM | |
| Choice of Viral Substrate | |||||
| WNV/ZIKV Substrate | 10000 | 10 | 5 | µM | |
| DENV Substrate | 10000 | 10 | 5 | µM | |
| Assay buffer | |||||
| HEPES pH=7.3 | 20 | 10 | 10 | mM | |
| NaCl | 100 | 50 | 50 | mM | |
| Glycerol | 50 | 5 | 5 | % | |
| Igepal | 10 | 0.05 | 0.05 | % | |
| TCEP | 1000 | 1 | 1 | mM | |
Prepare 384-well Plate for experiment
Prepare 384-well Plate for experiment
2h 31m
2h 31m
OPEN the EQUIcon Software and SELECT the "Flavivirus dispense 7,8 C" Program
PRIME the GNF Washer/Dispenser II (GNF) with 3 mL Ehtanol and 3 mL Dionized Water
CONFIRM that the GNF had accurately dispensed Ethanol and Water
WEIGH the plate and RECORD
DISPENSE 3 mL Ehtanol and 3 mL Dionized Water into a plate
WEIGH the plate and RECORD. Determine if the GNF Washer/Dispenser II had accurately dispensed 3 g Dionized Water and 2.367 g Ethanol
CONNECT Assay Buffer to 7C and your Flavivirus NS2B/NS3 to position 8C of the GNF Washer/Dispenser II.
PRIME the GNF with 300 µL Assay Buffer and with 300 µL 200 nanomolar (nM) Flavivirus NS2B/NS3 respectively.
DISPENSE 10 µL Assay Buffer to columns 1 and 23 using the 7C position of the GNF
- Note: These columns will be the inhibitor control columns (Containing: substrate + assay buffer + DMSO, no compounds)
DISPENSE 10 µL 200 nanomolar (nM) Flavivirus NS2B/NS3 to columns 2 through 22 and column 24 using the 8C position of the GNF.
- Note: 200 nanomolar (nM) Flavivirus NS2B/NS3 is two times the assay concentration. The final concentration of the Flavivirus NS2B/NS3 is 100 nanomolar (nM) during the assay.
- Columns 2 and 24 are neutral control columns (Contain: Enzyme + substrate + DMSO, no compounds)
CENTRIFUGE 1500 rpm, Room temperature, 00:01:00 plate to remove bubbles
1m
INCUBATE plate 02:00:00 at Room temperature
⚠ Make sure the plate is protected from light!
During Incubation: PREPARE the GNF to dispense the Flavivirus Substrate
2h
EMPTY 7C of the GNF.
WASH 7C tubing in Assay Buffer. Discard used Assay buffer
PRIME 7C of the GNF with 300 µL 10 micromolar (µM) Flavivirus Substrate
DISPENSE 10 µL 10 nanomolar (nM) Flavivirus Substrate to Columns 1 through 23 (the full plate)
- Note: 10 nanomolar (nM) Flavivirus Substrate is two times the assay concentration. The final concentration of the Flavivirus Substrate is 5 nanomolar (nM) during the assay.
CENTRIFUGE 1500 rpm, Room temperature, 00:01:00 plate to remove bubbles
INCUBATE plate for 00:30:00 at Room temperature
⚠ Make sure the plate is protected from light!
Recommended: Clean GNF during incubation
30m
Read Plate Flourescence
Read Plate Flourescence
READ and RECORD the plate Relative fluorescence units (RFU) via the "Flavivirus protocol" on the PHERAstar FS Control Software.
Expected result
gain 300 should yield ~20,000 RFU in full reaction; 7000 RFU in Buffer control