Jun 20, 2025

Public workspaceFlaviviruses (West Nile, Zika, Dengue serotypes 1-4) NS2B-NS3 protease fluorescence dose response and single point assay V.4

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7yebkgwz/v4
  • Haim Barr1,2,
  • Noa Lahav1,2
  • 1Weizmann Institute of Science;
  • 2ASAP Discovery Consortium
  • ASAP Discovery
Noa Lahav
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7yebkgwz/v4
Protocol Citation: Haim Barr, Noa Lahav 2025. Flaviviruses (West Nile, Zika, Dengue serotypes 1-4) NS2B-NS3 protease fluorescence dose response and single point assay. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7yebkgwz/v4Version created by Mary-Ann Xavier
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: June 04, 2025
Last Modified: June 20, 2025
Protocol Integer ID: 219547
Keywords: Flaviviridae, Fluorescence assay, NS2B-NS3, ZIKV, WNV, DENV, ASAP, flavivirus infection, flavivirus, ns3 protease fluorescence dose response, compounds top assay concentration, biochemical assay, compounds assay concentration, dengue, top assay concentration, ns3 protein, treatments for viral infectious disease, assay buffer, direct enzyme activity measurement method, viral infectious disease, enzyme, zika, assay reliability, point assay, assay, total assay volume, stock to assay, ns3 protease, fluorescence intensity, fluorescence, protein
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 diseases related to Flavivirus infection, (specifically West Nile, Zika, and Dengue) and targets the conserved NS2B-NS3 protein. In this current version, we added the Addgne id.

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.
Initial screening was conducted with 2 compound concentrations to determine %inhibition, followed by dose response for IC50 measurement (as specified below). In each run a reference compound was added to verify the assay reliability and robustness.

Assay was developed at the Weizmann Institute of Science, as a part of the ASAP Discovery Consortium.
-------------------------------------------------------
Experiment Concentrations (From Stock to Assay)
ABCDE
ReagentStockConcentration Loaded into CERTUSFinal Concentration in Assay PlateUnits
Substrate10000105µM
DENV NS2B-NS3217000200100nM
ZIKV NS2B-NS3225000200100nM
WNV NS2B-NS3222000200100nM

Assay Buffer
ABCE
ReagentStock Final concentration Units
Tris pH=8.5100020mM
Glycerol5010%
Triton100.01%
-------------------------------------------------------
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 Stock Solution Concentrations)
  1. Concentration50 Mass / % volume ReagentGlycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
  2. Concentration1000 millimolar (mM) ReagentTris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259
  3. Concentration10 % (v/v) Triton ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
*Note: all components are added fresh to the assay buffer before each experiment
---------------------------------------------------------
Additional Reagents:
West Nile Virus (WNV) Reagents:
Concentration222000 nanomolar (nM) WNV NS2B/NS3 Enzyme
  • WNV NS2B/NS3 was originally Concentration222000 nanomolar (nM) and was diluted to Concentration200 nanomolar (nM) with freshly made Assay Buffer before each experiment
Concentration10000 nanomolar (nM) WNV Enzyme Substrate
  • Enzyme Substrate was ReagentBoc-Gly-Arg-Arg-AMC acetate saltBiosynthCatalog #FB110553
  • Substrate stock was created by dissolving the substrate powder in DMSO to create Concentration10 millimolar (mM) Substrate Stock aliquots and stored at Temperature-80 °C . Before each experiment, the Substrate Stock was diluted to Concentration10 micromolar (µM) Substrate with freshly made Assay Buffer.

Zika (ZIKV) Virus Reagents:
Concentration225000 nanomolar (nM) ZIKV NS2B/NS3 Enzyme
  • ZIKV NS2B-NS3 was originally Concentration225000 nanomolar (nM) and was diluted to Concentration200 nanomolar (nM) with freshly made Assay Buffer before each experiment
Concentration10000 nanomolar (nM) ZIKV Enzyme Substrate
  • Enzyme Substrate was ReagentBoc-Gly-Arg-Arg-AMC acetate saltBiosynthCatalog #FB110553
  • Substrate stock was created by dissolving the substrate powder in DMSO to create Concentration10 millimolar (mM) Substrate Stock aliquots and stored at Temperature-80 °C . Before each experiment, the Substrate Stock was diluted to Concentration10 micromolar (µM) Substrate with freshly made Assay Buffer.

Dengue (DENV) Reagents:
Concentration217000 nanomolar (nM) DENV NS2B/NS3 Enzyme
  • DENV NS2B/NS3was originally Concentration217000 nanomolar (nM) and was diluted to Concentration200 nanomolar (nM) with freshly made Assay Buffer before each experiment
Concentration10000 nanomolar (nM) DENV Enzyme Substrate
  • Enzyme Substrate was ReagentBz-Nle-KRR-AMC (hydrochloride)Cayman Chemical CompanyCatalog #27710
  • Substrate Stock was created by dissolving the substrate powder in DMSO to create Concentration10 millimolar (mM) Substrate Stock aliquots and stored at Temperature-80 °C . Before each experiment, the Substrate Stock was diluted to Concentration10 micromolar (µM) Substrate with freshly made Assay Buffer.

Expected result
A literature reference inhibitor (compound 83, Journal of Medicinal Chemistry 2015 58 (23), 9354-9370 https://doi.org/10.1021/acs.jmedchem.5b01441) was added to each run of NS2B-NS3 to give IC50 values of ~2 μM for DENV-2 and WNV and ~3 μM for ZIKA .


Protocol materials
ReagentZIKV VE_Ganxian2016 NS2B-NS3 protease fusion (non-cleavable linker, N-term Twin-Strep-tag)addgeneCatalog #228661
ReagentDENV-1 strain US/Hawaii/1944 NS2B-NS3 protease fusion with non cleavable linkeraddgeneCatalog #228648
ReagentDENV-2 Thailand/16681/84 NS2B-NS3 protease fusion (non-cleavable linker)addgeneCatalog #228650
ReagentWNV NY99 NS2B-NS3 protease fusion (non-cleavable linker) K→A mutation to reduce autoproteolysisaddgeneCatalog #228660
ReagentDENV-3 Philippines/H87/1956 NS2B-NS3 protease fusion (non-cleavable linker)addgeneCatalog #228651
ReagentDENV-4 strain H241 NS2B-NS3 protease fusion (non-cleavable linker)addgeneCatalog #228652
Troubleshooting
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Dummy plate: In each step of the protocol, dispense the reagents into an empty plate before dispensing them into the assay compounds plate (this will detect and prevent dispensing issues).

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

Flavivirus NS2B-NS3 protease expression and purification
Zika virus NS2B-NS3 fused protease expression and purification of a construct suitable for biochemical assay. Using the plasmid ReagentZIKV VE_Ganxian2016 NS2B-NS3 protease fusion (non-cleavable linker, N-term Twin-Strep-tag)addgeneCatalog #228661

Protocol
NAME

Zika NS2B-NS3 fusion construct for assay small scale expression and purification protocol

CREATED BY
Korvus Wang



West Nile virus NS2B-NS3 fused protease expression and purification of the construct suitable for biochemical assay.ReagentWNV NY99 NS2B-NS3 protease fusion (non-cleavable linker) K→A mutation to reduce autoproteolysisaddgeneCatalog #228660

Protocol
NAME

West Nile virus NS2B-NS3 protease fusion construct small scale expression and purification protocol

CREATED BY
Korvus Wang


Dengue virus serotype 1 virus NS2B-NS3 fused protease expression and purification of the construct suitable for biochemical assay. ReagentDENV-1 strain US/Hawaii/1944 NS2B-NS3 protease fusion with non cleavable linkeraddgeneCatalog #228648


Protocol
NAME

Dengue virus serotype 1 NS2B-NS3 protease fusion construct small scale expression and purification protocol

CREATED BY
Korvus Wang


Dengue virus serotype 2 virus NS2B-NS3 fused protease expression and purification of the construct suitable for biochemical assay. ReagentDENV-2 Thailand/16681/84 NS2B-NS3 protease fusion (non-cleavable linker)addgeneCatalog #228650


Protocol
NAME

Dengue virus serotype 2 NS2B-NS3 protease fusion construct small scale expression and purification protocol

CREATED BY
Korvus Wang



Dengue virus serotype 3 virus NS2B-NS3 fused protease expression and purification of a construct suitable for biochemical assay.
ReagentDENV-3 Philippines/H87/1956 NS2B-NS3 protease fusion (non-cleavable linker)addgeneCatalog #228651


Protocol
Dengue virus serotype 3 NS2B-NS3 protease fusion construct using parallel rapid protein expression and purification: 100 mL cultures
NAME

Dengue virus serotype 3 NS2B-NS3 protease fusion construct using parallel rapid protein expression and purification: 100 mL cultures

CREATED BY
Korvus Wang


Dengue virus serotype 4 virus NS2B-NS3 fused protease expression and purification of a construct suitable for biochemical assay.ReagentDENV-4 strain H241 NS2B-NS3 protease fusion (non-cleavable linker)addgeneCatalog #228652


Protocol
NAME

Dengue virus serotype 4 NS2B-NS3 protease fusion construct: 100 mL cultures

CREATED BY
Korvus Wang


Determine which Flavivirus is needed and prepare solutions
Prepare solutions based on the materials section.
ABCDE
ReagentStockLoaded into CERTUSFinal in assay plateunits
Choice of NS2B-NS3 Enzyme Protein
DENV NS2B-NS3217000200100nM
ZIKV NS2B-NS3225000200100nM
WNV NS2B-NS3222000200100nM
Choice of Viral Substrate
WNV/ZIKV Substrate10000 105µM
DENV Substrate10000 105µM
Assay buffer
Tris pH=8.510002020mM
Glycerol501010%
Triton100.010.01%

Prepare 384-well Plate for experiment
2h 31m
PRIME the dispenser with Assay Buffer by selecting the PRIME button 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 enzyme, no compounds)
EMPTY the dispenser tubes.
  • Discard Assay Buffer discharged from the dispenser tubes.
PRIME the dispenser with Concentration200 nanomolar (nM) Flavivirus NS2B/NS3 by selecting the PRIME button until the tubes are filled completely.
DISPENSE Amount10 µL Concentration200 nanomolar (nM) Flavivirus NS2B/NS3 to columns 2 through 22 and column 24.
Note:
  • Be sure to cycle dispensing on an empty plate first, to detect and avoid dispensing issues.
  • Concentration200 nanomolar (nM) Flavivirus NS2B/NS3 is two times the assay concentration. The final concentration of the Flavivirus NS2B/NS3 in the assay plate is Concentration100 nanomolar (nM) .
  • Columns 2 and 24 are neutral control columns (Contain: Enzyme + substrate + DMSO, no compounds)

EMPTY the dispenser tubes .
  • Discard the Concentration200 nanomolar (nM) Flavivirus NS2B/NS3 discharged from the tubes
CENTRIFUGE plate Centrifigation1500 rpm, 00:01:00 to remove bubbles

1m
INCUBATE plate Duration02:00:00 at TemperatureRoom temperature
⚠ Make sure the plate is protected from light!

During Incubation: wash and prepare the dispenser to the next step
2h
PRIME the dispenser with Amount200 µL Concentration10 micromolar (µM) Flavivirus Substrate
  • Note: Be sure to cycle dispensing on an empty plate first, to detect and avoid dispensing issues.
DISPENSE Amount10 µL of Concentration10 micromolar (µM) Flavivirus Substrate to Columns 1-24 (the full plate).
  • Note: Concentration10 micromolar (µM) Flavivirus Substrate is two times the assay concentration. The final concentration of the Flavivirus Substrate in the assay plate is Concentration5 micromolar (µM) .
CENTRIFUGE plate Centrifigation1500 rpm, Room temperature, 00:01:00 to remove bubbles

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

Recommended: Clean the dispenser during incubation
30m
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) via the "Flavivirus protocol" on the PHERAstar FS Control Software.
  • Software is a standard Fluorescence Assay set for Optimal excitation wavelength 360 nm, emission wavelength 470 nm, and a Gain of 200.
Equipment
PHERAstar FS
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
0471B0001A
SKU
https://www.bmglabtech.com/en/pherastar-fsx/?utm_term=pherastar%20plate%20reader&utm_campaign=usa.roi.products&utm_source=adwords&utm_medium&gclid=Cj0KCQjw8qmhBhClARIsANAtbodGRjigZtEYwcoMXUtxsLn25xp4gjKra3ZNt9jLh9-FwOoFR_5EUHUaAlkREALw_wcB
LINK

Expected result
gain 300 should yield ~20,000 RFU in full reaction; ~7000 RFU in Buffer control