Aug 04, 2025

Public workspaceAssay for screening of compounds that inhibit enzymatic activity of DENV-2 NS2B-NS3 protease

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg32m87v25/v1
  • Kateryna Horbatok1,2,
  • Oleksandr Zhadovets1,2
  • 1Enamine Ltd;
  • 2ASAP Discovery Consortium
  • ASAP Discovery
Nick Lynch
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg32m87v25/v1
Protocol Citation: Kateryna Horbatok, Oleksandr Zhadovets 2025. Assay for screening of compounds that inhibit enzymatic activity of DENV-2 NS2B-NS3 protease. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32m87v25/v1
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: October 07, 2024
Last Modified: August 04, 2025
Protocol Integer ID: 109692
Keywords: Fluorescence (RFU), Robust Coefficient of Variation (RCV), Protease, DENV-2, enzymatic, NS2B-NS3 protease, inhibitors, enzymatic activity of the dengue virus protease, dengue virus protease, enzymatic assay, direct quantification of enzyme activity, quantification of enzyme activity, enzyme activity, enzymatic activity, cleavage by the enzyme, ns3 protease, enzyme, assay, screening of compound, ns3 protease this protocol, fluorescence detection
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 protocol describes the enzymatic assay used to identify compounds that inhibit enzymatic activity of the dengue virus protease (DENV-2 NS2B-NS3 protease). Enzymatic assay is based on the conversion of substrate into product, which is used for quantification of enzyme activity. In case of using fluorogenic substrates, cleavage by the enzyme releases a fluorescent fragment, enabling the direct quantification of enzyme activity via fluorescence detection. This assay is run as single point and dose response.

Guidelines
Introduction:
AB
Target nameDENV-2 NS2B-NS3
Project AimTo identify compounds that inhibit enzymatic activity of the dengue virus protease DENV-2 NS2B-NS3.
Assay PrincipleUnder standard conditions the enzyme cleaves its substrate (Bz-Nle-Lys-Lys-Arg-AMC) with the release of a fluorogenic moiety 7-AMC, that was quenched before. In the presence of compounds that inhibit activity of the enzyme, substrate is not cleaved which results in a low fluorescent signal.
Assay readoutFluorescence (RFU)
Summary of Assay Conditions:

Figure 1. Plate loading for dose response format

Figure 2. Plate loading for single point format

AB
DescriptionReaction volume composition*, 10 µL (before 10 µL of stop-reaction reagent is added)
Negative control (100% of inhibition)0.5% of DMSO, 5 µM of Substrate
Positive control (0% of inhibition)0.5% of DMSO, 25 nM of DENV-2, 5 µM of Substrate
Reference 1 controlASAP-0000570, 3-fold dilution, 25 – 0.1 µM, 6 points (n = 2), 25 nM of DENV-2, 5 µM of Substrate
Reference 2 controlASAP-0015081, 3-fold dilution, 25 – 0.1 µM, 6 points (n = 2), 25 nM of DENV-2, 5 µM of Substrate
CompoundsCompounds in dose response format, 3-fold dilution, 100 – 0.4 µM, 6 points (n = 2)​, 25 nM of DENV-2, 5 µM of Substrate Compounds in single point format, at 4 concentrations 200, 150, 100, 50 µM (n = 1)​, 25 nM of DENV-2, 5 µM of Substrate
*The final concentrations of compounds, enzyme and substrate are calculated considering that enzymatic reaction volume is 10 µl, while total volume is 20 µl after the stop-reagent is added.

Materials
Tools/Equipment required:

ABC
№Tool NameTool Source
1. Manual single channel pipettes Finnpipette 1-10 µL Proline® Plus 10-100 µL MLine® 20-200 µL IKA PETTE 100-1000 µL Thermo Scientific Sartorius Sartorius IKA
2. Multichannel electronic pipettes (E1-ClipTip) 1-30 µL 2-125 µL ThermoFisher
3. Multidrop CombiThermoFisher
4. DLAB Levo plus (for stripettes)DLAB
5. Biomek Span-8Beckman Coulter
6. Labcyte Echo 650Beckman Coulter
7. SpectraMax ParadigmMolecular Devices
8. ASSAB  incubatorAssab
9. accuSpin 3 centrifugeFisher Scientific
Consumables:

ABCD
№Disposable NameDisposable SourceCatalogue Number
1. 384-well, round bottom, small volume, non-binding, black plateCorning4514
2. Multichannel Pipette tips (F1-ClipTip) 30 µL 125 µL Thermo Scientific94420103 94420153
3. Stripettes 5 mL 10 mL 25 mL 50 mLThermo Scientific11829660 11839660 11517752 11537752
4. 15 mL Polypropylene falcon tubesSente-LabSL50352/SG
5. 50 mL Polypropylene falcon tubesSente-LabSL65351/SG
6. Manual Pipette tips 30 µL 200 µL 1000 µLMatrix Biosigma Sente-Lab7611 597799 SL96153
7. Small tube metal tip dispensing cassette SN 837823692Thermo Scientific24073295
8. Silverseal Sealer, AluminiumGreiner676090
9. SealPlate filmExcel ScientificZ369659-100EA
10. Axygen® Ultra-Clear, Pressure-Sensitive Sealing Film for Real-Time PCRCorningUC-500
Reagent384-well Low Volume Black Round Bottom Polystyrene NBS Microplate, 10 per Bag, without Lid, NonsteriCorningCatalog #4514
ReagentClipTip™ Filtered 384-Format Pipette TipsThermo Fisher ScientificCatalog #94420103
ReagentClipTip™ Filtered 384-Format Pipette TipsThermo Fisher ScientificCatalog #94420153
Reagent Small tube metal tip dispensing cassetteThermo Fisher ScientificCatalog #24073295
ReagentSILVERSEAL SEALER, ALUMINIUMgreiner bio-oneCatalog #676090
ReagentAxygen® 70 µm Ultra Clear Pressure Sensitive Sealing Film for Real Time PCR, NonsterileCorningCatalog #UC-500


Reagents:
ABCDEF
№Reagent NameStock ConcentrationReagent SourceCatalogue No.Storage Conditions
1. DENV-2 NS2B-NS3 (DENV-2)217 µMClient Supplied--80°C
2. Bz-Nle-Lys-Lys-Arg-AMC (Substrate)-Cayman Chemicals27710-20°C
3. ASAP-0000570 (Reference 1)20 mMClient Supplied--80°C
4. ASAP-0015081 (Reference 2)20 mMClient Supplied--80°C
5. Tris hydrochloride (Tris-HCl)-Bio BasicTB0103RT
6. Glycerol100%ALLHIM2905450000RT
7. Triton 100%Sigma-Aldrich93443-100MLRT
8. Dimethyl sulfoxide (DMSO)100%Honeywell34869-2.5LRT
9. Trisodium citrate dihydrate (Stop-reaction reagent)-Klebrig1361407143RT
10. Tween 20100%PanReac AppliChem142312.2RT
ReagentBz-Nle-KRR-AMC (hydrochloride)Cayman Chemical CompanyCatalog #27710
ReagentTris hydrochlorideBio Basic Inc.Catalog #TB0103.SIZE.250G
ReagentTriton X-100 SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #93443-100ML
ReagentDimethyl sulfoxideHoneywell FlukaCatalog #34869
ReagentTween ® 20Panreac AppliChemCatalog #142312.1611


Standard Reagent Formulations:
ABCDEF
№Prepared Reagent NameComponent NamePrepared Reagent VolumeComponent QuantityStorage
1. 1 M Tris-HCl (adjusted to pH 8.5)Tris hydrochloride MQ H2O10 mL10 g 10 mL4°C
2. 10 mM SubstrateBz-Nle-Lys-Lys-Arg-AMC DMSO40x15 µL5 mg 600 µL-20°C
3. 1 M Trisodium citrate dihydrate pH 4.0Trisodium citrate dihydrate HCl MQ H2O20 mL5.88 g 3 mL 17 mLRT
4. 1% Tween 20100% Tween 20 MQ H2O2 L20 mL 1.98 LRT
5. 10% Triton100% Triton MQ H2O10 ml1 ml 9 mlRT
Daily Reagent Formulations*:
ABCDEF
№Prepared Reagent NameComponentsComponent QuantityFinal Concentration in solutionPrepared Reagent Volume
1. Assay Buffer1 M Tris-HCl pH 8.5 100% Glycerol 10% Triton MQ H2O800 µL 4 mL 40 µL 35.16 mL20 mM 10% 0.01% -40 mL
2. 21.7 µM DENV-2 217 µM DENV-2 Assay Buffer3 µL 27 µL21.7 µM (Intermediate stock)30 µL
3. 50 nM DENV-2 (2x solution)21.7 µM DENV-2 Assay Buffer29 µL 12.557 mL50 nM -12.586 mL
4. 10 µM Substrate (2x solution)10 mM Substrate Assay Buffer13.5 µL 13.486 mL10 µM -13.5 mL
5. 500 mM Stop-reaction reagent (2x solution)1 M Trisodium citrate dihydrate solution MQ H2O11 mL 11 mL500 mM -22 mL
*All calculations shown here are for a 4-plate HTS run. This consists of 4 assay plates with a dummy plate at the beginning of the dispense and dead volume of dispensing equipment.



Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Assay plate preparation
Plate loading for dose response format.

  • Prepare DMSO, tested compounds (a total of 24 compounds per plate) and reference compounds by Biomek Span-8 and printed onto assay-ready plates (assay plate — Corning #4514) using the Labcyte Echo 650.
  • Reference controls are formatted in row B in the following order:
  • Reference compound 1 – B1-B12 – 50 nL of ASAP-0000570 in dose response format, 3-fold dilution, Concentration5 millimolar (mM) – Concentration0.02 millimolar (mM) , 6 points (n = 2).
  • Reference compound 2 – B13-B24 – 50 nL of ASAP-0015081 in dose response format, 3-fold dilution, Concentration5 millimolar (mM) – Concentration0.02 millimolar (mM) , 6 points (n = 2).
  • DMSO is formatted in rows A, O and P – 50 nL in each well.
  • Compounds are formatted in rows C-N – 50 nL in each well in dose response format, 3-fold dilution, Concentration20 millimolar (mM) – Concentration0.08 millimolar (mM) , 6 points (n = 2).

Figure 1. Plate map for dose-response scheme prepared by liquid handling department.

Protein expression and purification
The assays performed here used the following protein.
Assay plate preparation
Plate loading for single point format.

  • Prepare DMSO, tested compounds (a total of 72 compounds per plate) and reference controls by Biomek Span-8 and printed onto assay-ready plates (assay plate — Corning #4514) using the Labcyte Echo 650.
  • Reference controls are formatted in row B in the following order:
  • Reference compound 1 – B1-B12 – 50 nL of ASAP-0000570 in dose response format, 3-fold dilution, Concentration5 millimolar (mM) – Concentration0.02 millimolar (mM) , 6 points (n = 2).
  • Reference compound 2 – B13-B24 – 50 nL of ASAP-0015081 in dose response format, 3-fold dilution, Concentration5 millimolar (mM) – Concentration0.02 millimolar (mM) , 6 points (n = 2).
  • DMSO is formatted in rows A, O and P – 50 nL in each well.


Format the compounds in rows C-N in the following order:

  • Compounds 1-24 – C1-F24 – 50 nL in each well at 4 concentrations 40, 30, 20, 10 mM (n = 1).
  • Compounds 25-48 – G1-J24 – 50 nL in each well at 4 concentrations 40, 30, 20, 10 mM (n = 1).
  • Compounds 49-72 – K1-N24 – 50 nL in each well at 4 concentrations 40, 30, 20, 10 mM (n = 1).

Figure 2. Plate map for single point scheme prepared by liquid handling department.

Seal the plates with Silverseal Sealer, Aluminium (no heating is applied) and store at Temperature-20 °C . On the day of the assay, remove the assay plates from the Temperature-20 °C freezer and left to warm up to TemperatureRoom temperature .
Temperature
After liquid handling dispensing and before the start of assay procedures plates must be centrifuged at Centrifigation1000 rpm, 00:01:00 .
1m
Centrifigation
Further assay procedures are identical for both screening formats: dose-response and single point, and summarized in "Assay performance" section.
Assay performance
2h 43m
Multidrop Combi settings:

  • Plate: 384 standard (15 mm)
  • Cassette type: Small tube dispensing cassette
  • Dispense volume: Amount5 µL / Amount10 µL
  • Predispense volume: Amount50 µL
  • Dispense speed: medium
  • Dispense direction: by rows

Note
Multidrop Combi small tube dispensing cassette should be checked before screening procedures using gravimetric accuracy verification and photometric precision verification, in accordance with the Thermo Scientific Multidrop Combi User Manual. Multidrop Combi cassette must have dispensing accuracy ± 5% & RCV ≤ 3% for each channel, and no obvious dispensing patterns.

Multidrop Combi dispense #1.
  • Fill the tubes with assay buffer, preincubate for Duration00:02:00 , then empty the tubes.
  • Fill tubes A-G with DENV-2 2x solution and tube H with assay buffer, prime ~Amount50 µL , preincubate the solutions in the tubes for Duration00:02:00 . Then empty the tubes and mix the solution in the falcon gently.
  • Fill the tubes with the working solution another time, prime ~Amount50 µL . Then dispense Amount5 µL to half of the dummy plate, followed by the assay plates (fig 3.).
  • Seal the plates with SealPlate film and centrifuge at Centrifigation1000 rpm, 00:01:00 and then left to incubate for Duration02:00:00 at Temperature25 °C in the incubator, while Multidrop Combi is flushed through with 1% Tween 20 and MQ H2O before the next dispense.

Figure 3. Multidrop Combi dispense #1. Rows A-N are dispensed with 5 µL of 50 nM DENV-2, Rows O-P are dispensed with 5 µL of assay buffer.

2h 5m
Incubation
Centrifigation
Mix
Multidrop Combi dispense #2.

  • Fill the tubes with assay buffer, preincubate for Duration00:02:00 then empty the tubes.
  • Fill the tubes with Substrate 2x solution, prime ~Amount50 µL , preincubate the Substrate in the tubes for Duration00:02:00 . Then empty the tubes and mix the solution in the falcon gently.
  • Fill the tubes with the working solution another time, prime ~Amount50 µL . Then dispense Amount5 µL to half of the dummy plate, followed by the assay plates (fig 4.).
  • Seal the plates with SealPlate film and centrifuge at Centrifigation1000 rpm, 00:01:00 and then left to incubate for Duration00:30:00 at Temperature25 °C in the incubator in the dark, while Multidrop Combi is flushed through with 1% Tween 20 and MQ H2O before the next dispense.

Figure 4. Multidrop Combi dispense #2. Full plate is dispensed with 5 µL of Substrate.

35m
Incubation
Centrifigation
Mix
Multidrop Combi dispense #3.

  • Fill the tubes with Stop-reaction reagent 2x solution, prime ~Amount50 µL , preincubate the solution in the tubes for Duration00:02:00 . Then empty the tubes and mix gently the solution in the falcon.
  • Fill the tubes with the working solution another time, prime ~Amount50 µL . Then dispense Amount10 µL to half of the dummy plate, followed by the assay plates (fig 5.).
  • Seal the plates with Axygen films and centrifuge at Centrifigation1000 rpm, 00:01:00 , while Multidrop Combi is flushed through with 1% Tween 20 and MQ H2O before shutdown.

Figure 5. Multidrop Combi dispense #3. Full plate is dispensed with 10 µL of 500 mM Stop-reaction reagent.

Read the plates using SpectraMax Paradigm.

3m
Incubation
Centrifigation
Mix
Reader settings:

  • Mode: FL
  • Ex./Em.: 360/460
  • Integration Time: 70 ms
  • Read Order: by columns
  • Read Temperature: TemperatureRoom temperature

Temperature
Data analysis
Export the raw data in .xls files as RFU signals (in plate format).
The data is analyzed using specifically designed scripts in Python.
Parameters for QC:
  • Robust Signal to Background (RS/B) = median of high control (0% of inhibition) / median of low control (100% inhibition).
  • Plate Robust Z’-factor (RZ’) = 1 – (3*(RSD of high control (0% of inhibition) + RSD of low control (100% inhibition))/(median of high control (0% of inhibition) – median of low control (100% inhibition)).
  • Robust Coefficient of Variation (RCV) = RSD of data / median of data*100%.

Note
NB: Plate must pass criteria:
RZ’ ≥ 0.5
RS/B ≥ 2
RCV < 20%
No obvious liquid handling-related patterns

Report the compound response as % inhibition based on 0% and 100% inhibition controls:
  • Compound response (% inhibition by median) = (median of high control (0% of inhibition) - data point median)/(median of high control (0% of inhibition) – median of negative control (100% of inhibition)) * 100.

Acknowledgements
Grateful for Haim Barr and Noa Lahav for sharing their protocol developed at Weizmann Institute of Science as part of ASAP Discovery Consortium