Assay for screening of compounds that inhibit enzymatic activity of ZIKV NS2B-NS3 protease

Oleksandr Zhadovets; Maksym Pavliuk

Aug 04, 2025

Assay for screening of compounds that inhibit enzymatic activity of ZIKV NS2B-NS3 protease

DOI

https://dx.doi.org/10.17504/protocols.io.36wgqn273gk5/v1

Oleksandr Zhadovets^1,2,
Maksym Pavliuk^1,2

¹Enamine Ltd;
²ASAP Discovery Consortium

ASAP Discovery

Nick Lynch

Curlew Research, ASAP Discovery Consortium

DOI: https://dx.doi.org/10.17504/protocols.io.36wgqn273gk5/v1

Protocol Citation: Oleksandr Zhadovets, Maksym Pavliuk 2025. Assay for screening of compounds that inhibit enzymatic activity of ZIKV NS2B-NS3 protease. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn273gk5/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 14, 2024

Last Modified: August 04, 2025

Protocol Integer ID: 110118

Keywords: Inhibitors, Fluorescence (RFU), Gravimetric accuracy, ZIKV, NS2B-NS3 protease, Robust Coefficient of Variation (RCV), enzymatic activity of the dengue virus protease, dengue virus protease, enzymatic activity of zikv ns2b, enzymatic assay, direct quantification of enzyme activity, quantification of enzyme activity, enzyme activity, cleavage by the enzyme, enzyme, ns3 protease, enzymatic activity, dengue, screening of compound, ns3 protease this protocol, fluorescence detection, zikv ns2b, fluorogenic substrate

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 (ZIKV 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.

Guidelines

Introduction:

AB
Target nameZIKV NS2B-NS3
Project AimTo identify the compounds that inhibit enzymatic activity of the ZIKV NS2B-NS3 protease
Assay PrincipleEnzymatic 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. In this assay ZIKV NS2B/NS3 cleaves its substrate (Boc-Gly-Arg-Arg-AMC) with the release of a fluorogenic moiety 7-AMC.
Assay readoutFluorescence (RFU)
 
Summary of Assay Conditions:

Figure 1. Plate loading for dose response 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, 50 nM of ZIKV NS2B/NS3, 5 µM of Substrate
Reference 1 controlASAP-0015081, 2-fold dilution, 50-0.02 µM, 12 points (n=2),  50 nM of ZIKV NS2B/NS3, 5 µM of Substrate
Reference 2 controlASAP-0000570, 2-fold dilution, 100-0.05 µM, 12 points (n=2), 50 nM of ZIKV NS2B/NS3, 5 µM of Substrate
CompoundsCompounds in dose response format, 2-fold dilution, 25 – 0.01 µM, 12 points (n = 2)​, 50 nM of ZIKV NS2B/NS3, 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-reaction 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 Combi nLThermoFisher
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 Scientific                                                     94420103                                 94420153
3.       
   Stripettes                                                               5 mL                                                                  10 mL                                                                 25 mL                                                                  50 mLThermo Scientific                                                    11829660                                 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 µL                                               Matrix                                       Biosigma                                 Sente-Lab                                                  7611                                       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
11.      Minisart® NY25 Syringe Filter, 0.2 µm Polyamide (Nylon)Sartorius17845-Q
 384-well Low Volume Black Round Bottom Polystyrene NBS Microplate, 10 per Bag, without Lid, NonsteriCorningCatalog #4514  
ClipTip™ Filtered 384-Format Pipette TipsThermo Fisher ScientificCatalog #94420103  
ClipTip™ Filtered 384-Format Pipette TipsThermo Fisher ScientificCatalog #94420153  
 Small tube metal tip dispensing cassetteThermo Fisher ScientificCatalog #24073295  
SILVERSEAL SEALER, ALUMINIUMgreiner bio-oneCatalog #676090  
Axygen® 70 µm Ultra Clear Pressure Sensitive Sealing Film for Real Time PCR, NonsterileCorningCatalog #UC-500  
Minisart® NY25 Syringe Filter, 0.2 µm Polyamide (Nylon)SartoriusCatalog #17845-Q  


Reagents:
 
ABCDEF
№Reagent NameStock ConcentrationReagent SourceCatalogue No.Storage Conditions
1.        ZIKV NS2B/NS3 (ZIKV)225 µMClient Supplied--80°C
2.        Boc-Gly-Arg-Arg-AMC (Substrate)-BiosynthFB110553-20°C
3.        ASAP-0015081 (Reference 1)20 mMClient Supplied--80°C
4.        ASAP-0000570 (Reference 2)20 mMClient Supplied--80°C
5.        Tris hydrochloride (Tris-HCl)-Bio BasicTB0103RT
6.        Glycerol100%ALLHIM2905450000RT
7.        Triton100%Sigma-Aldrich93443-100MLRT
8.        Dimethyl sulfoxide (DMSO)100%Honeywell34869-2.5LRT
9.        Sodium acetate (Stop-reaction reagent)-Klebrig127-09-3RT
10.     Sodium dodecyl sulfate (SDS)-Biotech151-21-3RT
Boc-Gly-Arg-Arg-AMC acetate saltBiosynthCatalog #FB110553  
Tris hydrochlorideBio Basic Inc.Catalog #TB0103.SIZE.250G  
Triton X-100 SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #93443-100ML  
Dimethyl sulfoxideHoneywell FlukaCatalog #34869  


Standard Reagent Formulations:
 
ABCDEF
№Prepared Reagent NameComponent NamePrepared Reagent VolumeComponent                    QuantityStorage
1.       
   1 M Tris-HCl                                       (adjusted to pH 8.5)Tris hydrochloride                      MQ H2O15 mL15 g                                 15 mL4°C
2.       
   10 mM SubstrateBoc-Gly-Arg-Arg-AMC DMSO15x10 µL                                         15x15 µL                                       20x20 µL5 mg                                775 µL-20°C
3.       
   20% Sodium acetate                   (adjusted to pH 4.0)Sodium acetate                             MQ H2O40 mL                                                   8 g                                    32 mLRT
4.       
   10% Triton100% Triton                                   MQ H2O10 ml1 ml                                          9 mlRT
5.       
   1% SDSSodium dodecyl sulfate                 MQ H2O2 L20 g                                  1.98 LRT
 
Daily Reagent Formulations*:
 
ABCDEF
№Prepared Reagent NameComponentsComponent        QuantityConcentration in solutionPrepared Reagent Volume
1.       
   Assay Buffer1 M Tris-HCl pH 8.5                 100% Glycerol                         10% Triton                              MQ H2O1.2 mL                                          6 mL                                              60 µL                                            52.74 mL20 mM                                                   10%                                               0.01%                                                 -60 mL
2.       
   22.5 µM ZIKV225 µM ZIKV                            Assay Buffer                         6 µL                                              54 µL22.5 µM                                           (Intermediate stock) 60 µL
3.       
   100 nM ZIKV                                                            (2x solution)22.5 µM ZIKV                              Assay Buffer55 µL                                                   12.320 mL100 nM                                        -12.375 mL
4.       
   10 µM Substrate                                            (2x solution)10 mM Substrate                       Assay Buffer12.5 µL                                      12.488 mL10 µM                                               -12.5 mL
5.       
   10% Stop-reaction reagent (2x solution)                                          MQ H2O20% Sodium acetate solution11 mL                                            11 mL10%                                                   -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.
 

Safety warnings

Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.

Protein expression and purification

Assay plate preparation

Plate loading for dose response format

Prepare DMSO, tested compounds (a total of 12 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 and O in the following order: 
Reference control 1 – B1-B12, O13-O24 – 50 nL of ASAP-0015081 in dose response format, 2-fold dilution, 10-0.005 mM, 12 points (n=2).
Reference control 2 – B13-B24, O1-O12 – 50 nL of ASAP-0000570 in dose response format, 2-fold dilution, 20-0.01 mM, 12 points (n=2).
DMSO is formatted in rows A and P – 50 nL in each well.  
Compounds are formatted in rows C-N – 50 nL in each well in dose response format, 2-fold dilution, 5-0.0025 mM, 12 points (n = 2).

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

Seal the plates with Silverseal Sealer, Aluminium (no heating is applied) and store at -20 °C  . On the day of the assay, remove assay plates from the -20°C freezer and left to warm up to Room temperature  . 

After liquid handling dispensing and before the start of assay procedures plates must be centrifuged at 1000 rpm, 00:01:00 .

Assay performance

2h 49m

Multidrop Combi nL settings:

Plate: 384 Corning lvol rnd
Dispense volume: 5 µL   / 10 µL  
Predispense volume: 50 µL  
Dispense direction: by columns
Speed: 3 (max is 5)
Correction factor: 1.031

Note
Multidrop Combi nL should be checked before screening procedures using gravimetric accuracy verification and photometric precision verification, in accordance with the Thermo Scientific Multidrop Combi nL User Manual. Multidrop Combi nL must have dispensing accuracy ± 2% & RCV ≤ 4% for each channel, and no obvious dispensing patterns.

Multidrop Combi nL dispense #1. 

Fill the tubes with freshly prepared filtered assay buffer, prime ~50 µL  , preincubate for 00:05:00  . Dispense  5 µL   of assay buffer to half of the dummy plate, followed by the assay plates (fig 2.). 
Cover the plates with plate lids and left at the bench before next dispense.

Figure 2. Multidrop Combi nL dispense #1. Row P is dispensed with 5 µL of assay buffer.

Multidrop Combi nL dispense #2. 

Immediately after the first dispense empty the tubes, filled with 100 nM ZIKV (2x solution) and primed ~50 µL  . 
Preincubate the solution in the tubes for 00:02:00  . Dispense 5 µL    to half of the dummy plate, followed by the assay plates (fig 3.). 
Seal the plates with SealPlate film and centrifuged at 1000 rpm, 00:01:00  and then left to preincubate for 02:00:00   at 25 °C   in the incubator, while Multidrop Combi nL is flushed through with 45 mL   of 1% SDS and 90 mL   of MQ H2O before the next dispense.

Figure 3. Multidrop Combi nL dispense #2. Rows A-O are dispensed with 5 µL of 100 nM ZIKV (2x solution).

2h 3m

Multidrop Combi nL dispense #3. 

Fill the tubes with assay buffer, preincubate for 00:05:00  , then empty the tubes. 
Fill the tubes with 10 micromolar (µM)   Substrate (2x solution), prime ~50 µL  , preincubate the Substrate in the tubes for 00:02:00  . Dispense 5 µL   to half of the dummy plate, followed by the assay plates (fig 4.). 
Seal the plates with SealPlate film and centrifuged at 1000 rpm, 00:01:00  and then left to incubate for 00:30:00   at 25 °C   in the incubator (protected from light), while Multidrop Combi nL is flushed through with 45 mL   of 1% SDS and 90 mL   of MQ H2O before the next dispense.

Figure 4. Multidrop Combi nL dispense #3. Full plate is dispensed with 5 µL of 10 µM Substrate (2x solution)

38m

Multidrop Combi nL dispense #4. 

Fill the tubes with 10% Stop-reaction reagent (2x solution), prime ~50 µL  , preincubate the solution in the tubes for 00:02:00  . Dispense 10 µL   to half of the dummy plate, followed by the 4 assay plates (fig 5.). 
Seal the plates with Axygen films and centrifuged at 1000 rpm, 00:01:00 , while Multidrop Combi nL is flushed through with 45 mL   of 1% SDS and 90 mL   of MQ H2O.

Figure 5. Multidrop Combi nL dispense #4. Full plate is dispensed with 10 µL of 10% Stop-reaction reagent (2x solution).

Read the plates using SpectraMax Paradigm.

Reader settings:

Mode: FL
Ex./Em.: 360/460
Integration Time: 140 ms
Read Order: by columns
Read Temperature: Room 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%.

The files with raw and analyzed data are uploaded to CDD Vault Protocol (ZIKV-NS2B-NS3_fluorescence-dose-response_bienta) for further analysis.

Protocol references

http://dx.doi.org/10.17504/protocols.io.q26g7yebkgwz/v3

Acknowledgements

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

	A	B
	Target name	ZIKV NS2B-NS3
	Project Aim	To identify the compounds that inhibit enzymatic activity of the ZIKV NS2B-NS3 protease
	Assay Principle	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. In this assay ZIKV NS2B/NS3 cleaves its substrate (Boc-Gly-Arg-Arg-AMC) with the release of a fluorogenic moiety 7-AMC.
	Assay readout	Fluorescence (RFU)

	A	B
	Description	Reaction 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, 50 nM of ZIKV NS2B/NS3, 5 µM of Substrate
	Reference 1 control	ASAP-0015081, 2-fold dilution, 50-0.02 µM, 12 points (n=2), 50 nM of ZIKV NS2B/NS3, 5 µM of Substrate
	Reference 2 control	ASAP-0000570, 2-fold dilution, 100-0.05 µM, 12 points (n=2), 50 nM of ZIKV NS2B/NS3, 5 µM of Substrate
	Compounds	Compounds in dose response format, 2-fold dilution, 25 – 0.01 µM, 12 points (n = 2), 50 nM of ZIKV NS2B/NS3, 5 µM of Substrate

A	B	C
№	Tool Name	Tool 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 Combi nL	ThermoFisher
4.	DLAB Levo plus (for stripettes)	DLAB
5.	Biomek Span-8	Beckman Coulter
6.	Labcyte Echo 650	Beckman Coulter
7.	SpectraMax Paradigm	Molecular Devices
8.	ASSAB incubator	Assab
9.	accuSpin 3 centrifuge	Fisher Scientific

A	B	C	D
№	Disposable Name	Disposable Source	Catalogue Number
1.	384-well, round bottom, small volume, non-binding, black plate	Corning	4514
2.	Multichannel Pipette tips (F1-ClipTip) 30 µL 125 µL	Thermo Scientific	94420103 94420153
3.	Stripettes 5 mL 10 mL 25 mL 50 mL	Thermo Scientific	11829660 11839660 11517752 11537752
4.	15 mL Polypropylene falcon tubes	Sente-Lab	SL50352/SG
5.	50 mL Polypropylene falcon tubes	Sente-Lab	SL65351/SG
6.	Manual Pipette tips 30 µL 200 µL 1000 µL	Matrix Biosigma Sente-Lab	7611 597799 SL96153
7.	Small tube metal tip dispensing cassette SN 837823692	Thermo Scientific	24073295
8.	Silverseal Sealer, Aluminium	Greiner	676090
9.	SealPlate film	Excel Scientific	Z369659-100EA
10.	Axygen® Ultra-Clear, Pressure-Sensitive Sealing Film for Real-Time PCR	Corning	UC-500
11.	Minisart® NY25 Syringe Filter, 0.2 µm Polyamide (Nylon)	Sartorius	17845-Q

A	B	C	D	E	F
№	Reagent Name	Stock Concentration	Reagent Source	Catalogue No.	Storage Conditions
1.	ZIKV NS2B/NS3 (ZIKV)	225 µM	Client Supplied	-	-80°C
2.	Boc-Gly-Arg-Arg-AMC (Substrate)	-	Biosynth	FB110553	-20°C
3.	ASAP-0015081 (Reference 1)	20 mM	Client Supplied	-	-80°C
4.	ASAP-0000570 (Reference 2)	20 mM	Client Supplied	-	-80°C
5.	Tris hydrochloride (Tris-HCl)	-	Bio Basic	TB0103	RT
6.	Glycerol	100%	ALLHIM	2905450000	RT
7.	Triton	100%	Sigma-Aldrich	93443-100ML	RT
8.	Dimethyl sulfoxide (DMSO)	100%	Honeywell	34869-2.5L	RT
9.	Sodium acetate (Stop-reaction reagent)	-	Klebrig	127-09-3	RT
10.	Sodium dodecyl sulfate (SDS)	-	Biotech	151-21-3	RT

A	B	C	D	E	F
№	Prepared Reagent Name	Component Name	Prepared Reagent Volume	Component Quantity	Storage
1.	1 M Tris-HCl (adjusted to pH 8.5)	Tris hydrochloride MQ H2O	15 mL	15 g 15 mL	4°C
2.	10 mM Substrate	Boc-Gly-Arg-Arg-AMC DMSO	15x10 µL 15x15 µL 20x20 µL	5 mg 775 µL	-20°C
3.	20% Sodium acetate (adjusted to pH 4.0)	Sodium acetate MQ H2O	40 mL	8 g 32 mL	RT
4.	10% Triton	100% Triton MQ H2O	10 ml	1 ml 9 ml	RT
5.	1% SDS	Sodium dodecyl sulfate MQ H2O	2 L	20 g 1.98 L	RT

A	B	C	D	E	F
№	Prepared Reagent Name	Components	Component Quantity	Concentration in solution	Prepared Reagent Volume
1.	Assay Buffer	1 M Tris-HCl pH 8.5 100% Glycerol 10% Triton MQ H2O	1.2 mL 6 mL 60 µL 52.74 mL	20 mM 10% 0.01% -	60 mL
2.	22.5 µM ZIKV	225 µM ZIKV Assay Buffer	6 µL 54 µL	22.5 µM (Intermediate stock)	60 µL
3.	100 nM ZIKV (2x solution)	22.5 µM ZIKV Assay Buffer	55 µL 12.320 mL	100 nM -	12.375 mL
4.	10 µM Substrate (2x solution)	10 mM Substrate Assay Buffer	12.5 µL 12.488 mL	10 µM -	12.5 mL

5.	10% Stop-reaction reagent (2x solution) MQ H2O	20% Sodium acetate solution	11 mL 11 mL	10% -	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.