Aug 10, 2025
Assay for Screening of Compounds that Inhibit Enzymatic Activity of EV-A71 2Apro
- Diana Alieksieieva1,
- Oleksandr Zhadovets1,
- Oleksii Stroganov1
- 1Enamine Ltd.
- ASAP Discovery
Protocol Citation: Diana Alieksieieva, Oleksandr Zhadovets, Oleksii Stroganov 2025. Assay for Screening of Compounds that Inhibit Enzymatic Activity of EV-A71 2Apro. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9k49l3p/v1
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: November 18, 2024
Last Modified: August 10, 2025
Protocol Integer ID: 112784
Keywords: EV-A71 2Apro, Enzymatic Activity, Assay screening, Enterovirus, biochemical assay, 2A protease, infectious disease, enzymatic activity of the ev, enzymatic activity of ev, biochemical assay, enzymatic activity, a71 2a protease, enzyme, adaptation of an assay, assay, screening of compound, fluorogenic moiety edan, a71 2apro this protocol, 2a protease, fluorescence, increasing fluorescence, a71, compound
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Abstract
This protocol describes a biochemical assay used in screening to identify compounds that inhibit the enzymatic activity of the EV-A71 2A protease.
Under standard conditions, the enzyme cleaves its substrate (DABCYL-RDKITTLGKFGQDE-EDANS), releasing the fluorogenic moiety EDANS, increasing fluorescence. Prior to cleavage, fluorescence is quenched by the DABCYL moiety. In the presence of compounds that inhibit the enzyme’s activity, the substrate remains uncleaved, resulting in a low fluorescent signal.
This enables the determination of % inhibition at a given compound concentration, and allows for subsequent IC₅₀ determination when a dose-response format is applied.
This protocol was established through the transfer and adaptation of an assay originally developed at the Weizmann Institute of Science, as a part of the ASAP Discovery Consortium.
Guidelines
Introduction
| A | B | |
| Target name | EV-A71 2A protease | |
| Project Aim | To identify compounds that inhibit enzymatic activity of the 2A protease of enterovirus A71. | |
| Assay Principle | Under standard conditions the enzyme cleaves its substrate (DABCYL-RDKITTLGKFGQDE-EDANS) with the release of a fluorogenic moiety EDANS. Before EDANS is released, it is quenched by DABCYL moiety on the substrate. In the presence of compounds that inhibit activity of the enzyme, substrate is not cleaved which results in a low fluorescent signal. | |
| Assay readout | Fluorescence (RFU) |
Summary of Assay Conditions
Figure 7. Plate loading for the test of compounds at 2 concentrations.
Figure 8. Plate loading for 6-point dose-response formats (options A and B).
Figure 9. Plate loading for 12-point dose-response format.
| A | B | |
| Description | Reaction volume composition, 10 µL | |
| Negative control (100% of inhibition) | 0.5% DMSO, 6 µM of Substrate | |
| Positive control (0% of inhibition) | 0.5% DMSO, 2 µM of EVA71 2Apro, 6 µM of Substrate | |
| Reference compound | ASAP-0027815, at 4 concentrations, 100 µM, 50 µM, 2xIC50 [2 µM] and IC50 [1 µM] (n = 4), 2 µM of EVA71 2Apro, 6 µM of Substrate | |
| ASAP- 0027815 in dose-response format, 2-fold dilution, 50 – 0.025 µM, 12 points (n = 4), 2 µM of EVA71 2Apro, 6 µM of Substrate | ||
| Compounds | Compounds tested at 2 concentrations, 100 and 50µM, (n = 2), 2 µM of EVA71 2Apro, 6 µM of Substrate | |
| Compounds in 6-point dose-response format, option A: 2-fold dilution, 100 – 3 µM, (n=2), option B: 2-fold dilution, 5 – 0.16 µM, (n=2), 2 µM of EVA71 2Apro, 6 µM of Substrate | ||
| Compounds in 12-point dose-response format, 2-fold dilution, 200 – 0.2 µM & point 150 µM, (n=2), 2 µM of EVA71 2Apro, 6 µM of Substrate |
Materials
Tools/Equipment required
| A | B | |
| Tool Name | Tool Source | |
| Labcyte Echo 650 | Beckman Coulter | |
| Biomek Span-8 | Beckman Coulter | |
| Manual single channel pipettes 0.5-10 µL 10-100 µL 20-200 µL 20-200 µL 100-1000 µL | Thermo Scientific Thermo Scientific Thermo Scientific Sartorius Thermo Scientific | |
| Multichannel electronic pipettes (E1-ClipTip) 1-30 µL 2-125 µL | Thermo Scientific | |
| Multidrop Combi nL | Thermo Scientific | |
| DLAB Levo plus (for stripettes) | DLAB | |
| SpectraMax Paradigm | Molecular Devices | |
| ASSAB incubator | Assab | |
| accuSpin 3 centrifuge | Fisher Scientific |
Consumables
| A | B | C | D | |
| № | Disposable Name | Disposable Source | Catalogue Number | |
| 1 | 384 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 | 170355N 170356N 170357N 170358N | |
| 4 | 15 mL Polypropylene falcon tubes | Sente-Lab | SL50352/SG | |
| 5 | 50 mL Polypropylene falcon tubes | Sente-Lab | SL65351/SG | |
| 6 | Manual Pipette tips 10 µL 200 µL 200 µL 1000 µL | Sente-Lab Sente-Lab Sartorius Sente-Lab | SL96102 SL96062 790202 SL96153 | |
| 7 | Silverseal Sealer, Aluminium | Greiner | 676090 | |
| 9 | SealPlate film | Excel Scientific | Z369659-100EA | |
| 9 | Axygen Ultra-Clear, Pressure-Sensitive Sealing Film for Real-Time PCR | Corning | UC-500 | |
| 10 | Minisart NY25 Syringe Filter, 0.2 µm Polyamide (Nylon) | Sartorius | 17845----------Q | |
| 11 | Syringe 50,0ml, Luer Lock | Medicare | S-3S50,0 |
384-well Low Volume Black Round Bottom Polystyrene NBS Microplate, 10 per Bag, without LidCorningCatalog #4514
ClipTip™ Filtered 384-Format Pipette TipsThermo ScientificCatalog #94420103
ClipTip™ Filtered 384-Format Pipette TipsThermo ScientificCatalog #94420153
Nunc™ Serological PipettesThermo ScientificCatalog #170355N
Nunc™ Serological PipettesThermo ScientificCatalog # 170356N
Nunc™ Serological PipettesThermo ScientificCatalog # 170357N
Nunc™ Serological PipettesThermo ScientificCatalog #170358N
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
| A | B | C | D | E | F | |
| № | Reagent Name | Stock Concentration | Reagent Source | Catalogue No. | Storage Conditions | |
| 1 | EVA71 2Apro | 1.2 mM | Client Supplied | AddGene Catalog #228632 | -80°C | |
| 2 | DABCYL-RDKITTLGKFGQDE-EDANS | - | GenScript | - | -20°C | |
| 3 | ASAP-0027815 Telaprevir | 20 mM | Enamine | EN300-26212550 | -80°C | |
| 4 | Tris hydrochloride | - | Bio Basic | TB0103 | RT | |
| 5 | Glycerol | 100% | ALLHIM | 2905450000 | RT | |
| 6 | TCEP, Hydrochloride | - | Santa Cruz | 51805-45-9 | +4°C | |
| 7 | DMSO | 100% | Honeywell | 34869-2.5L | RT | |
| 8 | Tween 20 | 100% | Sigma-Aldrich | P7949-500ML | RT | |
| 9 | Bovine Serum Albumin (BSA) | - | Sigma-Aldrich | A3608-50G | +4°C | |
| 10 | SDS | - | Bio Basic | 151-21-3 | RT | |
| 11 | Sodium Chloride | - | Bio Basic | DB0483 | RT |
Tris hydrochlorideBio Basic Inc.Catalog #TB0103.SIZE.250G
TCEP, Hydrochloride Santa Cruz
Dimethyl sulfoxideHoneywell FlukaCatalog #34869
Sodium chlorideBio Basic Inc.Catalog #DB0483.SIZE.500G
TWEEN 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3608
SDSBio Basic Inc.Catalog #151-21-3
Standard Reagent Formulations
| A | B | C | D | E | F | |
| № | Prepared Reagent Name | Component Name | Prepared Reagent Volume | Component Quantity | Storage | |
| 1 | 1 M Tris-HCl pH 7.5 | Tris-HCl MQ H2O | 10 mL | 10 g 10 mL | 4°C | |
| 2 | 10 mM DABCYL-RDKITTLGKFGQDE-EDANS | DABCYL-RDKITTLGKFGQDE-EDANS DMSO | 10x15 µL 9x10 µL | 5 mg 237 µL | -20°C | |
| 3 | 1 M Sodium chloride | Sodium chloride MQ H2O | 500 mL | 29.22 g 500 mL | RT | |
| 4 | 10% Tween 20 | Tween 20 MQ H2O | 50 mL | 5 mL 45 mL | RT | |
| 5 | 1% SDS | Sodium dodecyl sulfate MQ H2O | 2 L | 20 g 2 L | RT | |
| 6 | 0.5 M TCEP | TCEP MQ H2O | 20x 1.5 mL | 4.30 g 30 mL | -20°C |
Daily Reagent Formulations*
| A | B | C | D | E | F | |
| № | Prepared Reagent Name | Components | Component Quantity | Concentration in solution | Prepared Reagent Volume | |
| 1 | 50 mg/mL BSA | Bovine Serum Albumin MQ H2O | 5 mg 100 μL | 50 mg/mL - | 100 µL | |
| 2 | Assay Buffer | 1 M Tris-HCl pH 7.5 Glycerol 0.5 Μ TCEP 1 Μ NaCl 10% Tween 20 50 mg/mL BSA Milli-Q H2O | 2.5 mL 5 mL 50 µL 7.5 mL 500 µL 50µL 34.4 mL | 50 mM 10% 1 mM 150 mM 0.1% 0.05 mg/mL - | 50 mL | |
| 4 | 4 µM EVA71 2Apro (2x solution) | 1.2 mM EVA71 2Apro Assay Buffer | 30 µL 8970 µL | 4 µM - | 9000 µL | |
| 5 | 12 µM Substrate solution (2x solution) | 10 mM DABCYL-RDKITTLGKFGQDE-EDANS Assay Buffer | 12 µL 9988 µL | 12 µM - | 10000 µL | |
| *All calculations shown here are for a 3-plate HTS run. This consists of 3 assay plates with a dummy plate at the beginning of the dispense and dead volume of dispensing equipment. | ||||||
Protocol materials
Enterovirus Coxsackievirus A16 2A proteaseaddgeneCatalog #228632
SDSBio Basic Inc.Catalog #151-21-3
Sodium chlorideBio Basic Inc.Catalog #DB0483.SIZE.500G
TWEEN 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3608
ClipTip™ Filtered 384-Format Pipette TipsThermo ScientificCatalog #94420103
Nunc™ Serological PipettesThermo ScientificCatalog # 170357N
Axygen® 70 µm Ultra Clear Pressure Sensitive Sealing Film for Real Time PCR, NonsterileCorningCatalog #UC-500
Dimethyl sulfoxideHoneywell FlukaCatalog #34869
TCEP, Hydrochloride Santa Cruz
384-well Low Volume Black Round Bottom Polystyrene NBS Microplate, 10 per Bag, without LidCorningCatalog #4514
Nunc™ Serological PipettesThermo ScientificCatalog # 170356N
Nunc™ Serological PipettesThermo ScientificCatalog #170358N
Tris hydrochlorideBio Basic Inc.Catalog #TB0103.SIZE.250G
SILVERSEAL SEALER, ALUMINIUMgreiner bio-oneCatalog #676090
ClipTip™ Filtered 384-Format Pipette TipsThermo ScientificCatalog #94420153
Nunc™ Serological PipettesThermo ScientificCatalog #170355N
Minisart® NY25 Syringe Filter, 0.2 µm Polyamide (Nylon)SartoriusCatalog #17845-Q
Assay Plate Preparation
1m
Preparation of compounds for test at 2 concentrations:
DMSO, tested compounds, and reference compound are prepared by Biomek Span-8 and printed onto assay-ready plates (assay plate - Corning #4514) using the Labcyte Echo 650.
Reference compound is formatted in the following order:
- Reference compound 1st concentration – C1-C2 and M23-M24 - 50 nL of ASAP-0027815 (20 millimolar (mM) , n=4)
- Reference compound 2nd concentration – D1-D2 and N23-N24 - 50 nL of ASAP-0027815 (10 millimolar (mM) , n=4)
- Reference compound 3rd concentration – A1-A2 and O23-O24 – 50 nL of ASAP-0027815 (400 micromolar (µM) , n = 4)
- Reference compound 4th concentration – B1-B2 and P23-P24 – 50 nL of ASAP-0027815 (200 micromolar (µM) , n = 4)
Figure 1. Plate Map of the test of compounds at 2 concentrations prepared by liquid handling department.
DMSO is formatted into wells E1-P2 and A23-L24.
Compounds are formatted in columns 3-22 in the following order:
- Compounds 1st concentration – rows A, C, E, G, I, K, M, O – 50 nL in each well at 20 millimolar (mM) (n = 2).
- Compounds 2nd concentration – rows B, D, F, H, J, L, N, P – 50 nL in each well at 10 millimolar (mM) (n = 2).
Preparation of compounds for test at 6-point dose-response format, option A:
DMSO, tested compounds and reference compound are prepared by Biomek Span-8 and printed onto assay-ready plates (assay plate – Corning #4514) using the Labcyte Echo 650.
Reference compound – rows B and O – 50 nL of ASAP-0027815 2-fold dilution, 10 millimolar (mM) – 5 micromolar (µM) , 12 points (n = 4)
Figure 2A. Plate Map of 6-point dose-response scheme prepared by liquid handling department, option A.
DMSO is formatted into rows A and P.
Compounds are formatted into all columns, in rows C-N in dose-response format, 2-fold dilution, 20 millimolar (mM) – 0.6 millimolar (mM) , 6 points (n=2), 50 nL in each well.
Preparation of compounds for test at 6-point dose-response format, option B:
DMSO, tested compounds and reference compound are prepared by Biomek Span-8 and printed onto assay-ready plates (assay plate – Corning #4514) using the Labcyte Echo 650.
Reference compound – rows B and O – 50 nL of ASAP-0027815 2-fold dilution, 10 millimolar (mM) – 5 micromolar (µM) , 12 points (n = 4)
Figure 2B. Plate Map of 6-point dose-response scheme prepared by liquid handling department, option B.
DMSO is formatted into rows A and P.
Compounds are formatted into all columns, in rows C-N in dose-response format, 2-fold dilution, 1 millimolar (mM) – 0.03 millimolar (mM) , 6 points (n=2), 50 nL in each well.
Preparation of compounds for test at 12-point dose-response format:
DMSO, tested compounds and reference compound are prepared by Biomek Span-8 and printed onto assay-ready plates (assay plate – Corning #4514) using the Labcyte Echo 650.
Reference compound – rows B and O – 50 nL of ASAP-0027815 2-fold dilution, 10 millimolar (mM) – 5 micromolar (µM) , 12 points (n = 4)
Figure 3. Plate Map of 12-point dose-response scheme prepared by liquid handling department.
DMSO is formatted into A and P.
Compounds are formatted into all columns, in rows C-N in dose-response format, 2-fold dilution, 40 millimolar (mM) – 0.04 micromolar (µM) & point 30 millimolar (mM) , 12 points (n=2), 50 nL in each well.
The plates are sealed with Silverseal Sealer, Aluminium (no heating is applied) and stored at -20 °C . On the day of the assay, assay plates are removed 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 .
1m
Further assay procedures are similar for both screening formats: dose-response and compounds at 2 concentrations. Summarized in “ Assay performance” section.
Assay Performance
1h 28m
Multidrop Combi nL settings:
- Plate type: 384 wells standard (16 mm)
- Predispense (per channel): 50 µL
- Dispense volume: 5 µL
- Correction factor: 1.031 (at RT)
- Dispense speed: medium (3)
- Dispense direction: by columns
Note
Multidrop Combi nL should be checked before screening procedures using gravimetric accuracy verification and photometric precision verification, in accordance to 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 . 5 µL of assay buffer are then dispensed to half of the dummy plate, followed by the 3 assay plates. The plates are covered with plate lids and left at the table before next dispense.
Select the following wells for dispense, based on a plate map format (fig. 4A, 4B):
2 concentrations format:
Dispensing of 5 µL into wells E2-P2 and A24-L24.
Figure 4A. Plate Map of dispensing 5 µL of assay buffer for 2 concentrations format.
Dose-response formats (all options):
Dispensing of 5 µL into row P.
Figure 4B. Plate Map of dispensing 5 µL of assay buffer for dose-response format.
We used the following plasmid in the protein expression and purification protocol.
Enterovirus Coxsackievirus A16 2A proteaseaddgeneCatalog #228632
Protocol
CREATED BY
korvus.wang
5m
Multidrop Combi nL dispense #2. Immediately after the first dispense the tubes are emptied, filled with 2x EVA71 2Apro enzyme solution and primed ~50 µL . Preincubate the solution in the tubes for 00:02:00 . 5 µL are then dispensed to half of the dummy plate, followed by the 3 assay plates. The plates are then sealed with SealPlate film and centrifuged at 1000 rpm, 00:01:00 and then left to incubate for 00:30:00 at Room temperature in the incubator, while Multidrop Combi nL is flushed through with ~40 mL of 1% SDS and ~80 mL of MQ H2O before the next dispense.
Select the following wells for dispense, based on a plate map format (fig. 5A, 5B):
2 concentrations format:
Dispensing of 5 µL into columns 1, 3-23 and wells A2-D2, M24-P24.
Figure 5A. Plate Map of dispensing 5 µL of 4 µM EVA71 2Apro for 2 concentrations format.
Dose-response formats (all options):
Dispensing of 5 µL into rows A-O.
Figure 5B. Plate Map of dispensing 5 µL of 4 µM EVA71 2Apro for dose-response format.
33m
Multidrop Combi nL dispense #3. Fill the tubes with assay buffer, prime ~50 µL , preincubate for 00:02:00 , then empty the tubes. Fill the tubes with 2x Substrate solution, prime ~50 µL , preincubate the solution in the tubes for 00:02:00 . 5 µL are then dispensed to half of the dummy plate, followed by the 3 assay plates (select dispensing of 5 µL of full plate). The plates are sealed with Axygen film and centrifuged at 1000 rpm, 00:01:00 and then left to incubate for 00:45:00 at Room temperature in the incubator, while Multidrop Combi nL is flushed through with ~40 mL of 1% SDS and ~80 mL of MQ H2O.
Figure 6. Plate Map of dispense #3 with Multidrop Combi. Full plate is dispensed with 5 µL of 12µM of Substrate.
50m
Reader settings:
- Mode: FL
- Ex./Em.: 360/510
- Integration Time: 140 ms
- Read Order: by rows
- 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 Protocols (EVA-71-2A_fluorescence-dose-response_bienta and EVA-71-2A_fluorescence-single-point_bienta) for further analysis.
Protocol references
This protocol was established through the transfer and adaptation of an assay originally developed at the Weizmann Institute of Science, as a part of the ASAP Discovery Consortium.
dx.doi.org/10.17504/protocols.io.j8nlk8kb5l5r/v1
- Haim Barr and Noa Lahav
- 1Weizmann Institute of Science;
- 2ASAP Discovery Consortium
Acknowledgements
Grateful for Haim Barr and Noa Lahav for sharing their protocol developed at Weizmann Institute of Science as part of ASAP Discovery Consortium