Aug 04, 2025
WNV NS2B-NS3 fluorescence single point
- Haim Barr1,2,
- Noa Lahav1,2,
- Jiyun Zhu3,2
- 1The Weizmann Institute of Science;
- 2ASAP Discovery Consortium;
- 3Stanford University
- Haim Barr: General acknowledgement: The Wohl Drug Discovery institute, The Nancy and Stephen Grand Israel National Center for Personalized Medicine.;
- ASAP Discovery
Protocol Citation: Haim Barr, Noa Lahav, Jiyun Zhu 2025. WNV NS2B-NS3 fluorescence single point. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1dy37vr2/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: November 03, 2024
Last Modified: August 04, 2025
Protocol Integer ID: 111445
Keywords: Fluorescence assay, Assay, Inhibitor, Fragment, Screening, WNV, NS2B-NS3 protease, single point, fluorescent assays for wnv ns2b, protease inhibitors against wnv ns2b, ns3 fluorescence, ns3 fluorescence single point purpose, ns3 cleavage of substrate bz, fluorescent assay, protease inhibitor, wnv ns2b, ns3 cleavage, peptide substrate, screening assay, enzyme, fluorescence, containing serine, fluorescence signal, candidates for medicinal optimization general description, ns3, medicinal optimization general description, enzymatic reaction, excitation of amc, result of lower enzyme activity, lower enzyme activity
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
Purpose: performing single point test of selected compounds from screening experiment libraries containing serine-targeting fragments and serine protease inhibitors against WNV NS2B/NS3 to find warheads and candidates for medicinal optimization
General description: This protocol details the fluorescent assays for WNV NS2B/NS3 cleavage of substrate Bz-Nle-KRR-AMC peptide. This method measures the fluorescence from the released AMC product as a result of the enzymatic reaction. When hydrolyzed, AMC is liberated from the peptide substrate. Excitation of AMC at 350 nm emits a resonant energy of 450 nm. When the enzyme is inhibited, the fluorescence signal will decrease as a result of lower enzyme activity. The screening method is validated by calculating the Z prime number of each plate, and the percentage of inhibition is calculated and then evaluated for inhibitory efficacy.
Outcome: hits were selected from screening assays with more than 50% inhibition
Materials
Assay Buffer Reagents (Concentration listed are from Stock Solutions)
- Tris-HCl (Fisher Scientific). Dissolving Tris powder in MilliQ water and adjust pH to 8.6 for a final concentration of 1 M. Filter with 0.2 um filter
- Triton (Sigma Aldrich) dissolving one part of Triton in 4 part of MilliQ water for a final concentration of 20 %
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Additional Reagents:
84090 nanomolar (nM) WNV NS2B/NS3 Enzyme
- The Enzyme stock was originally 242140 nanomolar (nM) and was diluted to 10000 nanomolar (nM) before every experiment in freshly made Assay Buffer. The final assay concentration is 12.5 nanomolar (nM)
20000000 nanomolar (nM) Substrate Bz-Nle-KRR-AMC
- Substrate stock was dissolved in DMSO to the stock concentration. Before each experiment, the substrate stock was diluted to 10000 nanomolar (nM) in freshly made Assay Buffer.The final assay concentration is 500 nanomolar (nM)
Troubleshooting
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Thaw TCEP solution on ice to make sure it is fresh
WNV NS2B/NS3 expression and purification
The protein used in this assay was expressed and purified and received from QQ01WNVNS2B -c003 -p005 from Centre for Medicines Discovery
Prepare Reagents
PREPARE all of the reagents/buffers required for this experiment.
Assay Buffer
| A | B | C | D | E | |
| Reagent | Stock | Final | Units | Note | |
| Tris pH 8.6 | 1000 | 10 | mM | ||
| Triton | 20 | 0.01 | % | ||
| glycerol | 100 | 20 | % v/v |
Reagents (dilute reagents in assay buffer for required volume)
| A | B | C | D | E | |
| Reagent | Stock | Prep | Final in assay plate | Units | |
| WNV-NS2B/NS3 | 1735000 | 83.3 | 50 | nM | |
| Substrate (Bz-Nle-KRR-AMC) | 20000000 | 100000 | 20000 | nM | |
Prepare 384-well Plate
16m
DILUTE Dilute protein and substrate using the assay buffer
- Protein dilution: 150 µL protein stock solution is added into 3480 µL Assay Buffer
- Substrate dilution: 1 µL x 20 mM substrate is added into 2 mL Assay buffer
MIX Add 10 µL enzyme stock solution into 384-well plates containing inhibitor stocks (column 2-22)
add 20 µL reaction buffer to A1-H1, I24-P24 (blank control)
add 10 µL reaction buffer to I1-P1, A24-H24 (substrate control)
MIX 180 µL diluted enzyme solution with 3.6 µL x 10 mM DCI (3,4-Dichloroisocoumarin) and aliquot 10 µL into I2-P2, A23-H23 (positive control inhibitor)
MIX 180 µL diluted enzyme solution with 3.6 µL DMSO and aliquot 10 µL into A2-H2, I23-P23 (no inhibitor control)
Incubate at room temperature for 1 hour
REACT Add 10 µL substrate solution into enzyme solution in the plate and mix well.
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) using Cytation 3 Multi-Mode Reader (BioTek, Winooski, VT)
Expected result
AMC product will give RFU signal at ex 350/em 450
Experimental Design
Plate Layout
DATA PROCESSING
Calculate the Z prime number of the screening plates to validate the screening:
Z’ = 1 – 3 *(Std.Dev of positive + Std.Dev of negative )/ |(Average of positive – Average of negative)|
Calculate the percentage of inhibition:
Inhibition % = ([RFU no inhibitor]-[RFU with compounds])/(RFU no inhibitor) * 100%
PLOT DATA
In Prism software, plot percentage of inhibition with corresponding number of compounds
Result
Results shown are exemplar