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BioAssay: AID 651876

Competitive SPR to confirm the inhibition of the compound to keap1-Nrf2 interaction Measured in Biochemical System Using Plate Reader - 2119-08_Inhibitor_Dose_DryPowder_Activity

The Keap1, an oxidative stress "sensor" protein, forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and proteosomal degradation. Under oxidative stress condition, Nrf2 dissociates from Keap1 and translocates into the nucleus. In the nucleus, Nrf2 dimerizes with small Maf proteins and binds to an antioxidant response element (ARE) in the promoter to increase the transcription of cytoprotective genes. This response plays an important role in several diseases, including cancer, inflammation, and neurodegeneration. ..more
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 Tested Compounds
 Tested Compounds
All(75)
 
 
Active(31)
 
 
Inactive(46)
 
 
 Tested Substances
 Tested Substances
All(79)
 
 
Active(31)
 
 
Inactive(48)
 
 
AID: 651876
Data Source: Broad Institute (2119-08_Inhibitor_Dose_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-12-06
Hold-until Date: 2013-07-17
Modify Date: 2013-07-17

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 31
Related Experiments
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AIDNameTypeProbeComment
504540Broad Institute HTS fluorescence polarization assay for inhibitors of Keap1-Nrf2 interaction Inhibitor Probe ProjectSummary1 depositor-specified cross reference: Summary assay
504523Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_SinglePoint_HTS_ActivityScreening same project related to Summary assay
588683Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651798Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651801Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651806Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651807The inhibition of Keap1-Nrf2 interaction in cells by beta-lactamase reporter assay Measured in Cell-Based System Using Plate Reader - 2119-03_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
651823Thermal shift assay to identify compound binding to Kelch domain of Keap1 protein Measured in Biochemical System Using RT-PCR - 2119-02_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651829Thermal shift assay to identify compound binding to Kelch domain of Keap1 protein Measured in Biochemical System Using RT-PCR - 2119-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651833Nrf2 PathHunter Nuclear Translocation assay in U2OS cells Measured in Cell-Based System Using Plate Reader - 2119-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651834Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
651858Counterscreen to eliminate false positive from thermal shift assay Measured in Biochemical System Using Plate Reader - 2119-07_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651860HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651863HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
Description:
Keywords:
Keap1-Nrf2 interaction, biotinylated peptide, SPR, binding, competition


Assay Overview:
The Keap1, an oxidative stress "sensor" protein, forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and proteosomal degradation. Under oxidative stress condition, Nrf2 dissociates from Keap1 and translocates into the nucleus. In the nucleus, Nrf2 dimerizes with small Maf proteins and binds to an antioxidant response element (ARE) in the promoter to increase the transcription of cytoprotective genes. This response plays an important role in several diseases, including cancer, inflammation, and neurodegeneration.

We conducted a high throughput screen to identify the inhibitors to disrupt the interaction between Keap1 and Nrf2 using a biochemical fluorescence polarization assay. To further confirm that the hits can bind to Keap1 and compete with Nrf2 for Keap1 binding, we employed a solution based competitive SPR assay. In this assay, the biotinylated 16mer peptide from Keap1 binding region of Nrf2 is captured by streptavidin on CM5 chip surface. The binding signal of Keap1 to Nrf2 was recorded using BIAcore 3000.

Expected Outcome:
Any compound competing with Nrf2 for binding to Keap1 shows a decrease of binding signal. The data is expressed as a percentage of inhibition using the ratio of unbound Keap1 in competition with the tested sample and total Keap1.
Protocol
Protocol for solution based competitive SPR
1. Immobilize 200ug/mL of Streptavidin (SA) diluted in acetate acid buffer on Fc1 and Fc2 of a conditioned CM5 Sensor Chip on Biacore 3000 (GE Healthcare Life Sciences) following the general procedure. The immobilization level is ~7000RU.
2. Dilute 16mer Nrf2 peptide with running buffer to a final concentration to 10nM for capture.
3. Inject the peptide solution over Fc2 at a flow rate of 10uL/min to saturate SA surface. The capture level of Fc2 is 300RU.
4. Incubate 40nM Kelch domain of Keap1 protein with the compounds at 5 or 50uM at room temperature
5. Inject the mixture solution of protein and compound over Fc1 and Fc2 for Keap1 binding to Nrf2 surface with a flow rate of 50uL/min for a 1 minute association and a 3 minutes dissociation at 25 degree Celsius
6. The chip surface is regenerated with 1M NaCl solution with a flow rate of 100uL/min for 1 minute followed by two consecutive 1-min washes with the running buffer at a flow rate of 100 uL/min.
7. The sensorgram is double referenced by subtracting the signal from the Fc1 and the buffer injection
8. The concentration of unbound Kelch domain is calculated based on its standard curve. The data was expressed as a percentage of inhibition using the ratio of unbound Kelch domain in competition sample and total Kelch domain.
Standard curve
1. Dilute Kelch domain in the running buffer started at 125nM for 6 points with a dilution factor of 2
2. Inject each sample over Fc1 and Fc2 with a flow rate of 50uL/min for a 1 minute association and a3 minutes dissociation at 25 degree Celsius
3. The slopes of the initial association phase from the corrected SPR sensorgrams were calculated by fitting to the linear model in BIAevaluation software 4.1 and plotted against the concentration of Kelch domain
Comment
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the percentage of inhibition due to the tested compound at a concentration of 50uM. The higher the PUBCHEM_ACTIVITY_SCORE, the more potent the compound is. Note that the score was not normalized
PUBCHEM_ACTIVITY_OUTCOME:
Compounds that gave greater than 20% inhibition at 50uM were declared as "Active".
Activity_Outcome = 2 (active)
Activity_Outcome = 1 (inactive)
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Kd (uM) Dissociation constant of the tested compoundString
2%Inhibition at 50 uM (50μM**)percent inhibition versus neutral controlFloat%
3%Inhibition at 5 uM (5μM**)percent inhibition versus neutral controlFloat%

** Test Concentration.
Additional Information
Grant Number: R03 MH093197-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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