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BioAssay: AID 651829

Thermal shift assay to identify compound binding to Kelch domain of Keap1 protein Measured in Biochemical System Using RT-PCR - 2119-02_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 subsequent proteosomal degradation. Under oxidative stress conditions, Keap1 liberates Nrf2 from its repression, resulting in an increased oxidative stress response. This response plays an important role in several diseases, including cancer, inflammation, and neurodegeneration. ..more
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 Tested Compounds
 Tested Compounds
All(86)
 
 
Active(26)
 
 
Inactive(62)
 
 
 Tested Substances
 Tested Substances
All(92)
 
 
Active(27)
 
 
Inactive(65)
 
 
AID: 651829
Data Source: Broad Institute (2119-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-30
Hold-until Date: 2013-07-17
Modify Date: 2013-07-17

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 26
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
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
651876Competitive SPR to confirm the inhibition of the compound to keap1-Nrf2 interaction Measured in Biochemical System Using Plate Reader - 2119-08_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
Description:
Keywords:
Kelch domain of Keap1 protein, inhibitor, binding, thermal shift assay

The Keap1, an oxidative stress "sensor" protein, forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and subsequent proteosomal degradation. Under oxidative stress conditions, Keap1 liberates Nrf2 from its repression, resulting in an increased oxidative stress response. This response plays an important role in several diseases, including cancer, inflammation, and neurodegeneration.
We conducted a high throughput screen to identify inhibitor to disrupt the interaction between Keap1 and Nrf2 using biochemical fluorescence polarization assay. To confirm that the identified compounds directly bind to keap1, we developed a thermal shift assay. The thermal shift assay relays on the differential bind of a fluorescent dye Sypro Orange to the native Keap1 and to the thermal denatured Keap1. When Sypro orange is in aqueous solution and do not bind to the native Keap1, its fluorescence is largely quenched. When Sypro orange binds to the hydrophobic domain of Keap1 exposed by thermal denaturing, it emits strong fluorescence. The fluorescence signal is recorded over the time of thermal denature and the Tm is derived. When a compound is directly bound to Keap1, it stabilizes Keap1, which results in an increase of Tm of Keap1.

Expected Outcome:
The compound which directly binds to Keap1 shows an increase of Tm with Keap1, but does not give a detectable Tm in the compound alone system.
Protocol
Protocol for thermal shift assay
Reagents and Instrument
1. Kelch domain of Keap1 protein: human recombinant Kelch domain expressed and purified from E.coli from Xtal Biostructures (X090-173)
2. Positive control: Ac-9mer-Nrf2, an acetylated 9-mer peptide from Keap1 binding region of Nrf2 from Rutgers
3. Dye: Sypro Orange 5000X from Sigma (Cat# S5692-50uL)
4. Buffer: 10mM HEPES pH7.4, 3.4mM EDTA, 150mM NaCl, 0.005% Tween-20
5. Instrument: LightCycler 480 from Roche
6. Plate: 384-well qPCR plate from Roche (Cat# 05102430001)
Solution preparation: (for a run of 22 plates)
1. Prepare 3X SyproOrange dye in buffer
Add 36uL of 5000X stock solution into 54mL buffer
2. Prepare Kelch-Dye mix solution
Add 6mL 0.83mg/mL Kelch domain protein into 54ml 3X SyproOrange dye, Mix
3. Positive control: 40uM Ac-9mer-Nrf2
Add 40uL of 10mM Ac-9mer-Nrf2 in 100% DMSO into 10mL of Kelch-Dye mixture
4. Add 200uL 100% DMSO in 50mL of Kelch-Dye mixture
Screen:
1. Dispense 5uL/well of positive control to assay plate based on D39 plate map by Combi-NL (Thermo Scientific)
2. Dispense 5uL/well of Kelch-Dye solution with DMSO to the other wells of assay plate by Combi-NL (Thermo Scientific)
3. Centrifuge plate for 1min at 1,000 rpm
4. Pin 25nL/well of compound into assay plate
5. Mix with plate shaker for 5 seconds at 2,000 rpm
6. Incubate Kelch domain protein with the compounds for 10 mins at room temperature
7. Run a protein melting protocol on LightCycler 480 at temperature range of 25~85 degrees C with a ramping rate of 3.6 degrees C/min, monitoring fluorescence at the excitation of 465nm and the emission of 580nm
8. Tm value for each well was derived through Roche protein-melting analysis software.
Final screening concentration:
Kelch: 0.083mg/mL
Sypro Orange: 3x
Ac-9mer-Nrf2: 40uM
Compound concentration: 38-0.16 uM, 9 points
DMSO=0.4%
Assay volume=5ul/well
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the highest mean of the delta Tm at any tested compound concentration, multiplied by ten and rounded to the closest integer. The higher the Tm shift, the higher the PUBCHEM_ACTIVITY_SCORE. Note that the score is not normalized and can be greater than 100.
PUBCHEM_ACTIVITY_OUTCOME:
Compounds that gave greater than 1 degree Celcius of delta_Tm at any compound concentration 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
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Max_Delta_Tm_(degrees C)Maximum observed positive change in melting temperature of the protein when exposed to the compound in any concentrationFloatdegrees C
2Max_Delta_Tm_Conc_uM*Micromolar concentration at which the highest delta Tm was observedFloatμM
3Max_Concentration_uM Highest concentration at which the compound was testedFloatμM
4Delta_Tm_at_0.038uM_(degrees C) (0.038μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
5Delta_Tm_at_0.08uM_(degrees C) (0.08μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
6Delta_Tm_at_0.16uM_(degrees C) (0.16μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
7Delta_Tm_at_0.3uM_(degrees C) (0.3μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
8Delta_Tm_at_0.6uM_(degrees C) (0.6μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
9Delta_Tm_at_1.2uM_(degrees C) (1.2μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
10Delta_Tm_at_2.6uM_(degrees C) (2.6μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
11Delta_Tm_at_5uM_(degrees C) (5μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
12Delta_Tm_at_10uM_(degrees C) (10μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
13Delta_Tm_at_19.5uM_(degrees C) (19.5μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C
14Delta_Tm_at_38uM_(degrees C) (38μM**)Observed shift in melting temperature at the indicated concentrationFloatdegrees C

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

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