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BioAssay: AID 504444

Nrf2 qHTS screen for inhibitors

Nrf2 is a transcription factor that maintains cellular redox homoestasis and protects cells from xenobiotics [1,2]. Nrf2 binds to the antioxidant response element (ARE) to induce gene expression of a broad spectrum of genes that encode for antioxidants. Hence this Nrf2 pathway provides a first line of defense against stress caused by exposure to radiation, electrophiles, and xenobiotics. In many more ..
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 Tested Compounds
 Tested Compounds
All(360868)
 
 
Active(7438)
 
 
Inactive(283489)
 
 
Inconclusive(70821)
 
 
 Tested Substances
 Tested Substances
All(364195)
 
 
Active(7472)
 
 
Inactive(285618)
 
 
Inconclusive(71105)
 
 
AID: 504444
Data Source: NCGC (NRF2002)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 7438
Depositor Specified Assays
AIDNameTypeComment
493153Nrf2 qHTS screen for inhibitors: Validationconfirmatorylopac validation
493163Nrf2 qHTS screen for inhibitors: Summarysummarysummary
720522Nrf2 qHTS screen for inhibitors: Purified Fluc Biochemical Counterscreen for Hit Validationconfirmatory
720524Nrf2 qHTS screen for inhibitors: Nrf2 A549 ARE-Fluc Confirmation Assay for Hit Validationconfirmatory
720523Nrf2 qHTS screen for inhibitors: A549 ARE-Fluc Cytotoxicity Counterscreen for Hit Validationconfirmatory
504648Nrf2 qHTS screen for inhibitors: counterscreen for cytotoxicityconfirmatory
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: R03-MH092170-01
Assay Provider: Shyam Biswal, Johns Hopkins University

Assay Overview:
Nrf2 is a transcription factor that maintains cellular redox homoestasis and protects cells from xenobiotics [1,2]. Nrf2 binds to the antioxidant response element (ARE) to induce gene expression of a broad spectrum of genes that encode for antioxidants. Hence this Nrf2 pathway provides a first line of defense against stress caused by exposure to radiation, electrophiles, and xenobiotics. In many cancers it has been found that tumor cells have manipulate the Nrf2 pathway for their survival against cytotoxic chemotherapeutics and radiotherapeutic agents. Finding a small molecule that act as an inhibitor of Nrf2 function would represent a novel therapeutic target that could lead to improvement in survival of patients undergoing chemo- and/or radio- therapy.

This is the Nrf2 activity measured in the luminescent read.

1. Shibata, T., et al., Genetic Alteration of Keap1 Confers Constitutive Nrf2 Activation and Resistance to Chemotherapy in Gallbladder Cancer. Gastroenterology, 2008.
2. Sjoblom, T., et al., The consensus coding sequences of human breast and colorectal cancers. Science, 2006. 314(5797): p. 268-74.
Protocol
NCGC Assay Protocol Summary:

A concentration-response assay was performed with A549 ARE_Fluc cells which are suspended in 5% FBS OPTI-MEM. Five uL were dispensed into 1536-well white TC-treated plate using the Kalypsys dispenser to achieve a density of 5000 cells/well in all columns except column 1. The plate is incubated at 37deg C at 5% CO2 for 2 hours before 23 nL of compounds are added with the Kalypsys pin tool equipped with a 1,536-pin array containing 10-nL slotted pins (FP1S10, 0.457-mm diameter, 50.8 mm long; V&P Scientific). The control compounds Budesonide and Staurosporine are both added at a concentration of 2mM and Budesonide is also added as a 1:10 titration, starting at 200uM, to achieve dose-response. After an 18-24 hour incubation at 37deg C at 5% CO2, 5ul of medium is added to column 1. Afterwards 1ul of 5x of CellTiter-Fluor (Promega) is added to every well. The plate is then incubated at 37deg C at 5% CO2 for 30 minutes before they are read on the ViewLux (Perkin Elmer) for a fluorescent read Ex=405(10)/Em=525(10) for a 3 second exposure, 2x binning, and high gain. Then 3 uL of 3x BugLite is dispensed into all wells and the plate is incubated at ambient room temperature for 15 minutes. Afterwards another ViewLux read is performed using the clear filter to get a luminescent read with the settings of 40 second exposure, 2x binning, and high gain. Cytotoxicity is assessed through the first fluorescent read and then biochemical activity against Nrf2 is measured in the luminescent read.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0007370000 uM (0.000737μM**)% Activity at given concentration.Float%
15Activity at 0.00147 uM (0.00147μM**)% Activity at given concentration.Float%
16Activity at 0.00294 uM (0.00294001μM**)% Activity at given concentration.Float%
17Activity at 0.00372 uM (0.00371585μM**)% Activity at given concentration.Float%
18Activity at 0.00590 uM (0.0059μM**)% Activity at given concentration.Float%
19Activity at 0.016 uM (0.0164463μM**)% Activity at given concentration.Float%
20Activity at 0.037 uM (0.0369μM**)% Activity at given concentration.Float%
21Activity at 0.073 uM (0.0734922μM**)% Activity at given concentration.Float%
22Activity at 0.101 uM (0.101148μM**)% Activity at given concentration.Float%
23Activity at 0.158 uM (0.158423μM**)% Activity at given concentration.Float%
24Activity at 0.295 uM (0.295005μM**)% Activity at given concentration.Float%
25Activity at 0.370 uM (0.370044μM**)% Activity at given concentration.Float%
26Activity at 0.661 uM (0.660708μM**)% Activity at given concentration.Float%
27Activity at 1.091 uM (1.09059μM**)% Activity at given concentration.Float%
28Activity at 1.837 uM (1.83708μM**)% Activity at given concentration.Float%
29Activity at 2.529 uM (2.52859μM**)% Activity at given concentration.Float%
30Activity at 3.974 uM (3.97421μM**)% Activity at given concentration.Float%
31Activity at 7.963 uM (7.96268μM**)% Activity at given concentration.Float%
32Activity at 9.266 uM (9.26596μM**)% Activity at given concentration.Float%
33Activity at 18.40 uM (18.4μM**)% Activity at given concentration.Float%
34Activity at 27.26 uM (27.2562μM**)% Activity at given concentration.Float%
35Activity at 46.02 uM (46.0238μM**)% Activity at given concentration.Float%
36Activity at 65.29 uM (65.2932μM**)% Activity at given concentration.Float%
37Activity at 92.20 uM (92.2μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03-MH092170-01

Data Table (Concise)
Classification
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