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BioAssay: AID 651597

qHTS of Nrf2 Activators: Hit Validation in Secondary FLuc Assay

Many diseases have some form of oxidative stress injury and ties to inflammation, causing a host of problems for the patient. The antioxidant response element (ARE) plays an important role in alleviating the harmful effects of oxidative stress. The antioxidant response element (ARE) is a transcriptional regulatory element involved in the activation of genes coding for a number of antioxidant more ..
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 Tested Compounds
 Tested Compounds
All(1103)
 
 
Active(388)
 
 
Inactive(383)
 
 
Inconclusive(332)
 
 
 Tested Substances
 Tested Substances
All(1106)
 
 
Active(390)
 
 
Inactive(384)
 
 
Inconclusive(332)
 
 
AID: 651597
Data Source: NCGC (Nrf2201)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-09-24

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 388
Related Experiments
AIDNameTypeComment
624153qHTS of Nrf2 Activators: SummarySummarydepositor-specified cross reference: Summary
624149qHTS of Nrf2 Activators: LOPAC ValidationConfirmatorysame project related to Summary assay
624171qHTS of Nrf2 ActivatorsConfirmatorysame project related to Summary assay
651593qHTS of Nrf2 Activators: Hit Validation in Primary AssayConfirmatorysame project related to Summary assay
651595qHTS of Nrf2 Activators: Hit Validation in Cytotox AssayConfirmatorysame project related to Summary assay
652013qHTS of Nrf2 Activators: Hit Validation in NQO-1 Induction AssayOthersame project related to Summary assay
652014qHTS of Nrf2 Activators: Hit Validation in GCLC Induction AssayOthersame project related to Summary assay
Description:
Many diseases have some form of oxidative stress injury and ties to inflammation, causing a host of problems for the patient. The antioxidant response element (ARE) plays an important role in alleviating the harmful effects of oxidative stress. The antioxidant response element (ARE) is a transcriptional regulatory element involved in the activation of genes coding for a number of antioxidant proteins and detoxifying enzymes. These enzymes work in concert to protect tissues from oxidative insults and chemical toxicities in human hepatocytes and immune cells. The protective effects of ARE activation are primarily triggered through Nrf2 (NF-E2-related factor) binding to and activating AREs. It is known that Nrf2 controls the production of over 250 antioxidant and detoxification proteins (Hu et al., 2006), thereby protecting tissues by increasing cellular antioxidant content and suppressing inflammatory signaling pathways. The transcription factor Nrf2 is a central link between oxidative chemicals, such as phenolic antioxidants and electrophilic compounds, and the activation of ARE. Nrf2 levels are constitutively low as a consequence of its interaction with Keap1, which targets its degradation (Zipper and Mulcahy, 2002). Electrophiles react with key cysteine residues in Keap1, releasing Nrf2 and allowing its translocation to the nucleus. Once within the nucleus, Nrf2 complexes with coactivators such as p300, and binds to the AREs to induce gene transcription of cytoprotective enzymes, resulting in the prevention of toxicity. Activation of Nrf2 protects tissues by increasing cellular antioxidant content and suppressing inflammatory signaling pathways. Identifying novel and potent Nrf2 activators by using the AREc32 cell line to detect ARE activation by small molecules will lead to the identification and development of probes to study protective pathways in multiple tissues. These specific probes are essential to study the ARE pathway, and eventually to determine whether this pathway does activate genes that could protect against a host of diseases, including cardiovascular diseases, obesity, diabetes, Alzheimer's, and Parkinson's disease.

This project's aim is to identify novel and potent Nrf2 activators by using the AREc32 cell line to detect ARE activation by small molecules, which will lead to the identification and development of probes to study the ARE pathway. In this counter assay, we aim to identify false positive as identified from this primary assay using an FLuc assay with an inhibiting read-out.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: DK081461
Assay Submitter (PI): Curtis Klaassen, University of Kansas Medical Center
Protocol
Assess whether an activator in the primary assay potentially causes such activation by inhibitor-induced stabilization of the firefly luciferase reporter enzyme by determining whether it inhibits firefly luciferase in the presence of Km concentrations of its substrates (ATP and D-luciferin).
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
16Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
17Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
18Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
19Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
20Activity at 11.50 uM (11.5μM**)% Activity at given concentration.Float%
21Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DK081461

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