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BioAssay: AID 652014

qHTS of Nrf2 Activators: Hit Validation in GCLC Induction 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(10)
 
 
Inactive(10)
 
 
 Tested Substances
 Tested Substances
All(11)
 
 
Inactive(11)
 
 
 Related BioAssays
 Related BioAssays
AID: 652014
Data Source: NCGC (NRF2A201)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-02-04

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
624153qHTS of Nrf2 Activators: SummarysummarySummary AID
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. Compounds that were identified in the primary screen were tested in this secondary assay to assess whether a hit also activates Nrf2 transcription of the target gene GCLC.

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
Hepac1c7 cells were seeded into 24-well plates at a density of 30,000 cells/well in 1 mL complete media per well and cultured overnight to allow attachment. Cells were treated the next day with compounds (1 uM)or positive control for 24 h. Total RNA samples were then isolated by using RNAzol B reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. Total RNAs were reverse-transcribed into cDNA by High Capacity cDNA Archive Kits (Applied Biosystems, Foster City, CA) and the resulting cDNA was used for real-time PCR analysis using SYBR(R) Green PCR Master Mix in a 7900HT Fast Real-Time PCR System.
Comment
Compounds that show "relative transcription levels at 1uM" below 200% are considered inactive and are assigned a score of 10; compounds that show activity > 200% and < 250% are considered inconclusive and are assigned a score of 50; relative transcription >= 250% are considered active and are assigned a score of 90.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Relative transcription levels at 1uMInteger%
Additional Information
Grant Number: DK081461

Data Table (Concise)
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