qHTS of Nrf2 Activators: Hit Validation in Primary 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 ..
BioActive Compounds: 854
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.
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
Quantification of ARE activation after compound treatment was measured by the Steady-Glo Luciferase (R) assay system (Promega) in AREc32_fluc stable cells. The Steady-Glo reagent acts by cell lysis and generation of a luminescent signal proportional to ARE activation, through the luciferase reporter in the AREc32 stable cell line. AREc32_fluc cells were harvested from T225 flask and resuspended in phenol red free OPTI-MEM medium with 5% FBS. Then 5 microL of 200,000 cells/mL resuspended cells were dispensed into each well of white, solid bottom, 1536-well tissue culture-treated plates using a Multidrop Combi dispenser. After overnight culture at 37 degrees C with 5% CO2, a total of 23 nL of compounds at 5 selected concentrations from the MLSMR/Sytravon or positive control (40 uM stock of CDDO-IM and 2 mM of tBHQ) in DMSO was transferred to each well of the assay plate using a pintool (Kalypsys, San Diego, CA), and the plates were further incubated at 37 degrees C with 5% CO2 for 24 hours. After that, 2 microL of Steady Glo luminescent substrate mix (Promega) was added to each well. The plate was incubated at room temperature for 30 minutes. The plates were measured on a ViewLux plate reader (PerkinElmer) with clear filter.
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 activators are ranked higher than compounds that showed apparent inhibition.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)