Nrf2 qHTS screen for inhibitors: counterscreen for cytotoxicity
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 ..
BioActive Compounds: 608
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03-MH092170-01
Assay Provider: Shyam Biswal, Johns Hopkins University
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.
As we have noticed in the pilot screen of small bioactive libraries, many compounds showing good inhibition to Nrf2 activity also induced cell death. So here we implemented a counterscreen to weed out the cytotoxic compounds.
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.
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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)