Luminescence-based cell-based high throughput dose response assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1)
Name: Luminescence-based cell-based high throughput dose response assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1). ..more
BioActive Compound: 1
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Enzo Lalli, CNRS
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03 DA030558-01
Grant Proposal PI: Enzo Lalli, CNRS
External Assay ID: DAX1_INH_LUMI_1536_3XIC50 DRUN
Name: Luminescence-based cell-based high throughput dose response assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1).
Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding (DBD) and ligand-binding domains (LBD). Small pharmacological compounds able to bind to the cleft of the ligand-binding domain could alter its conformation and subsequently modify transcription of target genes. Such ligand agonists and/or antagonists have already been successfully designed for 23 nuclear receptors among the 48 previously identified in the human genome (1-3). Of interest, DAX-1 (NR0B1; dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1) has been shown to act as a robust transcriptional repressor, inhibiting genes involved in steroidogenesis through interaction with corepressors and regulating the pluripotency of stem cells (4-8). The human DAX-1 gene encodes a protein whose C terminus is similar to the LBD of nuclear hormone receptors, while its N terminus is composed of three cysteine-rich 70 amino acids with little similarity to known proteins (4, 7). Mutations in Dax-1 have been shown to cause the X-linked form of adrenal hypoplasia congenita (AHC), associated with hypogonadotropic hypogonadism (HHG). AHC-HHG-associated mutations share an altered DAX-1 C-terminal domain (5, 9), resulting in loss of transcriptional repression activity (5, 7, 9). This finding suggests that the impairment of the DAX-1 transcriptional activity is directly linked to the pathogenesis of AHC-HHG. In addition, Dax-1 has also been shown to be highly expressed in pediatric Ewing tumors (10). As a result, the identification of selective inhibitors of Dax-1 will serve as useful tools to elucidate the developmental and tumorigenic roles of Dax-1, and its maintenance of stem cell pluripotency.
1. Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol 19:1429-1438, 2005.
2. Kliewer SA, Lehmann JM, and Willson TM. Orphan nuclear receptors: shifting endocrinology into reverse. Science 284: 757-760, 1999.
3. Li Y, Lambert MH, and Xu HE. Activation of nuclear receptors: a perspective from structural genomics. Structure (Camb) 11: 741-746., 2003.
4. Lalli, E., M. H. Melner, D. M. Stocco, and P. Sassone-Corsi. 1998. DAX-1 blocks steroid production at multiple levels. Endocrinology 139:4237-4243. 22. Lalli, E., and P. Sassone-Corsi. 1999. DAX-1 and the adrenal cortex. Curr. Opin. Endocrinol. Diabetes 6:185-190.
5. Ito, M., R. Yu, and J. L. Jameson. 1997. DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol. Cell. Biol. 17:1476-1483.
6. Zazopoulos, E., E. Lalli, D. M. Stocco, and P. Sassone-Corsi. 1997. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311-315.
7. Lalli, E., B. Bardoni, E. Zazopoulos, J.-M. Wurtz, T. M. Strom, D. Moras, and P. Sassone-Corsi. 1997. A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita. Mol. Endocrinol. 11: 1950-1960.
8. Lalli E, Alonso J. Targeting DAX-1 in embryonic stem cells and cancer. Expert Opin Ther Targets. 2010 Feb;14(2):169-77.
9. Zanaria, E., F. Muscatelli, B. Bardoni, T. M. Strom, S. Guioli, W. Guo, E. Lalli, C. Moser, A. P. Walker, E. R. B. McCabe, T. Meitinger, A. P. Monaco, P. Sassone-Corsi, and G. Camerino. 1994. An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635-641.
10. Mendiola M, Carrillo J, Garcia E, Lalli E, Hernandez T, de Alava E, Tirode F, Delattre O, Garcia-Miguel P, Lopez-Barea F, Pestana A, Alonso J. The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors. Int J Cancer. 2006 Mar 15;118(6):1381-9.
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The purpose of this assay is to determine dose response curves for compounds identified as active in a set of previous experiments entitled, "Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1)" (AID 504766). This assay monitors the ability of compounds to inhibit the activity of the DAX-1 nuclear receptor (NR0B1), a robust transcriptional repressor. This assay employs HEK293 cells co-transfected with a GAL4DBD-DAX1 C-terminal construct (pG4D 207-470) and a luciferase reporter under control of the thymidine kinase minimal promoter preceded by yeast Gal4 binding sites (pGAL4-tk-luc). In this assay, transfected cells are incubated with test compounds, followed by measurement of well luminescence. DAX1 activity represses expression of the luciferase reporter plasmid. As designed, a DAX1 inhibitor will prevent or reduce DAX-1-mediated transcriptional repression, leading to increased expression of the luciferase reporter gene, and increased well luminescence. Compounds are tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 67.5 uM.
HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix. Flasks were then incubated for 48 hours at 37 C, 5% CO2 and 95% relative humidity (RH). The day of the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and resuspended in fresh media at a density of 1 million cells per mL. Next, 115 mL of the cell suspension were dispensed into 5-layer culture flasks and allowed to attach for 1 hour at 37 C, 5% CO2 and 95% RH. Cells were then transfected with 5 mL of serum-free OptiMEM containing 90 ug of the pGal4-tk-luc reporter plasmid, 50 ug of the Gal4 DBD-DAX1 fusion expression vector pG4D207-470, and 400 uL of transfection reagent. Four hours post transfection, cells were harvested using 25 mL of preheated TrypLE and resuspended at a concentration of 750,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, DAX-1 inhibition was mimicked by transfecting cells with the empty vector pG4MpolyII instead of the pG4D207-470 vector.
The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora). The first three columns received control cells whereas the rest of the plate was dispensed with DAX1-transfected cells. The plates were then treated with 35 nL/well of test compounds or DMSO (final concentration 0.76%) on DAX-1 cells and Control cells using a PinTool transfer unit (GNF). Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95%RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of OneGlo reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer). The percent inhibition of each test compound was calculated as follows:
%_Inhibition = ( 1 -( median_positive_control - test_compound ) / ( median_positive_control - median_negative_control ) * 100
Positive control is defined as wells containing control cells treated with DMSO.
Negative control is defined as wells containing DAX-1 cells treated with DMSO.
Test compound is defined as wells containing DAX-1 cells containing test compound.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 67.5 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 67.5 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 1-0.
List of Reagents:
pGAL4-tk-luc reporter plasmid (supplied by Assay Provider)
pG4D207-470 DAX-1 expression plasmid (supplied by Assay Provider)
pG4MpolyII empty plasmid (supplied by Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
ONE-Glo (Promega, part E6130)
1536-well plates (Greiner part 789173)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
Assay: Dictionary: Version: 0.1
BAO: assay design: inducible reporter: luciferase induction
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: primary
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of gene expression
BAO: meta target: molecular target: protein target: receptor: nuclear receptor
BAO: version: 1.4b1090
Assay Format: Cell-based
Assay Cell Type: HEK293
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)