Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): luminescence-based cell-based high throughput dose response assay to identify compounds that interfere with the UAS/Gal4 system and/or luciferase reporter
Name: Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): luminescence-based cell-based high throughput dose response assay to identify compounds that interfere with the UAS/Gal4 system and/or luciferase reporter. ..more
BioActive Compounds: 13
Depositor Specified Assays
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: GAL4_INH_LUMI_1536_3XIC50 DCSRUN DAX1
Name: Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): luminescence-based cell-based high throughput dose response assay to identify compounds that interfere with the UAS/Gal4 system and/or luciferase reporter.
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.
Counterscreen, artifact, GAL4, DAX-1, Dax, Dax1, NR0B1, dose response, dose, titration, orphan, nuclear, nuclear receptor, NR, reporter, luciferase, lumi, luminescence, luminescent, chemiluminescence, inhibit, inhibitor, antagonist, adrenal, gonad, steroidogenic, hormone, AHC, stem cell, Ewing, tumor, cancer, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether 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) inhibit the activity of Gal4 DBD binding and act as luminescence-based assay artifacts. This assay will determine dose response curves. This assay employs HEK293T cells cotransfected with two plasmids: an empty GAL4DBD expression vector and a plasmid containing 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. As designed, compounds that inhibit Gal4 DNA binding activity or the general transcriptional machinery will interfere with expression of the luciferase gene, leading to reduced luciferase transcription and decreased well luminescence. In contrast, compounds that enhance or stabilize Gal4 DNA binding or the transcriptional machinery, or are luminescent artifacts will lead to an increase in 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 empty Gal4 DBD expression vector pG4MpolyII, 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, inhibition was mimicked by using untransfected cells.
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 untransfected cells whereas the rest of the plate was dispensed with transfected cells. The plates were then treated with 35 nL/well of test compounds or DMSO (final concentration 0.76%) 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 ONE-Glo 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 transfected cells treated with DMSO.
Test compound is defined as wells containing transfected cells treated with 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 100-94, and for inactive compounds 66-0.
List of Reagents:
HEK293 cells (ATCC, part CRL-1573)
pGAL4-tk-luc reporter plasmid (Assay Provider)
pG4MpolyII (Assay Provider)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
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.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: counter screening
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: cell-based format
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: meta target: molecular target: protein target: receptor: nuclear receptor
BAO: meta target: biological process target: regulation of gene expression
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: assay design: inducible reporter: luciferase induction
BAO: detection technology: luminescence: chemiluminescence
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)