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)
Name: 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). ..more
BioActive Compounds: 3417
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_1X%INH PRUN
Name: 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).
Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding (DBD) and ligand-binding domains (LBD) (1-3). Of interest, DAX-1 (NR0B1; nuclear receptor subfamily 0, group B, member 1; dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1) is an orphan nuclear receptor 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), which suggests that impairment of the DAX-1 transcriptional activity is directly linked to AHC-HHG pathogenesis. In addition, DAX-1 is 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 its roles of in steroidogenesis, tumorigenesis, and maintenance of stem cell pluripotency (8).
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 11: 741-746, 2003.
4. Lalli, E., M. H. Melner, D. M. Stocco, and P. Sassone-Corsi. DAX-1 blocks steroid production at multiple levels. Endocrinology 139: 4237-4243, 1998.
5. Ito, M., R. Yu, and J. L. Jameson. 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, 1997.
6. Zazopoulos, E., E. Lalli, D. M. Stocco, and P. Sassone-Corsi. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390: 311-315, 1997.
7. Lalli, E., B. Bardoni, E. Zazopoulos, J.-M. Wurtz, T. M. Strom, D. Moras, and P. Sassone-Corsi. A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita. Mol. Endocrinol. 11: 1950-1960, 1997.
8. Lalli E, Alonso J. Targeting DAX-1 in embryonic stem cells and cancer. Expert Opin Ther Targets 14: 169-77, 2010.
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.
DAX-1, Dax, Dax1, NR0B1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1, orphan, nuclear, nuclear receptor, NR, X chromosome, Gal4, reporter, luciferase, lumi, luminescence, luminescent, chemiluminescence, inhibit, inhibitor, antagonist, adrenal, gonad, steroidogenic, hormone, AHC, stem cell, Ewing, tumor, cancer, primary screen, primary, singlicate, 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 identify compounds that 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 singlicate at a final nominal concentration of 6.75 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 prior to run 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 SteadyLite 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.
A mathematical algorithm was used to determine nominally active compounds. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-16, and for inactive compounds 16-0.
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)
SteadyLite Reagent (PerkinElmer, part 6016989)
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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)