Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): Luminescence-based cell-based high throughput assay for nonselective inhibitors/assay artifacts using AP2 mutant SF-1 (NR5A1) Transactivation Assay
Name: Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): Luminescence-based cell-based high throughput assay for nonselective inhibitors/assay artifacts using AP2 mutant SF-1 (NR5A1) Transactivation Assay. ..more
BioActive Compounds: 1895
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: SF1-AF2MUT_INH_LUMI_1536_ 3X%INH CSRUN for DAX1-FULL INH
Name: Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): Luminescence-based cell-based high throughput assay for nonselective inhibitors/assay artifacts using AP2 mutant SF-1 (NR5A1) Transactivation Assay.
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.
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The purpose of this counterscreen is to ascertain whether compounds identified as active in the primary screen entitled "Luminescence-based cell-based primary high throughput screening assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): repression of SF-1 (NR5A1) activated StAR promoter by full-length DAX-1" (AID 652010) are artifacts or act non-selectivity via a non-DAX1 dependent manner.
This assay employs HEK cells co-transfected with the StAR luciferase promoter reporter together with expression plasmids for wildtype SF-1 and an AF-2 mutant SF-1, followed by treatment with test compounds. SF-1 AF-2 mut is a dominant-negative SF-1 mutant bearing the double point mutation L451A/L452A. It has been described in Mol. Endocrinol. 21: 2968-2987 (2007).
As designed, a compound increasing well luminescence in this assay lacking the DAX1-expresssion plasmid will be considered an assay artifact and will not be pursued. Compounds will be tested in triplicate at a nominal concentration of 6.8 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 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 seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 5 mL of serum-free OptiMEM containing 28 ug of the StAR promoter luciferase reporter plasmid, 14 ug of the SF-1 expressing plasmid, 14 ug of the SF-1-AF2 mutant expressing plasmid and 100 uL of transfection reagent. Four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 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 an empty vector in place of the DAX-1 expressing 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 (no DAX-1 expressed) whereas the rest of the plate was dispensed with DAX1-transfected cells. The plates were then treated with 34 nL/well of test compounds or DMSO (final concentration 0.68%) 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 (no DAX-1 expressed) 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.
PubChem Activity Outcome and Score:
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.
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 100-4, and for inactive compounds 4-0.
List of Reagents:
pGL2_1.3 kb_StAR luciferase reporter plasmid (Assay Provider)
pSG.SF-1 plasmid (Assay Provider)
pSG.SF-1-AF2mut plasmid (Assay Provider)
pSG5 empty vector (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. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)