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BioAssay: AID 652136

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
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 Tested Compounds
 Tested Compounds
All(2441)
 
 
Active(1895)
 
 
Inactive(546)
 
 
 Tested Substances
 Tested Substances
All(2441)
 
 
Active(1895)
 
 
Inactive(546)
 
 
AID: 652136
Data Source: The Scripps Research Institute Molecular Screening Center (SF1-AF2MUT_INH_LUMI_1536_ 3X%INH CSRUN for DAX1-FULL INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-03-19
Modify Date: 2013-06-04

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1895
Depositor Specified Assays
AIDNameTypeComment
652010Luminescence-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-1screeningPrimary screen (full-length DAX1 inhibitors in singlicate)
652012Summary of the probe development effort to identify 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-1summarySummary (full-length DAX1 inhibitors)
687017Luminescence-based cell-based high throughput dose response 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-1confirmatory
687018Counterscreen for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1): Luminescence-based cell-based high throughput dose response assay for nonselective inhibitors/assay artifacts using AP2 mutant SF-1 (NR5A1) Transactivation Assayconfirmatory
Description:
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.

Description:

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).

References:

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.

Keywords:

Counterscreen, CSRUN, artifacts, nonselective, non-selective, transactivation, co-transfection, reporter, luc, lumi, luminescence, luciferase, promoter, StAR, counterscreen, SF-1, AF-2 mut, NR5A1, 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, inhibit, inhibitor, antagonist, adrenal, gonad, steroidogenic, hormone, AHC, stem cell, Ewing, tumor, cancer, primary screen, singlicate, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

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.

Protocol Summary:

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

Where:

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)
Comment
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 6.8 uM (6.8μM**)Normalized percent inhibition of the primary screen at a compound concentration of 6.8 uM.Float%
2Standard DeviationStandard deviation of the percent inhibition at 6.8 uMFloat
3Inhibition at 6.8 uM [1] (6.8μM**)Percent inhibition of the primary screen at a compound concentration of 6.8 uM.Float%
4Inhibition at 6.8 uM [2] (6.8μM**)Percent inhibition of the primary screen at a compound concentration of 6.8 uM.Float%
5Inhibition at 6.8 uM [3] (6.8μM**)Percent inhibition of the primary screen at a compound concentration of 6.8 uM.Float%

** Test Concentration.
Additional Information
Grant Number: R03 DA030558-01

Data Table (Concise)
Classification
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