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BioAssay: AID 687017

Luminescence-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-1

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): repression of SF-1 (NR5A1) activated StAR promoter by full-length DAX-1. ..more
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 Tested Compounds
 Tested Compounds
All(249)
 
 
Active(55)
 
 
Inactive(194)
 
 
 Tested Substances
 Tested Substances
All(249)
 
 
Active(55)
 
 
Inactive(194)
 
 
AID: 687017
Data Source: The Scripps Research Institute Molecular Screening Center (DAX1-FULL_INH_LUMI_1536_3XIC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-05-31

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 55
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)
652134Luminescence-based cell-based primary high throughput confirmation 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-1screeningConfirmation assay (DAX-1 inhibitors in triplicate)
652136Counterscreen 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 AssayscreeningCounterscreen (SF-1 AP2 mutant assay in triplicate)
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: DAX1-FULL_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): repression of SF-1 (NR5A1) activated StAR promoter by full-length DAX-1.

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:

Dose response, DRUN, dose, triplicate, transactivation, co-transfection, reporter, luc, lumi, luminescence, luciferase, promoter, StAR, SF-1, 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 assay is to determine the IC50 of compounds that confirmed activity in a set of experiment 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 652134) and had no or little activity in the counterscreen assay entitled "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" (AID 652136).

SF-1 (steroidogenic factor 1; NR5A1) has been shown to function as a transcription factor for a variety of different steroidogenic enzyme genes in the adrenal gland and gonads. Specifically, SF-1 has been shown to induce differentiation of mesenchymal stem cells into steroidogenic cells with concomitant strong induction of StAR expression. DAX-1 has been shown to inhibit SF-1-mediated transactivation (5).

This assay employs HEK293T cells co-transfected with plasmids expressing SF-1 and a luciferase reporter under control of the StAR promoter together with an expression plasmid for DAX-1, followed by treatment with test compounds. Under normal conditions, DAX-1 represses the SF-1-induced expression of the luciferase reporter. As designed, a compound that inhibits DAX-1 activity will reduce DAX1-mediated repression of SF-1, thereby allowing SF-1 to increase StAR promoter activity, resulting in increased well luminescence. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 67.6 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 palsmid, 21 ug of the DAX-1 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:

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 (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 67.6 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 67.6 uM.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

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.

The PubChem Activity Score range for active compounds is 100-77, and for inactive compounds 71-0.

List of Reagents:

pGL2_1.3 kb_StAR luciferase reporter plasmid (Assay Provider)
pSG.SF-1 plasmid (Assay Provider)
pSV.DAX-1 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 fluorescence. 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
Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]

Assay: CurveFit [1]: Mask: Excluded Points

Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
8Response RangeThe range of Y.Float
9Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
10RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
11Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.0034 uM [1] (0.0034μM**)Value of %inhibition at 0.0034 micromolar inhibitor concentration; replicate [1]Float%
14Inhibition at 0.0034 uM [2] (0.0034μM**)Value of %inhibition at 0.0034 micromolar inhibitor concentration; replicate [2]Float%
15Inhibition at 0.0034 uM [3] (0.0034μM**)Value of %inhibition at 0.0034 micromolar inhibitor concentration; replicate [3]Float%
16Inhibition at 0.01 uM [1] (0.01μM**)Value of %inhibition at 0.01 micromolar inhibitor concentration; replicate [1]Float%
17Inhibition at 0.01 uM [2] (0.01μM**)Value of %inhibition at 0.01 micromolar inhibitor concentration; replicate [2]Float%
18Inhibition at 0.01 uM [3] (0.01μM**)Value of %inhibition at 0.01 micromolar inhibitor concentration; replicate [3]Float%
19Inhibition at 0.031 uM [1] (0.031μM**)Value of %inhibition at 0.031 micromolar inhibitor concentration; replicate [1]Float%
20Inhibition at 0.031 uM [2] (0.031μM**)Value of %inhibition at 0.031 micromolar inhibitor concentration; replicate [2]Float%
21Inhibition at 0.031 uM [3] (0.031μM**)Value of %inhibition at 0.031 micromolar inhibitor concentration; replicate [3]Float%
22Inhibition at 0.093 uM [1] (0.093μM**)Value of %inhibition at 0.093 micromolar inhibitor concentration; replicate [1]Float%
23Inhibition at 0.093 uM [2] (0.093μM**)Value of %inhibition at 0.093 micromolar inhibitor concentration; replicate [2]Float%
24Inhibition at 0.093 uM [3] (0.093μM**)Value of %inhibition at 0.093 micromolar inhibitor concentration; replicate [3]Float%
25Inhibition at 0.28 uM [1] (0.28μM**)Value of %inhibition at 0.28 micromolar inhibitor concentration; replicate [1]Float%
26Inhibition at 0.28 uM [2] (0.28μM**)Value of %inhibition at 0.28 micromolar inhibitor concentration; replicate [2]Float%
27Inhibition at 0.28 uM [3] (0.28μM**)Value of %inhibition at 0.28 micromolar inhibitor concentration; replicate [3]Float%
28Inhibition at 0.83 uM [1] (0.83μM**)Value of %inhibition at 0.83 micromolar inhibitor concentration; replicate [1]Float%
29Inhibition at 0.83 uM [2] (0.83μM**)Value of %inhibition at 0.83 micromolar inhibitor concentration; replicate [2]Float%
30Inhibition at 0.83 uM [3] (0.83μM**)Value of %inhibition at 0.83 micromolar inhibitor concentration; replicate [3]Float%
31Inhibition at 2.5 uM [1] (2.5μM**)Value of %inhibition at 2.5 micromolar inhibitor concentration; replicate [1]Float%
32Inhibition at 2.5 uM [2] (2.5μM**)Value of %inhibition at 2.5 micromolar inhibitor concentration; replicate [2]Float%
33Inhibition at 2.5 uM [3] (2.5μM**)Value of %inhibition at 2.5 micromolar inhibitor concentration; replicate [3]Float%
34Inhibition at 7.5 uM [1] (7.5μM**)Value of %inhibition at 7.5 micromolar inhibitor concentration; replicate [1]Float%
35Inhibition at 7.5 uM [2] (7.5μM**)Value of %inhibition at 7.5 micromolar inhibitor concentration; replicate [2]Float%
36Inhibition at 7.5 uM [3] (7.5μM**)Value of %inhibition at 7.5 micromolar inhibitor concentration; replicate [3]Float%
37Inhibition at 22.5 uM [1] (22.5μM**)Value of %inhibition at 22.5 micromolar inhibitor concentration; replicate [1]Float%
38Inhibition at 22.5 uM [2] (22.5μM**)Value of %inhibition at 22.5 micromolar inhibitor concentration; replicate [2]Float%
39Inhibition at 22.5 uM [3] (22.5μM**)Value of %inhibition at 22.5 micromolar inhibitor concentration; replicate [3]Float%
40Inhibition at 67.6 uM [1] (67.6μM**)Value of %inhibition at 67.6 micromolar inhibitor concentration; replicate [1]Float%
41Inhibition at 67.6 uM [2] (67.6μM**)Value of %inhibition at 67.6 micromolar inhibitor concentration; replicate [2]Float%
42Inhibition at 67.6 uM [3] (67.6μM**)Value of %inhibition at 67.6 micromolar inhibitor concentration; replicate [3]Float%

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

Data Table (Concise)
Classification
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