Bookmark and Share
BioAssay: AID 599

Counterscreen for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1): A cell-based dose-response assay for inhibition of the RAR-related orphan receptor A (RORA)

Counterscreen for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1): A cell-based dose-response assay for inhibition of the RAR-related orphan receptor A (RORA) ..more
_
   
 Tested Compounds
 Tested Compounds
All(357)
 
 
Active(219)
 
 
Inactive(138)
 
 
 Tested Substances
 Tested Substances
All(357)
 
 
Active(219)
 
 
Inactive(138)
 
 
AID: 599
Data Source: The Scripps Research Institute Molecular Screening Center (RORA_INH_Lumi_1536_CS_IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-21
Modify Date: 2007-03-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 219
Related Experiments
AIDNameTypeComment
525Primary Cell-based High Throughput Screening assay for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1)Screeningdepositor-specified cross reference
1844Summary of probe development efforts to identify inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1).Summarydepositor-specified cross reference
600Dose-response cell-based assay for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1)Confirmatorysame project related to Summary assay
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Orphagen Pharmaceuticals, San Diego, CA
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal number 1X01-MH077624-01

External Assay ID: RORA_INH_Lumi_1536_CS_IC50

Name:
Counterscreen for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1): A cell-based dose-response assay for inhibition of the RAR-related orphan receptor A (RORA)

Description:
Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding and ligand-binding domains.
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].
The nuclear receptor SF-1 (steroidogenic factor-1) belongs to the class of "unexplored" orphan nuclear receptors that have been poorly investigated at a pharmacological level.
SF-1 is expressed in the pituitary, testes, ovaries, and adrenal gland and regulates steroid hormone production at many levels, including direct regulation of expression of major P450 enzymes involved in steroid hormone synthesis [4].
To explore the potential of SF-1 as novel drug target, small molecule ligands have to be identified. To this end, we screened the MLSCN library via a cell-based assay developed by Orphagen Pharmaceuticals (San Diego, CA).
Ligands for SF-1 may have clinical applications as modulators of adrenal steroid synthesis. In particular, a properly designed antagonist to SF-1 is predicted to have therapeutic utility in the treatment of metastatic prostate cancer though suppression of both adrenal androgen and gonadal testosterone synthesis.

Another potential benefit of this effort could be the identification of SF-1 ligands that could become a novel class of centrally acting small molecules that regulate energy metabolism and obesity. Indeed, SF-1 is also a potential therapeutic target for the regulation of the ventromedial hypothalamus (VMH), known to process peripheral metabolic input and regulate energy homeostasis [5].

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 (Camb) 11: 741-746., 2003.
[4] Hammer GD and Ingraham HA. Steroidogenic Factor-1: Its role in endocrine organ development and differentiation. Frontiers in Neuroendocrinology 20: 199-223, 1999.
[5] Majdic G, Young M, Gomez-Sanchez E, Anderson P, Szczepaniak LS, Dobbins RL, McGarry JD, and Parker KL. Knockout mice lacking steroidogenic factor 1 are a novel genetic model of hypothalamic obesity. Endocrinology 143: 607-614., 2002.

Keywords:
steroidogenic factor, SF-1, SF1, nuclear receptor, NR5A1, FTZ1, FTZF1, ELP, AD4BP, transcriptional assay, cancer, obesity, CHO-K1, luciferase, luminescence, Scripps
Protocol
Assay Overview:
Compounds identified from a previously described set of experiments entitled "Primary Cell-based High Throughput Screening assay for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1)" (PubChem AID=525) were selected for further testing in this RAR-related orphan receptor A (RORA) assay. RORA is a nuclear receptor that does not share significant structural homology with SF-1 at the ligand-binding domain.
This counterscreen employs the same format as the assay used to select potential SF-1 inhibitors; except that here the biological target is RORA. A compound that appears as an inhibitor in both the SF-1 and RORA assays would be less desirable for follow-up, since the compound may be a non-selective inhibitor or format-specific artifact. On the other hand, compounds found inactive in this assay and confirmed active in SF-1 dose-response experiments are likely to be selective inhibitors of SF-1.
This dose-response counterscreen assay utilizes a fusion of the DNA-binding domain of the yeast transcriptional factor Gal4 with the ligand-binding domain of target receptor RORA (encoded by the pFA-hRORA plasmid, Orphagen Pharmaceuticals) to regulate a luciferase reporter containing 5xGal4 response elements at its promoter region (pG5-luc, Stratagene). Both pFA-hRORA and pG5-luc plasmids are transiently co-transfected in CHO-K1 (Chinese Hamster Ovary) cells. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated RORA nuclear receptors. Compounds that inhibit the basal transcription of luciferase are detected through the suppression of light emission using the SteadyLite luciferase detection kit (Perkin Elmer). Such compounds hence constitute false-positives inhibitors of the SF-1 nuclear receptor.
Three hundred and fifty nine (359) compounds selected during the primary screen were assessed in this dose-response experiment in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 100 micromolar. Dose response results for two substances have been invalidated and are not reported.
This assay was conducted in 1536-well format.
Protocol Summary:
Six million CHO-K1 cells were seeded in T-175 flasks (Corning part 431080) containing 20 milliliters of F12 media (Invitrogen part 31765-092) supplemented with 10% v/v fetal bovine serum (Gemini part 900-108) and 1% v/v penicillin-streptomycin-neomycin mix (Invitrogen part 15640-055). Flasks were then incubated for 20 hours at 37 degrees Celsius, 5%CO2 and 95% relative humidity.
The following day, CHO-K1 cells were transiently transfected with the pG5-luc reporter plasmid (Stratagene) and the RORA/Gal4 fusion expressing plasmid (pFA-hRORA, Orphagen Pharmaceuticals).
Transfection was performed using the TransIT-CHO transfection kit (Mirus part 2176) according to the manufacturer's protocol. Flasks were designated +RORA or -RORA. +RORA flasks received 1.2 milliliters of F12 media containing 54 microliters of TransIT CHO reagent (Mirus), 9 microliters of CHO Mojo reagent (Mirus), 9 ug of pG5-luc (Stratagene), 8.75 ug of empty pcDNA3.1 (Invitrogen), and 125 ng of pFA-hRORA plasmid (Orphagen Pharmaceuticals).
-RORA designated flasks received exactly the same transfection reagents and DNA excepting plasmid pFA-hRORA. Flasks were then placed back in the incubator at 37 degrees Celsius, 5% CO2 and 95% relative humidity.
Four hours after transfection, cells were trypsinized and suspended to a concentration of 800,000 cells per milliliter in F12 media (Invitrogen part 31765-092) supplemented with 10% v/v heat inactivated fetal bovine serum (Gemini part 900-108) and 1% v/v penicillin-streptomycin-neomycin mix (Invitrogen part 15640-055).
The assay began by dispensing 5 microliters of cell suspension to each well (i.e. 4,000 cells/well) of a white solid-bottom 1536-well plate. Cells from flasks designated -RORA were seeded in the first two columns of the 1536-well plate (mock-transfected positive control) and the remaining 46 columns were filled with +RORA cells.
One hour after seeding, +RORA cells were treated with 50 nL/well of test compounds or DMSO (negative control) and -RORA cells with 50nL/well of DMSO (positive control). Each compound dilution was assayed in triplicate, for a nominal total of 30 data points per dose-response. Plates were then placed in the incubator at 37 degrees Celsius, 5% CO2 and 95% relative humidity.
Twenty hours later, plates were equilibrated to room temperature for 20 minutes. A luciferase assay was performed by adding 5 microliters per well of the SteadyLite HTS reagent (Perkin Elmer part 6016989). After a 15 minutes incubation time, light emission was measured with the ViewLux reader (Perkin Elmer).
The percent inhibition of each compound has been calculated as follow:
%inhibition = (1-(median_positive_control - test_compound)/(median_positive_control - median_negative_control))*100
with positive control : -RORA cells treated with 1% DMSO
and negative control : +RORA cells treated with 1% DMSO
For each compound, percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept.
For each compound, percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept.
In cases where the highest concentration tested (100 micromolar) did not result in > 50% inhibition or where no curve fit was achieved, the IC50 was determined manually depending on the observed inhibition at the individual concentrations. Compounds with IC50 values of greater than 10 micromolar were considered inactive, compounds with IC50 equal or less than 10 micromolar are considered active.
The activity score was calculated based on pIC50 values for compounds for which an exact IC50 value was calculated and based on the observed pIC50 range, specifically the maximum lower limit of the pIC50 value as calculated from the lowest concentration for which greater than 50% inhibition is observed. This results in a conservative estimate of the activity score for compounds for which no exact IC50 value is given while maintaining a reasonable rank order of all compounds tested.
Comment
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to:
toxic compounds, compounds that inhibit luciferase activity, compounds that non-specifically inhibit or activate transcriptional activity.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: CHO-K1
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity QualifierActivity 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 concentrationString
2IC50Qualified IC50 in molar: The concentration at which 50% of the inhibition is observed (relative to 100% inhibition of DMSO-treated -RORA cells)FloatμM
3LogIC50Log10 of the qualified IC50 in M concentration.FloatLogIC50
4Hill CoefficientThe 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 shallower. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Curve Chi2A 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
9Curve R2This value indicates 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
10Excluded PointsNumber of excluded point in the dose-response curve (counting one data point per concentration).Integer
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 an outlier.Integer
12Inhibition at 5.0 nMNormalized percent inhibition at 5 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 15.1 nMNormalized percent inhibition at 15 nanomolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 45.3 nMNormalized percent inhibition at 45 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 135.8 nMNormalized percent inhibition at 136 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 407.4 nMNormalized percent inhibition at 407 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 1.2 uMNormalized percent inhibition at 1.2 micromolar inhibitor concentration; average of triplicate measurementFloat%
18Inhibition at 3.7 uMNormalized percent inhibition at 3.7 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 11.0 uMNormalized percent inhibition at 11 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 33.0 uMNormalized percent inhibition at 33 micromolar inhibitor concentration; average of triplicate measurement.Float%
21Inhibition at 99.0 uMNormalized percent inhibition at 99 micromolar inhibitor concentration; average of triplicate measurement.Float%

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
PageFrom: