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BioAssay: AID 610

Dose-response cell-based assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA)

Dose-response cell-based assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA) ..more
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 Tested Compounds
 Tested Compounds
All(273)
 
 
Active(228)
 
 
Inactive(45)
 
 
 Tested Substances
 Tested Substances
All(273)
 
 
Active(228)
 
 
Inactive(45)
 
 
AID: 610
Data Source: The Scripps Research Institute Molecular Screening Center (RORA_INH_Lumi_1536_IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2007-03-08

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 228
Depositor Specified Assays
AIDNameTypeComment
561Primary Cell-based High Throughput Screening assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA)screening
1901Summary of probe development efforts to identify inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA).summary
2139Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).summary
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)
Proposal number 1X01-MH077624-01

External Assay ID: RORA_INH_Lumi_1536_IC50

Name:

Dose-response cell-based assay for inhibitors of the Retinoic Acid Receptor-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].
RORA is one of three related orphan nuclear receptors, including RORB and RORC, known as "Retinoic Acid Receptor-related orphan receptors" [4].
RORA has unusual potential as a therapeutic target for "metabolic syndrome". This refers to a convergence of pathogenic factors, including insulin resistance, dyslipidemia, hypertension, and a proinflammatory state, that greatly elevate the risk of diabetes and atherosclerosis [5]. RORA has been shown to be implicated in several key aspects of this pathogenesis. For instance, the staggerer mouse, which carries a homozygous germline inactivation of RORA, shows low body weight, high food consumption [6-8], elevated angiogenesis in response to ischemia [9], susceptibility to atherosclerosis [8], and an abnormal serum lipid profile [10]. A combination of genetic and cellular studies also showed that RORA regulates lipoprotein levels and very likely has an impact on circadian rhythm and metabolism in peripheral tissue such as the liver.
Taken together, those observations highlight the need to identify specific ligands of RORA that could help understand its therapeutic potential and provide good chemical starting points for further drug development.

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]Jetten AM, Kurebayashi S, and Ueda E. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog Nucleic Acid Res Mol Biol 69: 205-247, 2001.
[5]Grundy SM, Brewer HB, Jr., Cleeman JI, Smith SC, Jr., and Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Arterioscler Thromb Vasc Biol 24: e13-18, 2004.
[6]Bertin R, Guastavino JM, and Portet R. Effects of cold acclimation on the energetic metabolism of the staggerer mutant mouse. Physiol Behav 47: 377-380, 1990
[7]Guastavino JM, Bertin R, and Portet R. Effects of the rearing temperature on the temporal feeding pattern of the staggerer mutant mouse. Physiol Behav 49: 405-409, 1991
[8]Mamontova A, Seguret-Mace S, Esposito B, Chaniale C, Bouly M, Delhaye-Bouchaud N, Luc G, Staels B, Duverger N, Mariani J, and Tedgui A. Severe atherosclerosis and hypoalphalipoproteinemia in the staggerer mouse, a mutant of the nuclear receptor RORalpha. Circulation 98: 2738-2743., 1998
[9]Besnard S, Silvestre J-S, Duriez M, Bakouche J, Lemaigre-Dubreuil Y, Mariani J, Levy BI, and Tedgui A. Increased ischemia-induced angiogenesis in the staggerer mouse, a mutant of the nuclear receptor RORa. Circ Res 89: 1209-1215, 2001.
[10] Raspe E, Duez H, Gervois P, Fievet C, Fruchart J-C, Besnard S, Mariani J, Tedgui A, and Staels B. Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha. J Biol Chem 276: 2865-2871, 2001.

Keywords:

RAR-related orphan receptor A, RORA, nuclear receptor, RZRA, ROR1, ROR2, ROR3, NR1F1, transcriptional assay, CHO-K1, luciferase, luminescence, Scripps, primary, inhibition
Protocol

Assay Overview:
Compounds identified from a previously described set of experiments entitled "Primary Cell-based High Throughput Screening assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA)" were selected for testing in this assay. Further information on the primary screen can be found by searching on this website for PubChem AID=561.
Among 278 compounds selected during the primary screening, 273 compounds were assessed in dose-response experiments in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 100 micromolar.
As with the primary screen, the dose-response 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 potential inhibitors of the RORA nuclear receptor.
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.

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.
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.Float
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 137 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 407.4 nMNormalized percent inhibition at 411 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 1.2 uMNormalized percent inhibition at 1.23 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 3.7 uMNormalized percent inhibition at 3.70 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 11.0 uMNormalized percent inhibition at 11.11 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 33.0 uMNormalized percent inhibition at 33.33 micromolar inhibitor concentration; average of triplicate measurement.Float%
21Inhibition at 99.0 uMNormalized percent inhibition at 100 micromolar inhibitor concentration; average of triplicate measurement.Float%

Data Table (Concise)
Classification
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