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BioAssay: AID 463143

Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify activators of the liver X receptor (LXR)

Name: Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify activators of the liver X receptor (LXR). ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Inactive(6)
 
 
AID: 463143
Data Source: The Scripps Research Institute Molecular Screening Center (LXR_ACT_LUMI_0384_FOLD-CHANGE MCSRUN Round 1)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-08-26
Hold-until Date: 2011-08-25
Modify Date: 2011-08-25

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
2139Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).Summary2 depositor-specified cross reference: Summary (ROR modulators)
2277Center Based Initiative to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR): luminescence-based high throughput cell-based assay to identify modulators of human nuclear receptors.Screening depositor-specified cross reference: Primary screen (ROR modulators in singlicate)
463078Late stage assay provider results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescent-based assay to identify RORA inhibitorsOther depositor-specified cross reference: Late stage assay (RORA inhibitors)
504899Late stage assay provider results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): Diet-Induced obesity (DIO) mouse model studies to assess the effect of probe candidate on hepatic glucose productionOther depositor-specified cross reference
504906Late stage assay provider results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): radioligand binding assay for RORa using [3H]25-hydroxycholesterol to determine whether probe candidates bind directly to RORaOther depositor-specified cross reference
504907Late stage assay provider results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): fluorescence-based real-time polymerase chain reaction assay to determine the effect of probe candidates on endogenous expression of glucose-6-phosphatase (G6PC)Other depositor-specified cross reference
561Primary Cell-based High Throughput Screening assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA)Screening same project related to Summary assay
610Dose-response cell-based assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA)Confirmatory same project related to Summary assay
1901Summary of probe development efforts to identify inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA).Summary same project related to Summary assay
2117Late stage results from the probe development effort to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).Screening same project related to Summary assay
463144Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify inhibitors of glucose-6-phosphatase (G6PC)Other same project related to Summary assay
463145Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify activators of the farnesoid X receptor (FXR)Screening same project related to Summary assay
463147Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify RORG inhibitorsScreening same project related to Summary assay
463148Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify inhibitors of the human herpes virus VP16 transcriptional activator protein (VP16)Screening same project related to Summary assay
463150Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based dose response assay to identify activators of the liver X receptor (LXR)Confirmatory same project related to Summary assay
463151Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescent-based dose response assay to identify RORG inhibitorsConfirmatory same project related to Summary assay
463152Late stage assay provider results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based dose response assay to identify RORA inhibitorsConfirmatory same project related to Summary assay
463172Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based dose response assay to identify inhibitors of glucose-6-phosphatase (G6PC)Confirmatory same project related to Summary assay
624276Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): radioligand binding for ROR gamma using Scintillation Proximity Assay (SPA)Confirmatory1 same project related to Summary assay
624277Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): Luminescence-based cell-based assays using RORE-Luc, IL-17-Luc, and ABCA1-Luc ReportersOther1 same project related to Summary assay
624278Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): Fluorescence-based cell-based QPCR assays to determine endogenous expression of ROR gamma target genes, proinflammatory IL-17 and IL-23ROther1 same project related to Summary assay
624279Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): luminescence-based cell-based dose response panel assay against ROR alpha, ROR gamma, LXR, FXR, and VP16Confirmatory1 same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patrick Griffin, TSRI
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: U54 MH084512
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: LXR_ACT_LUMI_0384_FOLD-CHANGE MCSRUN Round 1

Name: Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): luminescence-based cell-based assay to identify activators of the liver X receptor (LXR).

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). RORalpha (RORa ; RORA; NR1F1) is one of three related orphan nuclear receptors, including RORbeta (RORB ; RORB; NR1F2) and RORgamma (RORg; RORC; NR1F3), known as "Retinoic Acid Receptor-related orphan receptors" (4).

RORA has unusual potential as a therapeutic target for the "metabolic syndrome" which results in pathologies such as insulin resistance, dyslipidemia, hypertension, and a pro-inflammatory state, that greatly elevates the risk of diabetes and atherosclerosis (5).The related RORC demonstrates significant expression in metabolic tissues such as liver, adipose, and skeletal muscle (6). These two receptors are implicated in several key aspects of this metabolic pathogenesis. For instance, the staggerer mouse, which carries a homozygous germline inactivation of RORA, shows low body weight, high food consumption (7-9), elevated angiogenesis in response to ischemia (10), susceptibility to atherosclerosis (9), and an abnormal serum lipid profile (11). RORG null mice exhibit normal plasma cholesterol levels, but when bred with the RORA staggerer mice, the resulting RORalpha/gamma knockout exhibits hypoglycemia not found in the single mutant animals. These studies reveal the functional redundancy of RORa and RORg in regulating blood glucose levels and highlight the need for RORalpha/gamma ligands that can bind to these receptors and modulate their transcriptional activity (12,13).

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. Medvedev A, Yan ZH, Hirose T, Giguere V, Jetten AM. Cloning of a cDNA encoding the murine orphan receptor RZR/ROR gamma and characterization of its response element. Gene. 1996 Nov 28;181(1-2):199-206.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, Shan B. Role of LXRs in control of lipogenesis. Genes Dev. 2000 Nov 15;14(22):2831-8.
13. The benzenesulfoamide T0901317 is a novel ROR / Inverse Agonist. Kumar N, Solt LA, Conkright JJ, Wang Y, Istrate MA, Busby SA, Garcia-Ordonez R, Burris TP, Griffin PR. Mol Pharm. Feb;77(2):228-36. Epub 2009 Nov 3.

Keywords:

Late stage, late stage AID, assay provider, purchased, synthesized, RAR-related orphan receptor A, ROR alpha, RORa, RORA, RORG, counterscreen, fold change, LXR, nuclear receptor, library, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, inverse agonist, transcriptional assay, assay provider, center based initiative, center-based, luciferase, luminescence, 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 whether powder samples of RORA inverse agonist probe candidates are nonselective due to activation of LXR. This assay employs the LXR-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-LXRLBD construct (GAL4-LXR) and a GAL4UAS-luciferase reporter construct are incubated for 18-24 hours with test compounds. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated LXR nuclear receptors. As designed, compounds that activate LXR activity will activate the GAL4-LXR construct, thereby increasing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to an increase in well luminescence. Compounds were tested in singlicate at a final nominal concentration of 10 uM. Six replicates were performed for each assay.

Protocol Summary:

Luciferase reporter assays were conducted using a pBind GAL4DBD-LXRLBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4E6 cells in 10 cm plates, 9 ug of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and re-plated in 384 well plates at a density of 10,000 cells/well. Following 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells.

The fold-change inhibition for each compound was calculated as follows:

[Cells_treated_with_Test_Compound] / [Cells_treated_with_Vehicle(DMSO)]

The average fold-change of each compound tested was calculated.

PubChem Activity Outcome and Score:

Any compound that exhibited a 2 fold change activation greater than the hit cutoff calculated (2 fold activation) was declared active.

Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for inactive compounds is 100-1. There are no active compounds.

List of Reagents:

384 well plates (PerkinElmer, part 6007688)
Britelite Plus (PerkinElmer, part 6016767)
DMEM (Mediatech Inc, Part 10 013 CV)
Fugene 6 (Roche Applied Science, part 11814443001)
Comment
This assay was performed by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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, or 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Fold Change at 10 uM (10μM**)Indicates normalized fold change at a nominal compound concentration of 10 micromolar.Float
2Standard DeviationStandard deviation derived from the normalized fold change of the replicate data for each compound.Float
3Fold Change at 10 uM [1] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate one.Float
4Fold Change at 10 uM [2] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate two.Float
5Fold Change at 10 uM [3] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate three.Float
6Fold Change at 10 uM [4] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate four.Float
7Fold Change at 10 uM [5] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate five.Float
8Fold Change at 10 uM [6] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate six.Float

** Test Concentration.
Additional Information
Grant Number: U54 MH084512

Data Table (Concise)
Data Table ( Complete ):     View All Data
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