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BioAssay: AID 463151

Late 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 inhibitors

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): luminescent-based dose response assay to identify RORG inhibitors. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Inactive(1)
 
 
AID: 463151
Data Source: The Scripps Research Institute Molecular Screening Center (RORG_INH_LUMI_0384_IC50 MDCSRUN Round 1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-08-26
Hold-until Date: 2011-08-25
Modify Date: 2011-08-25

Data Table ( Complete ):           All
Target
Tested Compound:
Depositor Specified Assays
AIDNameTypeProbeComment
2139Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).summary2 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 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 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
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
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
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: RORG_INH_LUMI_0384_IC50 MDCSRUN 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): luminescent-based dose response assay to identify RORG inhibitors.

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, dose response, 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 a powder sample of an RORA inverse agonist probe candidate is nonselective due to inhibition of RORG. This dose response assay employs the RORgexpressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-RORaLBD construct (GAL4-RORg) 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 RORg nuclear receptors. As designed, compounds that inhibit RORg activity will prevent activation of the GAL4-RORg construct, thereby preventing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to a decrease in well luminescence. Compounds were tested in eight replicates using a 10-point dilution series starting at a nominal concentration of 3E-10 M.

Protocol Summary:

Luciferase reporter assays were conducted using a pBind GAL4DBD-RORgLBD 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.

For each test compound, fold inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using GraphPad Prism. The reported IC50 values were calculated from GraphPad Prism software.

PubChem Activity Outcome and Score:

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.

Any compound with a fold change > 0.7 at all test concentrations was assigned an activity score of zero. Any compound with a fold change <= 0.7 at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for inactive compounds is 0-0. 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
1QualifierActivity Qualifier identifies if the resultant data EC50 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
3Fold change at 10 uM [1] (10μM**)Fold change at 10 uM; replicate one.Float
4Fold change at 10 uM [2] (10μM**)Fold change at 10 uM; replicate two.Float
5Fold change at 10 uM [3] (10μM**)Fold change at 10 uM; replicate three.Float
6Fold change at 10 uM [4] (10μM**)Fold change at 10 uM; replicate four.Float
7Fold change at 10 uM [5] (10μM**)Fold change at 10 uM; replicate five.Float
8Fold change at 10 uM [6] (10μM**)Fold change at 10 uM; replicate six.Float
9Fold change at 10 uM [7] (10μM**)Fold change at 10 uM; replicate seven.Float
10Fold change at 10 uM [8] (10μM**)Fold change at 10 uM; replicate eight.Float
11Fold change at 3 uM [1] (3μM**)Fold change at 3 uM; replicate one.Float
12Fold change at 3 uM [2] (3μM**)Fold change at 3 uM; replicate two.Float
13Fold change at 3 uM [3] (3μM**)Fold change at 3 uM; replicate three.Float
14Fold change at 3 uM [4] (3μM**)Fold change at 3 uM; replicate four.Float
15Fold change at 3 uM [5] (3μM**)Fold change at 3 uM; replicate five.Float
16Fold change at 3 uM [6] (3μM**)Fold change at 3 uM; replicate six.Float
17Fold change at 3 uM [7] (3μM**)Fold change at 3 uM; replicate seven.Float
18Fold change at 3 uM [8] (3μM**)Fold change at 3 uM; replicate eight.Float
19Fold change at 1 uM [1] (1μM**)Fold change at 1 uM; replicate one.Float
20Fold change at 1 uM [2] (1μM**)Fold change at 1 uM; replicate two.Float
21Fold change at 1 uM [3] (1μM**)Fold change at 1 uM; replicate three.Float
22Fold change at 1 uM [4] (1μM**)Fold change at 1 uM; replicate four.Float
23Fold change at 1 uM [5] (1μM**)Fold change at 1 uM; replicate five.Float
24Fold change at 1 uM [6] (1μM**)Fold change at 1 uM; replicate six.Float
25Fold change at 1 uM [7] (1μM**)Fold change at 1 uM; replicate seven.Float
26Fold change at 1 uM [8] (1μM**)Fold change at 1 uM; replicate eight.Float
27Fold change at 0.3 uM [1] (0.3μM**)Fold change at 0.3 uM; replicate one.Float
28Fold change at 0.3 uM [2] (0.3μM**)Fold change at 0.3 uM; replicate two.Float
29Fold change at 0.3 uM [3] (0.3μM**)Fold change at 0.3 uM; replicate three.Float
30Fold change at 0.3 uM [4] (0.3μM**)Fold change at 0.3 uM; replicate four.Float
31Fold change at 0.3 uM [5] (0.3μM**)Fold change at 0.3 uM; replicate five.Float
32Fold change at 0.3 uM [6] (0.3μM**)Fold change at 0.3 uM; replicate six.Float
33Fold change at 0.3 uM [7] (0.3μM**)Fold change at 0.3 uM; replicate seven.Float
34Fold change at 0.3 uM [8] (0.3μM**)Fold change at 0.3 uM; replicate eight.Float
35Fold change at 0.1 uM [1] (0.1μM**)Fold change at 0.1 uM; replicate one.Float
36Fold change at 0.1 uM [2] (0.1μM**)Fold change at 0.1 uM; replicate two.Float
37Fold change at 0.1 uM [3] (0.1μM**)Fold change at 0.1 uM; replicate three.Float
38Fold change at 0.1 uM [4] (0.1μM**)Fold change at 0.1 uM; replicate four.Float
39Fold change at 0.1 uM [5] (0.1μM**)Fold change at 0.1 uM; replicate five.Float
40Fold change at 0.1 uM [6] (0.1μM**)Fold change at 0.1 uM; replicate six.Float
41Fold change at 0.1 uM [7] (0.1μM**)Fold change at 0.1 uM; replicate seven.Float
42Fold change at 0.1 uM [8] (0.1μM**)Fold change at 0.1 uM; replicate eight.Float
43Fold change at 0.03 uM [1] (0.03μM**)Fold change at 0.03 uM; replicate one.Float
44Fold change at 0.03 uM [2] (0.03μM**)Fold change at 0.03 uM; replicate two.Float
45Fold change at 0.03 uM [3] (0.03μM**)Fold change at 0.03 uM; replicate three.Float
46Fold change at 0.03 uM [4] (0.03μM**)Fold change at 0.03 uM; replicate four.Float
47Fold change at 0.03 uM [5] (0.03μM**)Fold change at 0.03 uM; replicate five.Float
48Fold change at 0.03 uM [6] (0.03μM**)Fold change at 0.03 uM; replicate six.Float
49Fold change at 0.03 uM [7] (0.03μM**)Fold change at 0.03 uM; replicate seven.Float
50Fold change at 0.03 uM [8] (0.03μM**)Fold change at 0.03 uM; replicate eight.Float
51Fold change at 0.01 uM [1] (0.01μM**)Fold change at 0.01 uM; replicate one.Float
52Fold change at 0.01 uM [2] (0.01μM**)Fold change at 0.01 uM; replicate two.Float
53Fold change at 0.01 uM [3] (0.01μM**)Fold change at 0.01 uM; replicate three.Float
54Fold change at 0.01 uM [4] (0.01μM**)Fold change at 0.01 uM; replicate four.Float
55Fold change at 0.01 uM [5] (0.01μM**)Fold change at 0.01 uM; replicate five.Float
56Fold change at 0.01 uM [6] (0.01μM**)Fold change at 0.01 uM; replicate six.Float
57Fold change at 0.01 uM [7] (0.01μM**)Fold change at 0.01 uM; replicate seven.Float
58Fold change at 0.01 uM [8] (0.01μM**)Fold change at 0.01 uM; replicate eight.Float
59Fold change at 0.003 uM [1] (0.003μM**)Fold change at 0.003 uM; replicate one.Float
60Fold change at 0.003 uM [2] (0.003μM**)Fold change at 0.003 uM; replicate two.Float
61Fold change at 0.003 uM [3] (0.003μM**)Fold change at 0.003 uM; replicate three.Float
62Fold change at 0.003 uM [4] (0.003μM**)Fold change at 0.003 uM; replicate four.Float
63Fold change at 0.003 uM [5] (0.003μM**)Fold change at 0.003 uM; replicate five.Float
64Fold change at 0.003 uM [6] (0.003μM**)Fold change at 0.003 uM; replicate six.Float
65Fold change at 0.003 uM [7] (0.003μM**)Fold change at 0.003 uM; replicate seven.Float
66Fold change at 0.003 uM [8] (0.003μM**)Fold change at 0.003 uM; replicate eight.Float
67Fold change at 0.001 uM [1] (0.001μM**)Fold change at 0.001 uM; replicate one.Float
68Fold change at 0.001 uM [2] (0.001μM**)Fold change at 0.001 uM; replicate two.Float
69Fold change at 0.001 uM [3] (0.001μM**)Fold change at 0.001 uM; replicate three.Float
70Fold change at 0.001 uM [4] (0.001μM**)Fold change at 0.001 uM; replicate four.Float
71Fold change at 0.001 uM [5] (0.001μM**)Fold change at 0.001 uM; replicate five.Float
72Fold change at 0.001 uM [6] (0.001μM**)Fold change at 0.001 uM; replicate six.Float
73Fold change at 0.001 uM [7] (0.001μM**)Fold change at 0.001 uM; replicate seven.Float
74Fold change at 0.001 uM [8] (0.001μM**)Fold change at 0.001 uM; replicate eight.Float
75Fold change at 0.0003 uM [1] (0.0003μM**)Fold change at 0.0003 uM; replicate one.Float
76Fold change at 0.0003 uM [2] (0.0003μM**)Fold change at 0.0003 uM; replicate two.Float
77Fold change at 0.0003 uM [3] (0.0003μM**)Fold change at 0.0003 uM; replicate three.Float
78Fold change at 0.0003 uM [4] (0.0003μM**)Fold change at 0.0003 uM; replicate four.Float
79Fold change at 0.0003 uM [5] (0.0003μM**)Fold change at 0.0003 uM; replicate five.Float
80Fold change at 0.0003 uM [6] (0.0003μM**)Fold change at 0.0003 uM; replicate six.Float
81Fold change at 0.0003 uM [7] (0.0003μM**)Fold change at 0.0003 uM; replicate seven.Float
82Fold change at 0.0003 uM [8] (0.0003μM**)Fold change at 0.0003 uM; replicate eight.Float

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

Data Table (Concise)
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