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BioAssay: AID 463144

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 inhibitors of glucose-6-phosphatase (G6PC)

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 inhibitors of glucose-6 phosphatase (G6PC). ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
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Active(1)
 
 
AID: 463144
Data Source: The Scripps Research Institute Molecular Screening Center (GLUCOSE-6-PHOSPHATASE_INH_LUMI_0384_FOLD-CHANGE MCSRUN Round..)
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 ):           Active    All
Target
BioActive Compound: 1
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: GLUCOSE-6-PHOSPHATASE_INH_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 inhibitors of glucose-6 phosphatase (G6PC).

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, G6PC, G6P, glucose-6-phosphatase, counterscreen, fold change, 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 samples of compounds identified as possible probe candidates for RORA can inhibit the RORA target gene glucose-6-phosphatase (G6PC) in cells. In these assays 293T cells were co-transfected with pS6 control plasmid or pS6 containing full length RORa along with G6PC promoter. SRC-2 as a coactivator was also co-transfected with G6PC promoter. Transfected cells were treated with compound for 20 hours. As designed, compounds that inhibit G6PC activity will decrease G6PC promoter activity, leading to reduced production of luciferase and decreased well luminescence. Luciferase activity was measured and relative change was determined by normalizing to cells treated with vehicle only. Compounds were tested using six replicates at a nominal concentration of 10 uM.

Protocol Summary:

Luciferase reporter assays were conducted by transfecting a native G6PC promoter-linked to luciferase, SRC-2 as co-activator in the presence of full length RORa 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 fold change inhibition less than the hit cutoff calculated (0.7 fold inhibition) 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 active compounds is 100-100. There are no inactive 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 of the counterscreen assay derived from the normalized percent activation of 8 replicates 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
9Fold Change at 10 uM [7] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate seven.Float
10Fold Change at 10 uM [8] (10μM**)Indicates fold change at a nominal compound concentration of 10 micromolar; replicate eight.Float

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

Data Table (Concise)
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