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: Hugh Rosen, TSRI
External Assay ID: NUCLEAR-RECEPTOR_MOD_LUMI_384_1XFOLDCHANGE

Name: Center 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.

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 (RORβ; RORB; NR1F2) and RORgamma (RORγ; 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.

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 RORa/? Inverse Agonist. Kumar N, Solt LA, Conkright JJ, Wang Y, Istrate MA, Busby SA, Garcia-Ordonez R, Burris TP, Griffin PR. Mol Pharm. 2009 Nov 10. [Epub ahead of print].

Keywords:

Nuclear receptor, NR, profiling, primary, Center-based initiative, RAR-related orphan receptor A, ROR alpha, RORa, RORA, library, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, activator, modulator, transcriptional assay, luciferase, luminescence, ROR gamma, RORg, RORC, RORG, NR1F3, Scripps Florida, assay provider, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

NUCLEAR-RECEPTOR_MOD_LUMI_384_1XFOLDCHANGE

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1			3	11	AR protein [Homo sapiens] [gi:124375976]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 367
2			4	10	estrogen receptor alpha isoform 1 [Homo sapiens] [gi:62821794]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2099
3			1	13	estrogen receptor beta isoform 2 [Homo sapiens] [gi:94538325]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2100
4			1	13	estrogen-related receptor alpha [Homo sapiens] [gi:18860920]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2101
5			1	13	estrogen-related receptor beta [Homo sapiens] [gi:238550159]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2103
6			7	7	estrogen-related receptor gamma isoform 2 [Homo sapiens] [gi:197209821]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2104
7			6	8	hepatocyte nuclear factor 4 alpha isoform e [Homo sapiens] [gi:71725341]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 3172
8			4	10	hepatocyte nuclear factor 4, gamma [Homo sapiens] [gi:115583654]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 3174
9			1	13	nuclear receptor subfamily 0, group B, member 1 [Homo sapiens] [gi:5016090]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 190
10			1	13	nuclear receptor subfamily 0, group B, member 1 [Homo sapiens] [gi:5016090]	Full Length	Taxonomy id: 9606 Gene id: 190
11				14	short heterodimer partner [Homo sapiens] [gi:13259503]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 8431
12			1	13	nuclear receptor subfamily 1, group D, member 1 [Homo sapiens] [gi:13430848]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 9572
13				14	nuclear receptor subfamily 1, group D, member 2 isoform 1 [Homo sapiens] [gi:224177491]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 9975
14			9	5	nuclear receptor subfamily 1, group H, member 2 [Homo sapiens] [gi:85362735]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7376
15			2	12	nuclear receptor subfamily 1, group H, member 3 isoform c [Homo sapiens] [gi:194294519]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 10062
16			1	13	unnamed protein product [Homo sapiens] [gi:221041442]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 9971
17			2	12	nuclear receptor subfamily 1, group I, member 2 isoform 1 [Homo sapiens] [gi:34398348]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 8856
18			2	12	constitutive androstane receptor isoform 12 [Homo sapiens] [gi:117938738]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 9970
19			2	12	nuclear receptor subfamily 2, group C, member 1 isoform c [Homo sapiens] [gi:189491766]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7181
20			1	13	nuclear receptor subfamily 2, group C, member 2 [Homo sapiens] [gi:36950991]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7182
21				14	nuclear receptor subfamily 2, group E, member 1 [Homo sapiens] [gi:4507537]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7101
22				14	nuclear receptor subfamily 2, group E, member 1 [Homo sapiens] [gi:4507537]	Full Length	Taxonomy id: 9606 Gene id: 7101
23				14	photoreceptor-specific nuclear receptor isoform b [Homo sapiens] [gi:7657395]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 10002
24			6	8	nuclear receptor subfamily 2, group F, member 1 [Homo sapiens] [gi:5032173]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7025
25				14	nuclear receptor subfamily 2, group F, member 1 [Homo sapiens] [gi:5032173]	Full Length	Taxonomy id: 9606 Gene id: 7025
26			1	13	nuclear receptor subfamily 2, group F, member 2 isoform b [Homo sapiens] [gi:223555949]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7026
27				14	nuclear receptor subfamily 2, group F, member 2 isoform b [Homo sapiens] [gi:223555949]	Full Length	Taxonomy id: 9606 Gene id: 7026
28			2	12	nuclear receptor subfamily 2, group F, member 6 [Homo sapiens] [gi:216409732]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2063
29			4	10	glucocorticoid receptor isoform gamma [Homo sapiens] [gi:66528677]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2908
30			5	9	nuclear receptor subfamily 3, group C, member 2 isoform 2 [Homo sapiens] [gi:260656020]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 4306
31				14	nuclear receptor subfamily 4, group A, member 1 [Homo sapiens] [gi:21361342]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 3164
32			1	13	nuclear receptor subfamily 4, group A, member 1 [Homo sapiens] [gi:21361342]	Full Length	Taxonomy id: 9606 Gene id: 3164
33			4	10	nuclear receptor subfamily 4, group A, member 2 [Homo sapiens] [gi:5453822]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 4929
34			6	8	nuclear receptor subfamily 4, group A, member 2 [Homo sapiens] [gi:5453822]	Full Length	Taxonomy id: 9606 Gene id: 4929
35			5	9	nuclear receptor subfamily 4, group A, member 3 isoform a [Homo sapiens] [gi:27894357]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 8013
36			13	1	nuclear receptor subfamily 5, group A, member 1 [Homo sapiens] [gi:20070193]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2516
37			9	5	nuclear receptor subfamily 5, group A, member 1 [Homo sapiens] [gi:20070193]	Full Length	Taxonomy id: 9606 Gene id: 2516
38			11	3	nuclear receptor subfamily 5, group A, member 2 isoform 2 [Homo sapiens] [gi:4504343]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2494
39			1	13	nuclear receptor subfamily 6, group A, member 1 [Homo sapiens] [gi:55859655]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 2649
40			3	11	nuclear receptor subfamily 6, group A, member 1 [Homo sapiens] [gi:55859655]	Full Length	Taxonomy id: 9606 Gene id: 2649
41			2	12	progesterone receptor isoform B [Homo sapiens] [gi:110611914]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5241
42			1	13	peroxisome proliferative activated receptor, alpha [Homo sapiens] [gi:50348666]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5465
43			3	11	peroxisome proliferative activated receptor, delta isoform 2 [Homo sapiens] [gi:29171750]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5467
44				14	peroxisome proliferative activated receptor gamma isoform 1 [Homo sapiens] [gi:116284368]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5468
45				14	peroxisome proliferative activated receptor gamma isoform 1 [Homo sapiens] [gi:116284368]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5468
46			6	8	retinoic acid receptor, alpha isoform 1 [Homo sapiens] [gi:4506419]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5914
47			6	8	retinoic acid receptor, alpha isoform 1 [Homo sapiens] [gi:4506419]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5914
48			4	10	retinoic acid receptor, beta isoform 1 [Homo sapiens] [gi:14916494]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5915
49			3	11	retinoic acid receptor, beta isoform 1 [Homo sapiens] [gi:14916494]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5915
50			5	9	retinoic acid receptor, gamma isoform 1 [Homo sapiens] [gi:4506423]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5916
51			2	12	retinoic acid receptor, gamma isoform 1 [Homo sapiens] [gi:4506423]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 5916
52			7	7	RAR-related orphan receptor A isoform c [Homo sapiens] [gi:4506577]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6095
53			2	12	RAR-related orphan receptor B [Homo sapiens] [gi:19743907]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6096
54			6	8	RAR-related orphan receptor C isoform b [Homo sapiens] [gi:48255918]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6097
55			4	10	retinoid X receptor, alpha [Homo sapiens] [gi:4506755]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6256
56			4	10	retinoid X receptor, alpha [Homo sapiens] [gi:4506755]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6256
57			7	7	retinoid X receptor, alpha [Homo sapiens] [gi:4506755]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6256
58			4	10	retinoid X receptor, alpha [Homo sapiens] [gi:4506755]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6256
59			3	11	retinoid X receptor, gamma isoform b [Homo sapiens] [gi:58331207]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6258
60				14	retinoid X receptor, gamma isoform b [Homo sapiens] [gi:58331207]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 6258
61			2	12	thyroid hormone receptor, alpha isoform 1 [Homo sapiens] [gi:40806160]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7067
62			3	11	thyroid hormone receptor, beta [Homo sapiens] [gi:40806162]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7068
63			1	13	vitamin D (1,25-dihydroxyvitamin D3) receptor [Homo sapiens] [gi:4507883]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7421
64			3	11	vitamin D (1,25-dihydroxyvitamin D3) receptor [Homo sapiens] [gi:4507883]	Ligand Binding Domain	Taxonomy id: 9606 Gene id: 7421
65			3	11	vitamin D (1,25-dihydroxyvitamin D3) receptor [Homo sapiens] [gi:4507883]	Variant 5' UTR	Taxonomy id: 9606 Gene id: 7421
66				14	transactivating tegument protein VP16 [Human herpesvirus 1] [gi:9629429]	Ligand Binding Domain	Taxonomy id: 10298 Gene id: 2703416

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§ Panel component ID.

Assay Overview:

The purpose of this assay is to identify compounds that act as modulators of human nuclear receptors and to demonstrate the utility of the GAL4 nuclear receptor library (13). This assay screens endogenous and synthetic ligands against a GAL4 nuclear receptor library which was built by replacing the endogenous N-terminus and DNA-binding domain (DBD) of all 48 receptors with a GAL4 DBD. The fusion constructs consist of the GAL4 DBD, the hinge domain, ligand binding domain (LBD), and F domain if applicable, of the human receptors. Plasmids coding for full-length receptors were also included for some receptors. In this assay HEK293T cells are co-transfected with a single GAL4 receptor and a luciferase reporter containing an upstream activating sequence (UAS) recognized by the GAL4 DBD, followed by treatment with test compounds. As designed, compounds that modulate activity of a particular nuclear receptor will modulate the binding of the GAL4 DBD to the UAS, thereby modulating luciferase production, resulting in an increase or decrease in well luminescence. Each compound was evaluated using two plates of the GAL4 NR library for a total of six replicates, at a nominal test concentration of 2 micromolar.

Protocol Summary:

The nuclear receptor library was plated into 384-well plates. HEK293T cells were reverse transfected with the well-specific construct and the UAS luciferase reporter pGL4.31 using Fugene6 transfection reagent in a final volume of 40 microliters. Control wells containing constructs encoding for the GAL4 DBD alone (pBind) or GAL4 fused to VP16 were also analyzed. After 24 hours, optimized compounds (2 micromolar final concentration) or DMSO was added to the plates and allowed to incubate for 20 hours. Next, 40 microliters of BriteLite was added to all wells and luciferase activity was measured on the PerkinElmer Envision 2104. Compounds that attenuate the GAL4-VP16-dependent luciferase activity are considered promiscuous or cytotoxic. To correct for plate-to-plate variance, sample data was normalized to wells containing vector only (69 wells) to determine Normalized Data Values (NDV). For each nuclear receptor, the average of NDV treated with a particular test compound was divided by the average NDV of wells treated with DMSO. This resulted in a fold-activation or fold-inhibition value for each receptor-compound pair. Because the activity of VP16 should not be impacted by these compounds and a change in this value is indicative of cell toxicity or general disruptions in transcription/translation, this fold-change value was then normalized to the VP16 fold-change. Compounds that induced in any receptor an average fold change that was three standard deviations from the VP16 value were considered active for that particular receptor.

Activity Score:

The PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for actives and 0 for inactives.

List of Reagents:

Fugene6 transfection reagent (Roche Applied Sciences)
BriteLite reagent (Perkin Elmer)
pGL4.31 construct (Promega)
384-well plates (Greiner, part 789176)

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, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal. The protein sequences encoded by the VDR and VDR-var2 plasmids are identical; there is an alternatively spliced exon in the 5'UTR in VDR-var2.

Grant Number: U54 MH084512