AID	Name	Type	Comment
2139	Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).	summary	Summary AID (EBNA1 inhibitors).
2277	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.	screening	Screening assay (Nuclear receptor profiling in singlicate).

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: ROR_MOD_PROBES_LATE_STAGE

Name: Late stage results from the probe development effort to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).

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 (ROR#; RORB; NR1F2) and RORgamma (ROR?; 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.

Summary of Probe Development Effort:

This AID reports the results of the Center-based effort to identify modulator probes of RORa and RORg. Following primary screening of a cell-based GAL4-human nuclear receptor profiling assay (Assay 1), certain compounds were identified as possible candidates for probe development. Compounds derived from these initial candidates were purchased as powders or synthesized at the SRIMSC and were tested for their ability to inhibit RORa and RORg in luciferase-based reporter assays performed in dose response assays starting at a nominal concentration of 20 micromolar (Assays 1 and 2). Compounds were subsequently counterscreened in dose response assays against the liver X receptor (LXR) and the farnesoid X receptor (FXR) to determine selectivity (Assays 3 and 4). Finally, compounds of interest were tested at a single concentration of 10 micromolar against VP16 to determine whether they were non-selective or cytotoxic (Assay 5), and probe candidates were tested against glucose-6-phosphatase to determine biologic efficacy on an ROR target gene.

The above probe development efforts resulted in the identification of two probes. The benzenesulfonamide compound SID 85257301 previously identified as a selective agonist of LXR (12) was identified here as a novel RORa/g inverse agonist probe that decreases the transcriptional activity of both ROR receptors (IC50 values = 2.0 and 1.73 micromolar, respectively). The second probe, SID 85257298 synthesized at the SRIMSC, was found to decrease RORa transcriptional activity (IC50 value = 2.47 micromolar). SID 85257298 represents an improvement over the prior art due to its lack of activity for LXR. Probe compound SID85257298 does not have activity against RORg (IC50 > 20 micromolar). These two probes are useful tools for examining ROR biology.

The results of our probe development efforts can be found at http://molscreen.florida.scripps.edu/probes.shtml. A manuscript has been published (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, Gigu#re 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 Pharmacol. 2010 Feb;77(2):228-36.

Keywords:

Late stage, probes, RAR-related orphan receptor A, ROR alpha, RORa, RORA, nuclear receptor, library, counterscreen, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, activator, transcriptional assay, assay provider, center based initiative, center-based, luciferase, luminescence, ROR gamma, RORg, RORC, RORG, LXR, FXR, NR1F3, Primary screen, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assays

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		RORA	5	1	retinoid-related orphan receptor alpha [Homo sapiens] [gi:216409696]		Taxonomy id: 9606 Gene id: 6095
2		RORG	4	2	retinoid-related orphan receptor gamma [Homo sapiens] [gi:216409700]		Taxonomy id: 9606 Gene id: 6097
3		LXR	3	3	nuclear receptor subfamily 1, group H, member 2 [Homo sapiens] [gi:85362735]		Taxonomy id: 9606 Gene id: 7376
4		FXR	1	5	nuclear receptor subfamily 1, group H, member 4 [Homo sapiens] [gi:4826980]		Taxonomy id: 9606 Gene id: 9971
5		VP16	1	6	transactivating tegument protein VP16 [Human herpesvirus 1] [gi:9629429]		Taxonomy id: 10298 Gene id: 2703416
6		G6Pase Promoter	2		glucose-6-phosphatase, catalytic subunit [Homo sapiens] [gi:4557599]		Taxonomy id: 9606 Gene id: 2538

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§ Panel component ID.

Please see AID 2277, the probe development assays below , reference(13), and Summary AID 2139 for all protocols performed in this Center-based probe development effort.

RORA Inhibition Assay (Assay 1):

The purpose of these assays is to identify compounds that inhibit RORa activity. This assay employs the RORa-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-RORaLBD construct (GAL4-RORa) 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 RORa nuclear receptors. As designed, compounds that inhibit RORa activity will prevent activation of the GAL4-RORa construct, thereby preventing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to a decrease in well luminescence. Compounds were tested in a 10-point dilution series starting at a nominal concentration of 20 micromolar. Six replicates were performed for each assay.

RORG Inhibition Assay (Assay 2):

The purpose of these assays is to identify compounds that inhibit RORg activity. This assay employs the RORg-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-RORgLBD 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 a 10-point dilution series starting at a nominal concentration of 20 micromolar. Six replicates were performed for each assay.

LXR Activation Assay (Assay 3):

The purpose of these assays is to identify compounds that increase liver X receptor (LXR) activity. 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 a 10-point dilution series starting at a nominal concentration of 20 micromolar. Six replicates were performed for each assay.

FXR Activation Assay (Assay 4):

The purpose of this assay is to identify compounds that increase farnesoid X receptor (FXR) activity. This assay employs the FXR-expressing cell line from a GAL4 nuclear receptor library. In this assay, HEK293T cells co-transfected with a GAL4DBD-FXRLBD construct (GAL4-FXR) 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 FXR nuclear receptors. As designed, compounds that activate FXR activity will activate the GAL4-FXR construct, thereby increasing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to an increase in well luminescence. Compounds were tested in a 10-point dilution series starting at a nominal concentration of 20 micromolar. Six replicates were performed for each assay.

VP16 Inhibition Counterscreen Assay (Assay 5):

In this counterscreen assay the nuclear receptor plasmid was replaced by the GAL4DBD-VP16LBD plasmid, which expresses the strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) fused to the GAL4 DBD. Cells are co-transfected with the 5xGAL4 response element (UAS) luciferase reporter to monitor GAL4DBD-VP16LBD activity, followed by incubation with test compounds for 18-24 hours. As designed, compounds that inhibit VP16 activity will decrease pGAL4DBD-VP16LBD activity, leading to reduced activation of the pG5-luc and decreased well luminescence. These compounds are likely to be nonselective inhibitors or cytotoxic. Compounds were tested in singlicate at a final nominal concentration of 10 micromolar. Six replicates were performed for each assay.

Glucose-6-Phosphatase (G6Pase) Promoter Assay (Assay 6):

The purpose of this assay is to determine whether probe candidates can modulate ROR target genes in cells. In these assays 293T cells were co-transfected with pS6 control plasmid or pS6 containing full length RORa along with G6Pase promoter. SRC-2 as a coactivator was also co-transfected with G6Pase promoter. Dose-response curve was determined by treating the transfected cells with varying concentrations of compound for 20 hours. Luciferase activity was measured and relative change was determined by normalizing to cells treated with vehicle only. Each data point was performed in eight replicates, n=8. Compounds were tested in a 10-point dilution series starting at a nominal concentration of 20 micromolar.

Probes were identified.

Grant Number: U54 MH084512