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: ENDOG-G6P_INH_LUMI_0384_Fold-Change MCSRUN Round 1

Name: Late 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).

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, 14).

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. Kumar N, Solt LA, Conkright JJ, Wang Y, Istrate MA, Busby SA, Garcia-Ordonez R, Burris TP, Griffin PR. The benzenesulfoamide T0901317 is a novel RORalpha/gamma Inverse Agonist. Mol Pharm. Feb;77(2):228-36. Epub 2009 Nov 3.
14. Kumar N, Kojetin DJ, Solt LA, Kumar KG, Nuhant P, Duckett DR, Cameron MD, Butler AA, Roush WR, Griffin PR, Burris TP.Identification of SR3335 (ML-176): A Synthetic RORalpha Selective Inverse Agonist. ACS Chem Biol. 2011 Mar 18;6(3):218-22. Epub 2010 Dec 6.

Keywords:

Late stage, late stage AID, assay provider, purchased, synthesized, RAR-related orphan receptor A, ROR alpha, RORa, RORA, nuclear receptor, G6PC, G6P, glucose-6-phosphatase, siRNA, fold-change, ML176, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, inverse agonist, transcriptional assay, selective, assay provider, center based initiative, center-based, RTPCR, RT-PCR, QPCR, PCR, SYBR, SYBR green, RNA, expression, endogenous, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assays

PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		Control siRNA			glucose-6-phosphatase [Homo sapiens] [gi:4557599]		Taxonomy id: 9606 Gene id: 2538
2		RORA + RORG siRNA			glucose-6-phosphatase [Homo sapiens] [gi:4557599]		Taxonomy id: 9606 Gene id: 2538

---

§ Panel component ID.

Assay Overview:

The purpose of this assay is to determine whether powder sample of a possible RORA inverse agonist probe candidate can inhibit the endogenous expression of RORA target gene (G6Pase; glucose-6-phosphatase). In this assay, HepG2 cells are incubated for 20 hours with test compound. Some cells were treated with siRNAs against RORs to mimic the ROR inhibition effect of the compound. As designed, a compound that inhibits RORa activity will reduce levels of the endogenous G6Pase mRNA. The compound was tested at a nominal concentration of 10 uM.

Protocol Summary:

HepG2 cells were treated with 10 uM compound (SID 85261497; ML176). Total RNA extraction and cDNA synthesis as well as the QPCR were performed as previously described (see PMID 20427485). Briefly, RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA) and quantified. Gene expression was determined using an ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA) and a SYBR Green detection system (Bio-Rad, Hercules, CA). A standard curve was generated using cDNA pooled from the experimental samples. Relative expression levels were determined by normalization to glyceraldehyde-3-phosphate dehydrogenase and expressed as arbitrary units. The expression of endogenous G6Pase in HepG2 cells was reduced by treatment with siRNA against human RORa (RORalpha) and RORg (RORgamma). siRNA oligos against RORs were transfected into HepG2 cells at 50 nM. Forty-two hours post transfection cells were treated for 6 h with vehicle (DMSO) or ML176 (10 mM). Total RNA was prepared from these cells and subjected to RT-PCR to measure the mRNA levels.

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

Cells_treated_with_Test_Compound / Cells_treated_with_Vehicle_(DMSO)

G6Pase gene levels were normalized to GAPDH gene levels.

The average fold change of each compound tested was calculated.

PubChem Activity Outcome and Score:

Any compound with a fold G6Pase mRNA level > 0.8 was considered inactive. Any compound with a fold G6Pase mRNA level < 0.8 was considered active.

Active compounds were given a score of 100 and inactives were scored 0.

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

List of Reagents:

RNeasy kit (QIAGEN, Valencia, CA)
384 well plates (PerkinElmer, part 6007688)
SYBR Green detection system (Bio-Rad, Hercules, CA)
DMEM (Mediatech Inc, Part 10 013 CV)
Fugene 6 (Roche Applied Science, part 11814443001).

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 fluorescence or general transcriptional mechanisms. 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.

Grant Number: U54 MH084512