Late 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 production
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): Diet-Induced obesity (DIO) mouse model studies to assess the effect of probe candidate on hepatic glucose production. ..more
BioActive Compound: 1
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: DIO-MOUSE-GLUCOSE_INH_ RORA MDRUN 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): Diet-Induced obesity (DIO) mouse model studies to assess the effect of probe candidate on hepatic glucose production.
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).
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.
Late stage, late stage AID, assay provider, purchased, synthesized, RAR-related orphan receptor A, ROR alpha, RORa, RORA, nuclear receptor, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, inverse agonist, mouse, DIO, diet-induced obesity, glucose, gluc, ML176, assay provider, center based initiative, center-based, selective, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether a powder sample of a possible RORA inverse agonist probe candidate reduce hepatic glucose production in animal models. In this assay, mice with diet-induced obesity are treated with probe candidate SID 85261497 and different measures of glucose production and body phenotypes are measured. As designed, a compound that reduces glucose production in the liver will reduce the expression of the liver genes G6Pase and PEPCK. The compound was given to animals at a nominal concentration of 15 mg/kg twice daily for 6 days.
This protocol has been previously described (see PMID 21090593).
Thirty-week-old diet-induced obese (DIO) C57BL/6 male mice were purchased from Jackson Laboratories and maintained on a 65% kcal high-fat diet from weaning. DIO mice were treated twice per day (07:00 h and 18:00 h) with 15 mg/kg SR3335 or vehicle for 6 days ip. Pyruvate tolerance test was conducted on day 6 of the treatment. Food was removed from mice in the morning after SR3335 injection, the mice were fasted for 6 h, and the pyruvate tolerance test was conducted at 13:00 h. Time 0 blood glucose was measured taken from the tail nip, and the pyruvate challenge was initiated by injection of 2 g/kg of pyruvate ip followed by measuring blood glucose at 15, 30, and 60 min following the injection. Blood glucose was measured by a one touch ultra glucose meter.
In 30 week old diet-induced-obesity mice were injected with the compound SID 85261497 (ML176) at the dose of 15 mg/kg twice daily showed significance reduction in hepatic glucose production in pyruvate tolerance test. Also, the expression of hepatic genes, G6Pase and PEPCK involved in the gluconeogenesis, was reduced by ~50%. Food intake and body weight were measured daily. At day 7 of the study, pyruvate tolerance tests and plasma insulin levels were determined in the fasted animals. The livers were isolated, followed by collection of mRNA, followed by determination of the expression of several LXR and RORA target genes.
See PMID 21090593 for all details.
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 reduced G6Pase and PEPCK mRNA levels to > 60% of control and or reduced blood glucose in the pyruvate tolerance test to levels to > 80% of control was assigned inactive. Any compound that reduced G6Pase and PEPCK mRNA levels to < 60% of control and reduced blood glucose in the pyruvate tolerance test to < 80% was assigned active.
Active compounds were given a score of 100, and inactives were given a score of 0.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
PMID 21090593 for all details.
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 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.
Data Table (Concise)