Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): luminescence-based cell-based dose response panel assay against ROR alpha, ROR gamma, LXR, FXR, and VP16
Name: Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): luminescent-based dose response panel assay against ROR alpha, ROR gamma, LXR, FXR, and VP16. ..more
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: RORG-RORA-LXR-FXR-VP16_INH_LUMI_0384_IC50 MDCSRUN (probe panel)
Name: Late stage assay provider counterscreen from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptor Gamma (RORC): luminescent-based dose response panel assay against ROR alpha, ROR gamma, LXR, FXR, and VP16.
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).
Here we describe the identification of a selective ROR gamma synthetic ligand, ML310, which functions as an inverse agonist. We show that ML310 can displace T1317 in a binding assay and does interact with ROR gamma protein to stabilize the protein in HDX-based experiments. In cotransfection assays, ML310 suppresses transcription activity in both GAL4-ROR gamma ligand binding domain (LBD) and full-length ROR gamma contexts. Furthermore, treatment of EL-4 cells with ML310 results in suppression of gene expression and production of IL-17. These data strongly suggest that ML310 is a potent and efficacious ROR gamma modulator and represses its activity. Thus, we have identified the first synthetic ROR gamma selective inverse agonist, and this compound can be utilized as a chemical tool to probe the function of this receptor both in vitro and in vivo. Additionally, our data suggests that ROR gamma inverse agonists may hold utility for the treatment of autoimmune disorders (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. 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.
14. Identification of SR2211: a potent synthetic ROR gamma-selective modulator.
Kumar N, Lyda B, Chang MR, Lauer JL, Solt LA, Burris TP, Kamenecka TM, Griffin PR. ACS Chem Biol. 2012 Apr 20;7(4):672-7. Epub 2012 Feb 13.
ML310, SR-03000002211, Late stage, late stage AID, assay provider, purchased, synthesized, RAR-related orphan receptor G, RORG, RORa, LXRa, FXR, LXR, panel, cell, cell-based, reporter, dose response, nuclear receptor, library, low throughput assay, NR1F3, inhibitor, inverse agonist, transcriptional assay, assay provider, center based initiative, center-based, luciferase, luminescence, GAL4, yeast, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
§ Panel component ID.
The purpose of this dose response assay is to determine whether a synthesized sample of a ROR gamma inverse agonist probe candidate can inhibit the activity of ROR gamma. This assay also determines the activity of the compound against ROR alpha, LXR, FXR, and VP16. In this assay, HEK293T cells co-transfected with a GAL4UAS-luciferase reporter construct and one each of the following plasmids: Gal4DBD-RORalpha, Gal4DBD-ROR gamma, Gal4DBD-LXRa, Gal4DBD-FXR, or Gal4DBD-VP16. Cells were next incubated for 20 hours with test compounds. As designed, compounds that inhibit ROR gamma (or other targets) will prevent activation of the GAL4- construct, thereby preventing GAL4DBD-mediated activation of the GAL4UAS-luciferase reporter, leading to a change in well luminescence. Relative change was determined by normalizing to cells treated with vehicle. Compounds were tested using 6 replicates.
Details of this assay can be found in Reference 14 Figure 3.
Luciferase reporter assays were conducted using a pBind GAL4DBD-RORgLBD construct and UAS luciferase reporter cotransfected 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.
For each test compound, fold inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using GraphPad Prism. The reported IC50 values were calculated from GraphPad Prism software.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Overall Outcome and Score:
Compounds that are only active against ROR gamma are considered active. If compounds are active against a target other than ROR gamma, then the compound is considered inactive.
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)
This assay was performed by the assay provider as described in Reference 14. 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. § Panel component ID.