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): radioligand binding for ROR gamma using Scintillation Proximity Assay (SPA)
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): radioligand binding for ROR gamma using Scintillation Proximity Assay (SPA). ..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_INH_RAD_0384_IC50 MDCSRUN (probe data: SPA assay)
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): radioligand binding for ROR gamma using Scintillation Proximity Assay (SPA).
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 ROR gamma (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 ROR gamma 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, SPA, Scintillation Proximity Assay, dose response, nuclear receptor, low throughput assay, NR1F3, inhibitor, inverse agonist, binding assay, assay provider, center based initiative, center-based, radioactivity, radiation, radioactive, binding, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this dose response assay is to determine whether a synthesized sample of a ROR Gamma inverse agonist probe candidate can bind to ROR gamma. This AID describes the results of the assay prvider's Scintillation Proximity Assay SPA).
Details of this assay can be found in Reference 14 Figure 10A.
SPA assays are competition assays which monitor the ability of test compounds (probe ML310) to compete with a radioligand for binding to ROR gamma. The ligand selected for these assays was T1317, a dual RORalpha/gamma inverse agonist that we identified several years ago as probe ML125. SPA assays contained 0.25 mg of beads (Glutathione YSi; PE no. RPNQ0033), 1 ug of GST-ROR gamma-LBD, 5 nM [3H]T1317 as radioligand and varying concentration of probe ML310 (SR-03000002211) in the assay buffer (50 mM HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mM NaCl and 5 mM MgCl2, 10% glycerol, 1 mM DTT, Complete protease inhibitor from Roche). All components were gently mixed, incubated for 20 h, and read in TopCount. The radioligand binding results were analyzed using GraphPad Prism software. These assays demonstrated that probe ML310 binds directly to the ROR gamma receptor, as it can displace (compete off) the radioligand [3H] T1317.
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:
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.
The PubChem Activity Score range for inactive compounds is 100-100. There are no active compounds.
List of Reagents:
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. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the test vial, or compounds that modulate binding. 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.
Data Table (Concise)