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 assays using RORE-Luc, IL-17-Luc, and ABCA1-Luc Reporters
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): Luminescence-based cell-based assays using RORE-Luc, IL-17-Luc, and ABCA1-Luc Reporters. ..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: RORE-IL17-ABCA1_INH_LUMI_0384_FOLD_CHANGE MDCSRUN (probe ML310)
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): Luminescence-based cell-based assays using RORE-Luc, IL-17-Luc, and ABCA1-Luc Reporters.
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, response element, full length, plasmid, RORE, IL-17, ABCA1, reporter, luc, lumi, luminescence, cell, cell-based, transfection, reporter, assay, dose response, nuclear receptor, low throughput assay, NR1F3, inhibitor, inverse agonist, binding assay, assay provider, center based initiative, center-based, 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 modulate the activity of several reporter plasmids. This assay determines the efficacy and selectivity for the compound. bind to ROR gamma.
Details of this assay can be found in Reference 14 Figure 10A.
To confirm probe candidate ML310 (SR-03000002211) can repress the ROR gamma transcriptional activity, we used a full length receptor along with a multimerized ROR response element (RORE, five repeats of RORE) driving luciferase gene expression. In the absence of ROR gamma, there was no change in the luciferase activity of 5XRORE with the treatment of SR-03000002211 as described in Reference 14 Figure 4a. SR-03000002211 significantly repressed the 5XRORE luciferase activity when full length ROR gamma was added during transfection as described in Reference 14 Figure 4b; however, there was no effect of SR-03000002211 on ROR alpha cotransfection with 5XRORE (data not shown). To further to examine the activity of SR-03000002211 in more native promoter based assay, we performed additional cotransfection assays where we transfected cells with full-length ROR alpha or ROR gamma and a luciferase reporter gene driven by a native promoter derived from a known ROR target gene, Il17. Il17 is a well-characterized ROR target gene that plays a critical role in the inflammatory pathway.1 As shown in Figure 4c, in a ROR alpha cotransfection assay, treatment of cells with SR-03000002211 did not alter the transcription driven by the Il17 promoter. We observed a significant, > 50% suppression of transcriptional activity of Il17 promoter in a ROR gamma-dependent manner (Figure 4d). As previously mentioned, there was no increase in the full length LXR alpha target gene, ABCA1, promoter activity (Figure 4e). These results confirm that we have been able to selectively target ROR gamma.
For each test compound, fold change 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 fold change values were calculated from GraphPad Prism software. Full dose response curves can be found in Reference 14.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds that induced a fold change in the reporter plasmid less than 20% at 1 uM were considered inactive (Fold change > 0.8 or < 1.2). Compounds that induced a fold change in the reporter plasmid greater than 20% at 1 uM were considered active (Fold change < 0.8 or > 1.2).
Overall Outcome and Score:
If a compound was active in panels: 5XRORE-Luc + ROR Gamma and IL-17-Luc + ROR gamma, and inactive in all other panels, then the compound was considered active. Otherwise, the compound was considered inactive.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
As described in Reference 14.
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 well, 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
** Test Concentration. § Panel component ID.