Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).
Name: Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR). ..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: ROR_MOD_PROBES_SUMMARY
Name: Summary of probe development efforts to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).
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 (RORalpha; RORA; NR1F1) is one of three related orphan nuclear receptors, including RORbeta (RORbeta]; RORB; NR1F2) and RORgamma (RORgamma; 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.
Summary of Probe Development Effort:
Following primary compound profiling of a cell-based GAL4-human nuclear receptor profiling assay, certain compounds were identified as possible candidates for probe development. Compounds derived from these initial candidates were purchased as powders or synthesized at the SRIMSC and were tested for their ability to inhibit RORa and RORg in luciferase-based reporter assays performed at a single concentration of 10 micromolar or in dose response assays starting at a nominal concentration of 20 micromolar. Compounds were subsequently counterscreened against the liver X receptor (LXR) and the farnesoid X receptor (FXR) to determine selectivity in singlicate and in dose response assays. Finally, compounds of interest were tested at a single concentration of 10 micromolar against VP16 to determine whether they were non-selective or cytotoxic. These late stage efforts have been described in AID 2117.
The above probe development efforts resulted in the identification of two probes. The benzenesulfonamide compound SID 85257301 previously identified as a selective agonist of LXR (12) was identified here as a novel RORa/g inverse agonist probe that decreases the transcriptional activity of both ROR receptors (IC50 values = 2.0 and 1.73 micromolar, respectively). The second probe, SID 85257298 synthesized at the SRIMSC, was found to decrease RORa transcriptional activity (IC50 value = 2.47 micromolar). SID 85257298 represents an improvement over the prior art due to its lack of activity for LXR. Probe compound SID 85257298 does not have activity against RORg (IC50 > 20 micromolar). These two probes are useful tools for examining ROR biology.
The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html. A probe report for SIDs 85257301 and 85257298 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML125 and ML124, respectively. A paper has been published (13).
(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. in press.
Late stage, probes, RAR-related orphan receptor A, ROR alpha, RORa, RORA, nuclear receptor, library, counterscreen, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, activator, transcriptional assay, luciferase, luminescence, ROR gamma, RORg, RORC, RORG, NR1F3, Primary screen, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Please see Related Bioassays and reference (13) for all protocols performed in this Center-based probe development effort.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 4
MLP Probe ML# for probe 1: ML125
PubChem Substance ID (SID) for probe 1: 85257301
PubChem Compound ID (CID) for probe 1: 447912
Probe type for probe 1: Modulator
IC50/EC50 (nM) for probe 1: 1730
Target for probe 1: ROR-gamma (gi: 19743903,19743909)
Disease relevance for probe 1: Metabolic disorders
Anti-target for probe 1: VP16
Fold selectivity for probe 1: inactive at 10 uM
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK56239/ (ID: 2510284)
Grant number for probe 1: MH084512-01
MLP Probe ML# for probe 2: ML176
PubChem Substance ID (SID) for probe 2: 85261497
PubChem Compound ID (CID) for probe 2: 2360837
Probe type for probe 2: Inverse Agonist
IC50/EC50 (nM) for probe 2: 482
Target for probe 2: RORA (gi: 19743903,19743909)
Disease relevance for probe 2: Diabetes
Anti-target for probe 2: RORG
Fold selectivity for probe 2: >20.7
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK143192/ (ID: 3033930)
Grant number for probe 2: MH084512
MLP Probe ML# for probe 3: ML310
PubChem Substance ID (SID) for probe 3: 117695958
PubChem Compound ID (CID) for probe 3: 51035449
Probe type for probe 3: Inverse Agonist
IC50/EC50 (nM) for probe 3: <320
Target for probe 3: RORgamma (gi: 19743903,19743909)
Disease relevance for probe 3: Multiple Sclerosis
Anti-target for probe 3: RORalpha
Fold selectivity for probe 3: >31.25
NCBI Book chapter link for probe 3: http://www.ncbi.nlm.nih.gov/books/NBK143543/ (ID: 3036466)
Grant number for probe 3: MH084512
MLP Probe ML# for probe 4: ML124
PubChem Substance ID (SID) for probe 4: 85257298
PubChem Compound ID (CID) for probe 4: 44237404
Probe type for probe 4: Modulator
IC50/EC50 (nM) for probe 4: 2470
Target for probe 4: ROR-alpha (gi: 19743903,19743909)
Disease relevance for probe 4: Metabolic disorders
Anti-target for probe 4: VP16
Fold selectivity for probe 4: inactive at 10 uM
PubMed Publication ID (PMID) for probe 1: 21499262,19887649
PubMed Publication ID (PMID) for probe 2: 21090593
PubMed Publication ID (PMID) for probe 3: 22769242,22292739
PubMed Publication ID (PMID) for probe 4: 19887649
NCBI Book chapter title for probe 1: Campaign to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR)
NCBI Book chapter title for probe 2: Identification of a novel selective inverse agonist probe and analogs for the Retinoic acid receptor-related Orphan Receptor Alpha (RORa)
NCBI Book chapter title for probe 3: Identification of a novel selective inverse agonist probe and analogs for the Retinoic acid receptor-related Orphan Receptor Gamma (ROR?)
Data Table (Concise)