Summary of probe development efforts to identify inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA).
Name: Summary of probe development efforts to identify inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA). ..more
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Xiaolin Li, Orphagen Pharmaceuticals, San Diego, CA
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 X01-MH077624-01
Grant Proposal PI: Xiaolin Li
External Assay ID: RORA_INH_LEADS_SUMMARY
Name: Summary of probe development efforts to identify inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA).
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).
RORA is one of three related orphan nuclear receptors, including RORB and RORC, known as "Retinoic Acid Receptor-related orphan receptors" (4). RORA has unusual potential as a therapeutic target for "metabolic syndrome". This refers to a convergence of pathogenic factors, including insulin resistance, dyslipidemia, hypertension, and a pro-inflammatory state, that greatly elevate the risk of diabetes and atherosclerosis (5). RORA has been shown to be implicated in several key aspects of this pathogenesis. For instance, the staggerer mouse, which carries a homozygous germline inactivation of RORA, shows low body weight, high food consumption (6-8), elevated angiogenesis in response to ischemia (9), susceptibility to atherosclerosis (8), and an abnormal serum lipid profile (10). A combination of genetic and cellular studies also showed that RORA regulates lipoprotein levels and very likely has an impact on circadian rhythm and metabolism in peripheral tissue such as the liver. Taken together, those observations highlight the need to identify specific ligands of RORA that could help understand its therapeutic potential and provide good chemical starting points for further drug development.
Summary of Probe Development Effort:
This AID serves as a summary of the High Throughput Screening (HTS) campaign and probe development effort to identify inhibitor probes of the Retinoic Acid Receptor-related orphan receptor A (RORA). Following primary HTS in singlicate (AID 561), counterscreening in singlicate against the nuclear receptor Steroidogenic Factor-1 (SF-1; NR5A1) (AID 525), counterscreening in triplicate against SF-1(AID 611), and titration assays in triplicate to determine potency (AID 610), two scaffolds were identified as possible candidates for probe development. A total of 27 compounds derived from these initial scaffolds generated either by purchase or synthesis were tested for their ability to inhibit RORA (Assay 5). These compounds were also subsequently tested in cell-based dose response assays against SF-1 (Assay 6) and VP16 (Assay 7) to determine selectivity. All compounds failed to meet selectivity criteria for RORA inhibitor probes: an IC50 in the RORA assay that was (1) lower than 10 μM and (2) at least 1 log lower than VP16 and/or SF-1 IC50 values.
As a result, the probe development effort based on the hits from the HTS screening campaign resulted in no improvement over the prior art. No probes were identified and the SRIMSC probe development effort for this RORA Inhibitors discovery grant proposal number has now stopped.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs, as detailed below and published in (11).
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. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. Madoux F, Li X, Chase P, Zastrow G, Cameron MD, Conkright JJ, Griffin PR, Thacher S, Hodder P. Potent, selective and cell penetrant inhibitors of SF-1 by functional ultra-high-throughput screening. Mol Pharmacol. 2008 Jun;73(6):1776-84.
Summary AID, Leads, RAR-related orphan receptor A, RORA, nuclear receptor, RZRA, ROR1, ROR2, ROR3, NR1F1, transcriptional assay, CHO-K1, luciferase, luminescence, primary, inhibition, 1536, Scripps, Scripps Florida, Molecular Library Probe Production Center Network, MLPCN.
Please see AIDs 561, 525, 611, and 610, and (11) for all protocols performed in this probe development effort.
RORA Inhibition Assay (Assays 1, 3 and 5). The purpose of this assay is to identify compounds that act as RORA inhibitors. The assay utilizes a fusion of the DNA-binding domain of the yeast transcriptional factor GAL4 with the ligand-binding domain of target receptor RORA (encoded by the pFA-hRORA plasmid, Orphagen Pharmaceuticals) to regulate a luciferase reporter containing 5xGAL4 response elements (upstream activating sequence; UAS) at its promoter region (pG5-luc, Stratagene). Both pFA-hRORA and pG5-luc plasmids are transiently co-transfected in CHO-K1 (Chinese Hamster Ovary) cells. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated RORA nuclear receptors. Compounds that inhibit the basal transcription of luciferase are detected through the suppression of light emission using the SteadyLite luciferase detection kit (Perkin Elmer). Such compounds constitute potential inhibitors of the RORA nuclear receptor. Compounds were tested in singlicate at a final nominal concentration of 10 micromolar and in triplicate in a 10-point 1:3 dilution series starting at a nominal concentration of 100 micromolar.
SF-1 Inhibition Counterscreen Assay (Assays 2, 4 and 6). The purpose of this counterscreen assay is to determine compound selectivity. This cell-based assay employs a fusion of the DNA-binding domain (DBD)of the yeast transcriptional factor GAL4 with the ligand-binding domain of the related nuclear receptor Steroidogenic Factor-1 (SF-1; NR5A1), encoded by the pFA-hSF-1 plasmid (Orphagen Pharmaceuticals), to regulate a luciferase reporter containing 5xGAL4 response elements (upstream activating sequence; UAS) at its promoter region (pG5-luc, Stratagene). Both pFA-hSF-1 and pG5-luc plasmids are transiently co-transfected in CHO-K1 cells. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated SF-1 nuclear receptors. Compounds that inhibit the basal transcription of luciferase are detected through the suppression of light emission using the SteadyLite luciferase detection kit (Perkin Elmer). Such compounds constitute potential inhibitors of the SF-1 nuclear receptor. Compounds were tested in singlicate at a final nominal concentration of 10 micromolar and in triplicate in a 10-point 1:3 dilution series starting at a nominal concentration of 100 micromolar.
VP16 Inhibition Counterscreen Assay (Assay 7). In this counterscreen assay the RORA plasmid was replaced by the GAL4DBD-VP16LBD plasmid, which expresses the strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) fused to the GAL4 DBD. Cells are co-transfected with the 5xGAL4 response element (UAS) luciferase reporter to monitor GAL4DBD-VP16LBD activity. As designed, compounds that inhibit VP16 activity will decrease pGAL4DBD-VP16LBD activity, leading to reduced activation of the pG5-luc and decreased well luminescence. These compounds are likely to be nonselective inhibitors or cytotoxic. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 100 micromolar.
No probes were identified and the SRIMSC probe development effort for this grant proposal number has now stopped.
** Test Concentration.
Data Table (Concise)