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BioAssay: AID 1954

Summary of probe development efforts to identify activators of the Retinoic Acid Receptor-related orphan receptor A (RORA).

Name: Summary of probe development efforts to identify activators of the Retinoic Acid Receptor-related orphan receptor A (RORA). ..more
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AID: 1954
Data Source: The Scripps Research Institute Molecular Screening Center (RORA_ACT_LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-09-23
Modify Date: 2013-06-11

Data Table ( Complete ):           View All Data
Target
Tested Compound:
Related Experiments
AIDNameTypeComment
522Primary Cell-based High Throughput Screening assay for activators of the nuclear receptor Steroidogenic Factor 1 (SF-1)Screeningdepositor-specified cross reference: Primary screen.
560Primary Cell-based High Throughput Screening assay for activators of the Retinoic Acid Receptor-related orphan receptor A (RORA)Screeningdepositor-specified cross reference: Primary screen.
681Dose-response cell-based assay for activators of the Retinoic Acid Receptor-related orphan receptor A (RORA)Confirmatorydepositor-specified cross reference: Dose response screen.
695Counterscreen for activators of the Retinoic Acid Receptor-related orphan receptor A (RORA): A cell-based dose-response assay for inhibition of the Steroidogenic Factor 1 (SF-1)Confirmatorydepositor-specified cross reference: Dose response screen.
Description:
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_ACT_LEADS_SUMMARY

Name: Summary of probe development efforts to identify activators of the Retinoic Acid Receptor-related orphan receptor A (RORA).

Description:

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 activator probes of the Retinoic Acid Receptor-related orphan receptor A (RORA). Following primary HTS in singlicate (AID 560), and primary screening in singlicate against the related nuclear receptor SF-1 (AID 522), certain compounds showing RORA selectivity were selected by applying the following criteria:

(1) RORA% >70% and S/I >4 OR
(2) 70>RORA % >50 and S/I >6 OR
(3) RORA% >50 and SF1% <0

Where:

RORA% indicates a test compound's percent activation calculated in the RORA primary screen (AID 560);
SF1% indicates that same test compound's percent activation calculated in the Steroidogenic Factor 1 (SF-1) Primary Screen (AID 522); and
S/I indicates the Selectivity Index ratio RORA% / SF1%.

Compounds that met the above criteria were selected for testing in triplicate in assays to determine potency (AID 681) and selectivity (AID 695). Compounds worth pursuing were selected by applying the following criteria:

(1) RORA EC50 value less than 10 μM
AND
(2) SF1EC50/RORA EC50 ratio greater than 10.

One compound that met these criteria was ordered for testing in SF-1, RORA, and VP16 titration assays (11), where it failed to demonstrate selectivity.

In summary, 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 project has now stopped.

Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below.

References:

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.

Keywords:

Summary AID, leads, RAR-related orphan receptor A, RORA, nuclear receptor, RZRA, ROR1, ROR2, ROR3, NR1F1, activation, activator, transcriptional assay, CHO-K1, luciferase, luminescence, primary, 1536, Scripps, Scripps Florida, Molecular Library Probe Production Center Network, MLPCN.
Protocol
Please see AIDs 560, 681, and 695 for all protocols performed in this probe development effort.

RORA Activation and EC50 Assays (Assays 1, 3, and 5).

The purpose of these assays is to determine the ability of compounds to activate SF-1. This assay employ CHO-K1 cells transiently transfected with a Gal4DBD::SF-1LBD plasmid that expresses a SF-1 nuclear receptor in which the SF-1 DNA binding domain (DBD) has been replaced with the Gal4 DBD. These cells also express a Gal4 response element UAS::luciferase reporter plasmid (11). As designed, compounds that activate SF-1 activity will enhance binding of the Gal4DBD to the Gal4 response element (upstream activating sequence; UAS) in the luciferase plasmid, thus increasing luciferase expression and well luminescence. Compounds are tested in singlicate at a nominal concentration of 10 micromolar and in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar.

SF-1 Activation and EC50 Assays (Assays 2, 4, and 6).

The purpose of these counterscreen assays is to determine compound activity against the related nuclear receptor, RORA. These assays employ CHO-K1 cells transiently transfected with a Gal4DBD::RORALBD plasmid that expresses a chimeric RORA nuclear receptor in which the RORA DNA binding domain (DBD) has been replaced with the Gal4 DBD. These cells also express a Gal4 UAS::luciferase reporter plasmid (11). As designed, compounds that activate RORA will enhance binding of the Gal4DBD to the Gal4 response element (upstream activating sequence; UAS) in the luciferase plasmid, thus increasing luciferase expression and well luminescence. Compounds active in these assays are considered nonselective. Compounds are tested in singlicate at a nominal concentration of 10 micromolar and in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar.

VP16 EC50 Assay (Assay 7).

The purpose of this counterscreen assay is to determine the promiscuity of compounds in transactivation reporter systems. This assay employs CHO-K1 cells which express a plasmid containing a fusion of the DNA binding domain (DBD) of the yeast transcription factor Gal4 fused to the ligand binding domain (LBD) of the herpes simplex virus protein VP16 (Gal4DBD::VP16LBD). The cells also express a Gal4 UAS::luciferase reporter plasmid (11). As designed, compounds that activate VP16 will enhance binding of the Gal4DBD to the Gal4 response element (upstream activating sequence; UAS) in the luciferase plasmid, thus increasing luciferase expression and well luminescence. Compounds active in this assay are considered nonselective. Compounds are tested in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar.

CHO Cytotoxicity Assay (Assay 8).

The purpose of this counterscreen assay is to determine the cytotoxicity of compounds identified as SF-1 activators. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP found in viable cells. The cell line used in this assay is the parental CHO cell line that has not been transfected with any plasmids. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP. Thus, well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that induce cell death will reduce ATP levels, and therefore reduce well luminescence. Compounds identified as active in this counterscreen assay are considered cytotoxic. Compounds are tested in triplicate in a 10-point, 1:3 serial dilution series, starting at a maximum concentration of 100 micromolar.
Comment
No probes were identified.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: CHO-K1
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML288
PubChem Substance ID (SID) for probe 1: 29220837
PubChem Compound ID (CID) for probe 1: 17759818
Probe type for probe 1: Activator
IC50/EC50 (nM) for probe 1: 1657
Target for probe 1: Small Heterodimer Partner (NR0B2) (gi: 548814)
Disease relevance for probe 1: Diabetes
Anti-target for probe 1: SF-1, VP16
Grant number for probe 1: MH077624-01
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Assay 1: RORA ACT Primary Assay (%ACT) (AID 560) (10μM**)Normalized percent activation of the primary screen at a compound concentration of 10 micromolar.Float%
2Assay 1: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
3Assay 2: SF-1 ACT Primary Assay (%ACT) (AID 522) (10μM**)Normalized percent activation of the primary screen at a compound concentration of 10 micromolar.Float%
4Assay 2: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
5Assay 3: RORA ACT Dose Response Assay (EC50) (AID 681)Qualified EC50 in micromolar: The concentration at which 50% of the activation is observed (relative to -100% activation of DMSO-treated -RORA cells).FloatμM
6Assay 3: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
7Assay 4: (Qualifier)Activity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
8Assay 4: SF-1 ACT Dose Response Counterscreen (EC50) (AID 695)Qualified EC50 in micromolar: The concentration at which 50% of the activation is observed (relative to -100% activation of DMSO-treated #SF-1 cells).FloatμM
9Assay 4: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
10Assay 5: RORA Dose Response Assay (EC50)The concentration at which 50 percent of the activity in the Activator assay is observed; (EC50) shown in uM.FloatμM
11Assay 5: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
12Assay 6: SF-1 Dose Response Counterscreen (EC50)The concentration at which 50 percent of the activity in the Activator assay is observed; (EC50) shown in uM.FloatμM
13Assay 6: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
14Assay 7: VP16 Dose Response Counterscreen (EC50)The concentration at which 50 percent of the activity in the Activator assay is observed; (EC50) shown in uM.FloatμM
15Assay 7: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String
16Assay 8: Cytotoxicity Counterscreen (EC50)The concentration at which 50 percent of the activity in the Cytotoxicity assay is observed; (EC50) shown in uM.FloatμM
17Assay 8: (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.String

** Test Concentration.
Additional Information
Grant Number: 1 X01-MH077624-01

Data Table (Concise)
Data Table ( Complete ):     View All Data
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