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BioAssay: AID 504735

Late stage assay provider counterscreen for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay for partial agonists of the peroxisome proliferator-activated receptor alpha (PPARA).

Name: Late stage assay provider counterscreen for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay for partial agonists of the peroxisome proliferator-activated receptor alpha (PPARA). ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(3)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(3)
 
 
Inactive(3)
 
 
AID: 504735
Data Source: The Scripps Research Institute Molecular Screening Center (PPARA_AG_LUMI_0384_3XEC50 MDRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-05-09

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 3
Depositor Specified Assays
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AIDNameTypeComment
631Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)screeningHTS Primary screen (SRC-1 recruitment by PPARg agonists in singlicate, HTRF)
1051Measurement of TR-FRET detection format artefact in the screen for agonists of steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)otherHTS Counterscreen (fluorescent artifacts in triplicate)
1300Confirmation biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)screeningHTS Confirmation (SRC-1 recruitment by PPARg agonists in triplicate, HTRF)
1319Dose response biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)confirmatoryHTS Dose response (SRC-1 recruitment by PPARg agonists in triplicate, HTRF)
1679TR-FRET dose response biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPAR gamma): non-selective agonistsconfirmatoryHTS Dose response (SRC-1 recruitment by PPARg agonists: non-selective agoniststs, HTRF)
504452Luminescence-based cell-based primary high throughput screening assay to identify partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)screeningPrimary screen (PPARg partial agonists in duplicate, LUMI)
504447Luminescence-based cell-based assay provider high throughput dose response assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)confirmatoryDose response (PPARg partial agonists in triplicate, LUMI)
504453Fluorescence-polarization-based biochemical polarscreen dose response binding assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)confirmatoryPolarscreen dose response (PPARg partial agonists in triplicate)
504446TR-FRET-based biochemical dose response competitive binding lanthascreen assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)confirmatoryLanthascreen dose response (PPARg partial agonists in triplicate)
1808Summary of probe development efforts to identify agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPAR gamma)summarySummary (SRC-1 recruitment by PPARg agonists)
Description:
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: MH079861-01
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: PPARA_AG_LUMI_0384_3XEC50 MDRUN

Name: Late stage assay provider counterscreen for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay for partial agonists of the peroxisome proliferator-activated receptor alpha (PPARA).

Description:

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and are lipid sensors functioning as ligand-dependent transcription factors regulating gene expression patterns of diverse biological processes (1, 2). PPARs play a critical role in metabolic processes such as glucose metabolism, lipid metabolism, and have been implicated in anti-atherogenic, anti-inflammatory as well as anti-hypertensive functions (3). Like other nuclear receptors, PPARs act as agonist-activated transcription factors, regulating specific target gene transcription. PPARs have been shown to respond to small molecules and are well-documented for therapeutic actions triggered by synthetic agonists (4-6). Among the three isoforms of PPAR identified, PPAR gamma (NR1C3) is implicated in several important disorders such as atherosclerosis, diabetes, obesity and cancer, providing strong justification for the search for specific PPARg agonists that can be used to treat these pathologies. However, the clinical use of PPARg agonists has been associated with adverse effects that are mainly caused by the concomitant activation of various target genes implicated in different physiological pathways. These side effects include weight gain through increased adipogenesis, renal fluid retention and plasma volume expansion, as well as toxic effects in the liver (7). To design safer and more selective PPARg agonists, the different physiological pathways triggered by PPARg activation have to be decoupled. Recently, new classes of PPARg ligands, the so called selective PPARg modulators (SPPARgMs), have been developed. These compounds respond as partial agonists in a GAL-4 luciferase assay and are assumed to display a different binding mode in the PPARg subunit compared to the full agonist, glitazones (8). Selective recruitment of transcriptional coactivators by partial agonists has also been demonstrated, suggesting that different PPARg binding mode leading to a distinct coactivator recruitment profile may explain the change in gene expression patterns compared to those of full agonists (glitazones). Further, due to their improved pharmacodynamic properties, there is substantial interest and need to develop insulin-sensitizing PPARg modulators with minimal classical activation of PPARg and reduced side effects, while maintaining robust antidiabetic efficacy (9-11).

References

1. Chawla, A., et al., Nuclear receptors and lipid physiology: Opening the X-files. Science, 2001. 294(5548): p. 1866-1870.
2. Krey, G., et al., Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Molecular Endocrinology, 1997. 11(6): p. 779-791.
3. Bishop-Bailey, D., T. Hla, and T.D. Warner, Intimal smooth muscle cells as a target for peroxisome proliferator-activated receptor-gamma ligand therapy. Circ Res, 2002. 91(3): p. 210-7.
4. Evans, R.M., G.D. Barish, and Y.X. Wang, PPARs and the complex journey to obesity. Nat Med, 2004. 10(4): p. 355-61.
5. Staels, B., et al., Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation, 1998. 98(19): p. 2088-93.
6. Barish, G.D., V.A. Narkar, and R.M. Evans, PPAR delta: a dagger in the heart of the metabolic syndrome. J Clin Invest, 2006. 116(3): p. 590-7.
7. Berger, J.P., T.E. Akiyama, and P.T. Meinke, PPARs: therapeutic targets for metabolic disease. Trends Pharmacol Sci, 2005. 26(5): p. 244-51.
8. Berger J, Leibowitz MD, Doebber TW, Elbrecht A, Zhang B, Zhou G, Biswas C, Cullinan CA, Hayes NS, Li Y, Tanen M, Ventre J, Wu MS, Berger GD, Mosley R, Marquis R, Santini C, Sahoo SP, Tolman RL, Smith RG, Moller DE. Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects. J Biol Chem. 1999 Mar 5;274(10):6718-25.
9. Berger JP, Petro AE, Macnaul KL, Kelly LJ, Zhang BB, Richards K, Elbrecht A, Johnson BA, Zhou G, Doebber TW, Biswas C, Parikh M, Sharma N, Tanen MR, Thompson GM, Ventre J, Adams AD, Mosley R, Surwit RS, Moller DE.Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator. Mol Endocrinol. 2003 Apr;17(4):662-76.
10. Minoura H, Takeshita S, Ita M, Hirosumi J, Mabuchi M, Kawamura I, Nakajima S, Nakayama O, Kayakiri H, Oku T, Ohkubo-Suzuki A, Fukagawa M, Kojo H, Hanioka K, Yamasaki N, Imoto T, Kobayashi Y, Mutoh S.
Eur J Pharmacol. 2004 Jun 28;494(2-3):273-81. Pharmacological characteristics of a novel nonthiazolidinedione insulin sensitizer, FK614.
11. Vidovic D, Busby SA, Griffin PR, SchC. A combined ligand- and structure-based virtual screening protocol identifies submicromolar PPARg partial agonists. ChemMedChem. 2011 Jan 3;6(1):94-103.

Keywords:

counterscreen, late stage, powder, PPARalpha, PPAR alpha, PPARa, NR1C1, hPPAR, MGC2237, MGC2452, alpha, PPARA, PPAR gamma, PPARg, PPARG1, PPARG2, PPAR, peroxisome proliferator-activated receptor gamma, partial agonist, agonist, transcriptional assay, assay provider, CBI, center based initiative, center-based, luciferase, sport6, COS, cell-based, RLU, luminescence, selective, nuclear receptor, tumor, cancer, dose response, triplicate, 384, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine the selectivity of the probe ML215 for PPARG (gamma) over PPARA (alpha). In this assay, Cos-1 cells co-transfected with a Gal4 PPARA LBD construct in a pBind vector backbone (pBind-Gal4-PPARA-LBD) and a UAS response element (UAS)-luciferase reporter construct, are incubated for 20 hours with test compound. As designed, a compound that activates PPARA activity will bind and activate the pBind-Gal4-PPARA-LBD construct, thereby stimulating PPARA-mediated activation of the UAS- luciferase reporter, leading to an increase in well luminescence. Compounds were tested in triplicate using an 8-point titration series starting at a nominal concentration of 3 uM (range 3 uM to 0.001 uM). The probe, ML215, was not active against PPARA in this assay demonstrating its selectivity as a probe for PPARG.

Protocol Summary:

Luciferase reporter assays were conducted using a pBind-Gal4PPARaLBD construct and UAS luciferase reporter cotransfected into Cos-1 cells. Reverse transfections were performed in bulk using 3E6 cells in 10 cm plates, 7 ug of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and replated in 384 well plates at a density of 10,000 cells/well. Following 4 hour incubation, compounds or DMSO vehicle were tested in triplicate using an 8-point titration series starting at a nominal concentration of 3 uM (range 3 uM to 0.001 uM). Cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells.

For every dose, fold change in activation over low control was determined as follows:

Fold_Change = Test_Compound_at_Given_Dose / Median_Low_Control

Fold change over low control was then plotted versus compound concentration and EC50s were calculated using GraphPad Prism.

FRET ratio was then plotted versus compound concentration and IC50s were calculated using GraphPad Prism. As a positive control, Rosiglitazone was tested in the same dose response experiment and calculated IC50 values were compared to values in literature as an assessment of assay robustness.

As a positive control, the PPARa specific agonist, GW7647 was tested in the same dose response experiment and calculated EC50 values were compared to values in literature as an assessment of assay robustness.

Where:

Low_Control is defined as wells containing DMSO.
Test_Compound is defined as wells containing test compound.

PubChem Activity Outcome and Score:

Compounds with a Calculated EC50 less than or equal to 1 uM were considered active in this assay.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.

List of Reagents:

Cells (Cos-1 cells (CRL-1650-ATCC))
Plasmids (pBind-Gal4PPARaLBD), (UAS luciferase (Addgene))
Britelite Plus (PerkinElmer, part 6016767)
DMEM (Mediatech Inc, Part 10 013 CV)
Fugene 6 (Roche Applied Science, part 11814443001).
384-well plates (Perkin Elmer, part 6007680)
Comment
This assay was performed by the assay provider. As a control for assay robustness, dose-response experiments using GW7647 were performed with every set of test compounds in the range 3 uM - 1 nM. Calculated EC50 values for GW7647 were compared to published values and were consistently 4.9 nM which is slightly better than the published EC50 of 6 nM.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity 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
2EC50*The concentration at which 50 percent of the activity in the agonsit assay is observed; (EC50) shown in micromolar.FloatμM
3Standard DeviationStandard deviation derived from the normalized percent inhibition of the data for each compound.Float
4Hill SlopeThe variable 'Hill Slope' describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
6Fold Change at 3 uM [1] (3μM**)Fold Change at 3 uM concentration; replicate [1]Float%
7Fold Change at 1 uM [1] (1μM**)Fold Change at 1 uM concentration; replicate [1]Float%
8Fold Change at 0.3 uM [1] (0.3μM**)Fold Change at 0.3 uM concentration; replicate [1]Float%
9Fold Change at 0.1 uM [1] (0.1μM**)Fold Change at 0.1 uM concentration; replicate [1]Float%
10Fold Change at 0.03 uM [1] (0.03μM**)Fold Change at 0.03 uM concentration; replicate [1]Float%
11Fold Change at 0.01 uM [1] (0.01μM**)Fold Change at 0.01 uM concentration; replicate [1]Float%
12Fold Change at 0.003 uM [1] (0.003μM**)Fold Change at 0.003 uM concentration; replicate [1]Float%
13Fold Change at 0.001 uM [1] (0.001μM**)Fold Change at 0.001 uM concentration; replicate [1]Float%
14Fold Change at 3 uM [2] (3μM**)Fold Change at 3 uM concentration; replicate [2]Float%
15Fold Change at 1 uM [2] (1μM**)Fold Change at 1 uM concentration; replicate [2]Float%
16Fold Change at 0.3 uM [2] (0.3μM**)Fold Change at 0.3 uM concentration; replicate [2]Float%
17Fold Change at 0.1 uM [2] (0.1μM**)Fold Change at 0.1 uM concentration; replicate [2]Float%
18Fold Change at 0.03 uM [2] (0.03μM**)Fold Change at 0.03 uM concentration; replicate [2]Float%
19Fold Change at 0.01 uM [2] (0.01μM**)Fold Change at 0.01 uM concentration; replicate [2]Float%
20Fold Change at 0.003 uM [2] (0.003μM**)Fold Change at 0.003 uM concentration; replicate [2]Float%
21Fold Change at 0.001 uM [2] (0.001μM**)Fold Change at 0.001 uM concentration; replicate [2]Float%
22Fold Change at 3 uM [3] (3μM**)Fold Change at 3 uM concentration; replicate [3]Float%
23Fold Change at 1 uM [3] (1μM**)Fold Change at 1 uM concentration; replicate [3]Float%
24Fold Change at 0.3 uM [3] (0.3μM**)Fold Change at 0.3 uM concentration; replicate [3]Float%
25Fold Change at 0.1 uM [3] (0.1μM**)Fold Change at 0.1 uM concentration; replicate [3]Float%
26Fold Change at 0.03 uM [3] (0.03μM**)Fold Change at 0.03 uM concentration; replicate [3]Float%
27Fold Change at 0.01 uM [3] (0.01μM**)Fold Change at 0.01 uM concentration; replicate [3]Float%
28Fold Change at 0.003 uM [3] (0.003μM**)Fold Change at 0.003 uM concentration; replicate [3]Float%
29Fold Change at 0.001 uM [3] (0.001μM**)Fold Change at 0.001 uM concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH079861-01

Data Table (Concise)
Classification
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