Bookmark and Share
BioAssay: AID 504453

Fluorescence-polarization-based biochemical polarscreen dose response binding assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)

Name: Fluorescence-polarization-based biochemical polarscreen dose response binding assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg). ..more
_
   
 Tested Compounds
 Tested Compounds
All(19)
 
 
Active(7)
 
 
Inactive(12)
 
 
 Tested Substances
 Tested Substances
All(19)
 
 
Active(7)
 
 
Inactive(12)
 
 
AID: 504453
Data Source: The Scripps Research Institute Molecular Screening Center (PPARG_AG_FP_0384_3XIC50 POLARSCREEN DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-16
Modify Date: 2011-04-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 7
Related Experiments
Show more
AIDNameTypeProbeComment
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)Summary depositor-specified cross reference
504735Late 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).Confirmatory depositor-specified cross reference
504938Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): chemiluminescence-based biochemical western blot assay for inhibitors of phosphorylation of PPARG protein levelsOther1 depositor-specified cross reference
504939Late stage assay provider results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay to identify transactivation inhibitor of PPRE (PPARG response element)Confirmatory depositor-specified cross reference
504943Late stage assay provider results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): TR-FRET-based biochemical dose response competitive binding lanthascreen assay for non-agonists of the PPARgConfirmatory depositor-specified cross reference
540282Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assessPPAR-gamma (PPARG) gene expression changes associated with osteoblast differentiationOther depositor-specified cross reference
540283Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess receptor activator of nuclear factor kappa-B ligand (RANKL) gene expression changes associated with osteoblast differentiationOther depositor-specified cross reference
540284Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess type I collagen (col1) gene expression changes associated with osteoblast differentiationOther depositor-specified cross reference
540285Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess alkaline phosphatase (Alp) gene expression changes associated with osteoblast differentiationOther depositor-specified cross reference
540286Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess AP2 (fatty acid binding protein 4) gene expression changes associated with adipocyte differentiation in 3T3-L1 cellsOther depositor-specified cross reference
540287Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess C/EBP-alpha gene expression changes associated with adipocyte differentiation in 3T3-L1 cellsOther depositor-specified cross reference
540289Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess PPAR-gamma gene expression changes associated with adipocyte differentiation in 3T3-L1 cellsOther depositor-specified cross reference
540290Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess CD36 gene expression changes associated with adipocyte differentiation in 3T3-L1 cellsOther depositor-specified cross reference
540291Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess Lpl gene expression changes associated with adipocyte differentiation in 3T3-L1 cellsOther depositor-specified cross reference
540292Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess Fasn gene expression changes associated with adipocyte differentiation in 3T3-L1 cells.Other depositor-specified cross reference
540293Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Leptin-deficient ob/ob mouse model studies to assess the effect of probe candidate on fasting blood glucoseOther1 depositor-specified cross reference
540294Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): fluorescence-based cell-based quantitative polymerase chain reaction assay to assess Glut4 gene expression changes associated with adipocyte differentiation in 3T3-L1 cellsOther depositor-specified cross reference
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)Screening same project related to Summary assay
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)Other same project related to Summary assay
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)Screening same project related to Summary assay
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)Confirmatory same project related to Summary assay
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 agonistsConfirmatory same project related to Summary assay
504446TR-FRET-based biochemical dose response competitive binding lanthascreen assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)Confirmatory same project related to Summary assay
504447Luminescence-based cell-based assay provider high throughput dose response assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)Confirmatory same project related to Summary assay
504452Luminescence-based cell-based primary high throughput screening assay to identify partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)Screening same project related to Summary assay
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: PPARG_AG_FP_0384_3XIC50 POLARSCREEN DRUN

Name: Fluorescence-polarization-based biochemical polarscreen dose response binding assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg).

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:

PPAR gamma, PPARg, PPARG1, PPARG2, PPAR, peroxisome proliferator-activated receptor gamma, partial agonist, agonist, competition, inhibit, polarscreen, Invitrogen, binding, assay provider, CBI, center based initiative, center-based, biochemical, fluorescence, FP, fluorescence polarization, polarization, 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 biochemical assay is to identify compounds that can directly bind to PPARg through competition with a fluorescently labeled high affinity PPARg compound. The fluorescent ligand when bound to the PPARg LBD protein has a constrained movement leading to a high fluorescence polarization value. When test compound displaces the fluorescent control compound, it causes this compound to tumble freely resulting in a low polarization value. This assay allows for the separation of compounds positive in the cell-based luminescence assays that are working through direct binding to PPARg versus compounds modulating PPARg transactivation activity through indirect mechanisms. Compounds are tested in triplicate using an 8-point titration series starting at a nominal concentration of 10 uM.
Protocol Summary:
This assay is a commercially available assay from Invitrogen and was conducted per manufacturers instructions (http://products.invitrogen.com/ivgn/product/PV3355). Compounds that were active in the dose-response cell-based luminescence assay were tested in this assay in dose response using concentrations in the range 10 uM to 0.001 uM.
For every dose, fold change in activation below high control was determined as follows:
Fold_Change = Test_Compound_at_Given_Dose / Median_High_Control
Where:
High_Control is defined as wells containing DMSO.
Test_Compound is defined as wells containing test compound.
Fold change under control was then plotted versus compound concentration and IC50 values 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.
PubChem Activity Outcome and Score:
Compounds with a Calculated IC50 less than or equal to 30 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:
Polar Screen PPARg Competitor Assay, Green (Invitrogen, Part: PV3355)
384-well plates (Greiner Bio-One CELLSTAR plate, Part: 655079)
Comment
This assay was performed by the assay provider. As a control for assay robustness, dose-response experiments using Rosiglitazone were performed with every set of test compounds in the range 5 uM to 200 pM. Calculated IC50 values for Rosiglitazone were compared to published values and were consistently 20 nM +/- 5 nM which is slightly better than the published EC50 of 47 nM.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the data for each compound.Float
3Hill SlopeThe variable HillSlope 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
4RsquareThis 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
5Fold Change at 10 uM [1] (10μM**)Value of fold change at 10 uM concentration; replicate one.Float%
6Fold Change at 3 uM [1] (3μM**)Value of fold change at 3 uM concentration; replicate one.Float%
7Fold Change at 1 uM [1] (1μM**)Value of fold change at 1 uM concentration; replicate one.Float%
8Fold Change at 0.300 uM [1] (0.3μM**)Value of fold change at 0.300 uM concentration; replicate one.Float%
9Fold Change at 0.100 uM [1] (0.1μM**)Value of fold change at 0.100 uM concentration; replicate one.Float%
10Fold Change at 0.030 uM [1] (0.03μM**)Value of fold change at 0.030 uM concentration; replicate one.Float%
11Fold Change at 0.010 uM [1] (0.01μM**)Value of fold change at 0.010 uM concentration; replicate one.Float%
12Fold Change at 0.003 uM [1] (0.003μM**)Value of fold change at 0.003 uM concentration; replicate one.Float%
13Fold Change at 0.001 uM [1] (0.001μM**)Value of fold change at 0.001 uM concentration; replicate one.Float%
14Fold Change at 10 uM [2] (10μM**)Value of fold change at 10 uM concentration; replicate two.Float%
15Fold Change at 3 uM [2] (3μM**)Value of fold change at 3 uM concentration; replicate two.Float%
16Fold Change at 1 uM [2] (1μM**)Value of fold change at 1 uM concentration; replicate two.Float%
17Fold Change at 0.300 uM [2] (0.3μM**)Value of fold change at 0.300 uM concentration; replicate two.Float%
18Fold Change at 0.100 uM [2] (0.1μM**)Value of fold change at 0.100 uM concentration; replicate two.Float%
19Fold Change at 0.030 uM [2] (0.03μM**)Value of fold change at 0.030 uM concentration; replicate two.Float%
20Fold Change at 0.010 uM [2] (0.01μM**)Value of fold change at 0.010 uM concentration; replicate two.Float%
21Fold Change at 0.003 uM [2] (0.003μM**)Value of fold change at 0.003 uM concentration; replicate two.Float%
22Fold Change at 0.001 uM [2] (0.001μM**)Value of fold change at 0.001 uM concentration; replicate two.Float%
23Fold Change at 10 uM [3] (10μM**)Value of fold change at 10 uM concentration; replicate three.Float%
24Fold Change at 3 uM [3] (3μM**)Value of fold change at 3 uM concentration; replicate three.Float%
25Fold Change at 1 uM [3] (1μM**)Value of fold change at 1 uM concentration; replicate three.Float%
26Fold Change at 0.300 uM [3] (0.3μM**)Value of fold change at 0.300 uM concentration; replicate three.Float%
27Fold Change at 0.100 uM [3] (0.1μM**)Value of fold change at 0.100 uM concentration; replicate three.Float%
28Fold Change at 0.030 uM [3] (0.03μM**)Value of fold change at 0.030 uM concentration; replicate three.Float%
29Fold Change at 0.010 uM [3] (0.01μM**)Value of fold change at 0.010 uM concentration; replicate three.Float%
30Fold Change at 0.003 uM [3] (0.003μM**)Value of fold change at 0.003 uM concentration; replicate three.Float%
31Fold Change at 0.001 uM [3] (0.001μM**)Value of fold change at 0.001 uM concentration; replicate three.Float%

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
PageFrom: