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BioAssay: AID 1319

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 (PPARgamma)

Name: 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 (PPARgamma) ..more
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 Tested Compounds
 Tested Compounds
All(349)
 
 
Active(10)
 
 
Inactive(339)
 
 
 Tested Substances
 Tested Substances
All(349)
 
 
Active(10)
 
 
Inactive(339)
 
 
AID: 1319
Data Source: The Scripps Research Institute Molecular Screening Center (PPARgSRC1_AG_TRFRET_1536_EC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2008-06-18

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 10
Depositor Specified Assays
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AIDNameTypeProbeComment
1032Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 2 (SRC-2) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)screening
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
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
731Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)screening
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Pat Griffin, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 X01 MH079861-01
Grant Proposal PI: Patrick Griffin, Scripps Florida

External Assay ID: PPARgSRC1_AG_TRFRET_1536_EC50

Name: 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 (PPARgamma)

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 that have been identified, PPARgamma (NR1C3) is known to be implicated in several important disorders such as atheroscelerosis, diabetes, obesity and cancer, providing strong justification for search of 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 that retain their efficacy without inducing unwanted side effects, the different physiological pathways triggered by PPARg activation have to be decoupled. This can be achieved by screening for agonists that favor specifically the association of a given cofactor.
For this campaign, the MLSCN compound library will be screened in multiple assays, each one probing PPARg association with a different SRC coactivator. The small molecule agonists selected and optimized in this screening program will provide useful chemical tools for probing PPARg/coactivator interactions, helping the design of safer PPARg agonists.


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.

Keywords:

PPARgamma, PPARg, SRC-1, SRC1, nuclear receptor coactivator 1, NCOA1, NCoA-1, PAX3/NCOA1 fusion protein, F-SRC-1, RIP160, KAT13A, steroid receptor coactivator-1, agonist, time-resolved fluorescence energy transfer, TR-FRET, FRET, 1536-well, HTS, High Throughput Screening, dose response, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, MLSCN.
Protocol
Assay Overview:

The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)" (PubChem AID 631). Additionally, compounds selected for testing in this AID were not active or exhibited at least a 3-fold lower % Activation value (compared to AID 631) in a set of experiments entitled, "Primary biochemical high throughput screening assay to identify agonists of the steroid receptor coactivator 2 (SRC-2) recruitment by PPARgamma" (PubChem AID 1032) and "Primary biochemical high throughput screening assay to identify agonists of the steroid receptor coactivator 3 (SRC-3) recruitment by PPARgamma" (PubChem AID 731).

This assay tests the ability of compounds to increase interactions between PPARg and its coactivator SRC-1, as measured by Time-Resolved Fluorescence Energy Transfer (TR-FRET) between a GST-PPARg ligand binding domain (LBD) fusion protein and a FLAG-tagged SRC-1 coactivator. The fusion protein and coactivator are each recognized by fluorophore-labeled antibodies: anti-GST Europium Kryptate (EuK) donor and anti-FLAG Allophycocyanin (APC) acceptor, respectively. Compounds that promote the association of SRC-1 with PPARg will shorten the distance between the two entities, allowing TR-FRET to occur between the associated antibodies. As designed, a compound that acts as an agonist will increase TR-FRET. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 80 micromolar.

Protocol Summary:

Prior to the start of the assay 5 ul of TR-FRET Assay Buffer (125 mM Potassium Fluoride, 100mM Sodium Phosphate, 0.5% w/v CHAPS, 0.1% w/v Bovine Serum Albumin, pH 7.0, filtered at 0.22 micrometer) were dispensed into columns 1 and 2 of 1536-well assay plates. The remaining 46 columns were filled with 5 ul of TR-FRET Assay Buffer supplemented with 150 ng/mL anti-GST EuK, 3 micrograms/mL anti-FLAG APC, 2 nM GST-tagged PPARg-LBD [aa 204-477] and 40 nM FLAG-tagged SRC-1 protein [aa 601-762]. Next, the plates were centrifuged for 30 seconds at 300g. The assay was started by dispensing 40 nL of GW1929 (10 micromolar final nominal concentration), test compounds, or DMSO alone (0.8% final concentration) into the appropriate wells. The plates were then incubated for 15 hours at 4 degrees Celsius and fluorescence was measured. After excitation at 340 nm, well fluorescence was monitored at 617 nm (EuK) and 671 nm (APC) with the ViewLux microplate reader (Perkin Elmer).

For each well, a fluorescence ratio was calculated according to the following mathematical expression:

Ratio = I671nm / I617nm x 10,000

Where I671nm represents the measured fluorescence emission at 671nm and I617nm represents the measured fluorescence emission at 617nm.
The percent activation for each compound was calculated using as follows:
% Activation = 100 x ((Ratio_TestCompound - Median_Ratio_LowControl) / (Median_Ratio_HighControl - Median_Ratio_LowControl))
Where:

TestCompound is defined as wells containing test compound.
LowControl is defined as wells containing DMSO.
HighControl is defined as wells containing GW1929.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 80 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 80 uM. Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

List of Reagents:

Potassium Fluoride (Sigma, part 449148-25G)
CHAPS (Sigma, part C5070-5G)
Sodium Phosphate (Fluka Biochemika, part 71505)
Bovine Serum Albumin (Sigma, part A3294-10G)
Anti-GST EuK (CisBio, part 61GSTKLB)
Anti-FLAG APC (SureLight APC, PerkinElmer, part AD0059F)
GST-tagged PPARg-LBD [aa 204-477] (ProteinOne, part P4036)
FLAG-tagged SRC-1 protein [aa 601-762] (produced by Dr. Scott Busby, Scripps Florida).
Reference agonist GW1929 (Sigma, part G5568)
Black solid-bottom polystyrene 1536 well plates (Greiner Bio-One, part K1536SBSN)
Comment
Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: compounds that perturb fluorescence at 617 nm and/or 671 nm, compounds that interfere with the association of the FRET complex, and the presence of lint or dust in the test well. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.

The inactive compounds of this assay have activity score range of 0 to 65 and active compounds range of activity score is 65 to 100.
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 agonist assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Log10 of the qualified EC50 (EC50) from the agonist assay in M concentrationFloat
4Hill 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
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis 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
10Number of DataPointsOverall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Activation at 4.0 nM (0.004μM**)Value of %activation at 4.0 nM activator concentration; average of triplicate measurement.Float%
12Activation at 12.1 nM (0.0121μM**)Value of %activation at 12.1 nM activator concentration; average of triplicate measurement.Float%
13Activation at 36.3 nM (0.0363μM**)Value of %activation at 36.3 nM activator concentration; average of triplicate measurement.Float%
14Activation at 108.9 nM (0.1089μM**)Value of %activation at 109 nM activator concentration; average of triplicate measurement.Float%
15Activation at 326.6 nM (0.3266μM**)Value of %activation at 327 nM activator concentration; average of triplicate measurement.Float%
16Activation at 1.0 uM (1μM**)Value of %activation at 1 uM activator concentration; average of triplicate measurement.Float%
17Activation at 2.9 uM (2.9μM**)Value of %activation at 2.9 micromolar activator concentration; average of triplicate measurement.Float%
18Activation at 8.8 uM (8.8μM**)Value of %activation at 8.8 micromolar activator concentration; average of triplicate measurement.Float%
19Activation at 26.5 uM (26.5μM**)Value of %activation at 26.5 micromolar activator concentration; average of triplicate measurement.Float%
20Activation at 79.4 uM (79.4μM**)Value of %activation at 79.4 micromolar activator concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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