|Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma) - BioAssay Summary
Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma) ..more
BioActive Compounds: 519
Depositor Specified Assays
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Scripps Florida
Network: Molecular Library Screening Center Network (MLSCN)
Proposal Number: 1 X01 MH079861-01
PI: Patrick Griffin
External Assay ID: PPARgSRC3_AG_TRFRET_1536_%ACT
Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)
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 . 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 .
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 project, 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, hence helping the design of safer PPARg agonists.
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.
PPARgamma, PPARg, PPARG1, PPARG2, NR1C3, SRC-3, SRC3, nuclear receptor coactivator 3, NCOA3, RAC3, AIB1, ACTR, p/CIP, TRAM-1, CAGH16, TNRC16, agonist, time-resolved fluorescence energy transfer, TR-FRET, FRET, 1536-well, HTS, High-Throughput Screening, primary, Scripps
A homogeneous coactivator assay able to measure interactions between PPARg and its coactivator SRC-3 using Time-Resolved Fluorescence Energy Transfer(TR-FRET) readout has been developed. This assay is based on the efficient Fluorescence Resonance Energy Transfer (FRET) between a GST-PPARg ligand binding domain (LBD) fusion protein and a FLAG-tagged SRC3 coactivator. The fusion protein and coactivator are recognized by the fluorophore-labeled antibodies, anti-GST Europium Kryptate (EuK) donor and anti-FLAG Allophycocyanin (APC) acceptor, respectively.
Agonist ligands that promote the association of the SRC-3 coactivator with the fusion protein will shorten the distance between the two entities, allowing FRET to occur between the associated antibodies complex. Therefore, agonist activity is detected as an increase in fluorescence at the acceptor emission wavelength.
The assay was run in black solid-bottom polystyrene 1536 well plate.
Five microliters of TR-FRET assay buffer (125 mM Potassium Fluoride, 100mM Sodium Phosphate, 0.5% w/v CHAPS, 0.1% w/v Bovine Serum Albumin, pH7.0, filtered at 0.22 micrometer) were dispensed column 1-2 of the plate.
The remaining 46 columns were filled with five microliters of TR-FRET assay buffer supplemented with 150 ng/mL of anti-GST EuK, 3 ug/mL of anti-FLAG APC, 4 nM of GST-tagged PPARg-LBD [aa 204-477] and 35 nM of FLAG-tagged SRC-3 protein [aa 601-762].
Microplates were then centrifuged for 30s at 300g. Forty nanoliters of controls or test compounds (8 micromolar final nominal concentration, 0.8% final DMSO concentration) were then distributed to each well.
Plates were then incubated for 15 hours at 4 degrees Celsius.
After this incubation time, microplates were allowed to equilibrate 30 minutes at room temperature before being centrifuged for 30 seconds at 300g.
After excitation at 340 nm, wells fluorescence was monitored at 617 nm (EuK) and 671 nm (APC) with the ViewLux microplate reader (Perkin Elmer). Values measured from both wavelengths were used to calculate a ratio for each well, accordingly to the following mathematical expression:
Ratio = I671nm / I617nm x 10,000
where I671nm represents the measured fluorescence emission at 671 nm and I617nm represents the measured fluorescence emission at 617nm.
The percent activation for each compound has then been calculated as follows:
%activation = 100 x ((Ratio_compound - Median_Ratio_LowControl) / (Median_Ratio_HighControl - Median_Ratio_LowControl))
with LowControl: test wells containing DMSO
and HighControl: test wells containing 10 micromolar of reference agonist GW1929 (Sigma, part#G5568)
A mathematical algorithm was used to determine nominally active compounds.
Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater %activation than the cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
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-3 protein [aa 601-762] (produced by Dr. Scott Busby, Scripps Florida).
Reference agonist GW1929 (Sigma, part#G5568)
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
- the presence of lint or dust in the test well.
** Test Concentration.
Data Table (Concise)