Dose response biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center http://molscreen.florida.scripps.edu/ ..more
BioActive Compounds: 6
Depositor Specified Assays
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center http://molscreen.florida.scripps.edu/
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: Pat Griffin, TSRI
External Assay ID: PPARgSRC3_AG_TRFRET_1536_EC50
Name: Dose response 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, dose response, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, MLSCN.
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 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)" (PubChem AID 731). Additionally, compounds selected for testing in this AID were not active or exhibited at least a 3-fold lower % Activation value (compared to AID 731) in a set of experiments entitled, "Primary biochemical high throughput screening assay to identify agonists of the steroid receptor coactivator 1 (SRC-1) recruitment by PPARgamma," (PubChem AID 631) and "Primary biochemical high throughput screening assay to identify agonists of the steroid receptor coactivator 2 (SRC-2) recruitment by PPARgamma," (PubChem AID 1032).
This assay tests the ability of compounds to increase the interactions between PPARg and its coactivator SRC-3, as measured using Time-Resolved Fluorescence Energy Transfer(TR-FRET) between a GST-PPARg ligand binding domain (LBD) fusion protein and a FLAG-tagged SRC-3 coactivator. The fusion protein and coactivator are 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-3 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.
Prior to the start of the assay, 5 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, 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 of anti-GST EuK, 3 micrograms/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]. Next, the plates were centrifuged for 30 seconds at 300g. The assay was started by dispensing 40 nL of GW1929 (8 uM final nominal concentration), test compounds, or DMSO (0.8% final DMSO concentration) to 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 as follows:
% Activation = 100 x ((Ratio_TestCompound - Median_Ratio_LowControl) / (Median_Ratio_HighControl - Median_Ratio_LowControl))
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 micromolar) 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-3 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)
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.
Active compounds of this assay fall into the activity score range of 86 to 100 and inactive compounds have range of activity score from 0 to 81.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)