Confirmation 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: Confirmation 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
BioActive Compounds: 340
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: Hugh Rosen, TSRI
External Assay ID: PPARgSRC1_AG_TRFRET_1536_3X%ACT
Name: Confirmation biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 1 (SRC-1) 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 atherosclerosis, 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 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.
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, 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, confirmation screen, Scripps, Scripps Research Institute Molecular Screening Center.
The purpose of this assay is to confirm PPARgamma/SRC1 activity of compounds identified as active in at least two of the following experiments: "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), "Primary biochemical High Throughput Screening assay for agonists of the steroid receptor coactivator 2 (SRC-2) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)" (PubChem AID 1032), and "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).
This assay tests the ability of compounds to modulate interactions between PPARg and its coactivator SRC1, as measured by Time-Resolved Fluorescence Energy Transfer (TR-FRET) between a GST-PPARg ligand binding domain (LBD) fusion protein and a FLAG-tagged SRC1 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. Agonist ligands that promote the association of SRC1 with PPARg will shorten the distance between the two entities, allowing 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.
Prior to the start of the assay 5 microliters of TR-FRET Assay Buffer (125 mM Potassium Fluoride, 100 mM 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 microliters of TR-FRET Assay Buffer supplemented with 150 ng/mL anti-GST EuK, 3 ug/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 uM final nominal concentration), test compounds (8 uM final nominal concentration), or DMSO alone (0.8% final concentration) into the appropriate wells. Immediately after addition of test compounds fluorescence (t0) was measured by exciting at 340 nm, and reading fluorescence emission at 617 nm (EuK) and 671 nm (APC) with the ViewLux microplate reader (Perkin Elmer). The plates were then incubated for 15 hours at 4 degrees Celsius and fluorescence was measured again (t15). 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 for each timepoint, according to the following mathematical expressions:
t15_Ratio = t15_I671nm / t15_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.
Data were evaluated using % Activation. Any compound that exhibited an average % Activation greater than the hit cutoff calculated for the Primary screen was declared active. The reported PubChem_Activity_Score has been normalized to 100% of the highest observed 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-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)
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. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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 nonspecifically 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 10 and active compounds range of activity score is 10 to 100.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)