Measurement of TR-FRET detection format artefact in the screen for agonists of steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma)
Name: Measurement of TR-FRET detection format artefact in the screen for agonists of steroid receptor coactivator 3 (SRC-3) recruitment by the peroxisome proliferator-activated receptor gamma (PPARgamma) ..more
BioActive Compounds: 390
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
Grant Proposal PI: Pat Griffin, TSRI
External Assay ID: PPARgSRC3_ARTEFACT_TRFRET_1536_RAW RATIO
Name: Measurement of TR-FRET detection format artefact in the screen for agonists of 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, steroid receptor coactivator-3, nuclear receptor coactivator 3, NCOA3, RAC3, AIB1, ACTR, p/CIP, TRAM-1, CAGH16, TNRC16, agonist, primary, counterscreen, time-resolved fluorescence energy transfer, TR-FRET, HTRF, 1536-well, HTS, High-Throughput Screening, fluorescence, artefact, Scripps, Scripps Molecular Screening Center.
This assay complements 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). Since the positive control agonist, GW1929, exhibited time resolved-fluorescence resonance energy transfer (TR-FRET) activity only after several hours of incubation with the SRC-3/PPARgamma complex, a sample well that exhibited TR-FRET activity immediately after initiation of the SRC-3/PPARgamma assay most likely was the result of format-specific artefact. Therefore, this assay's specific purpose is to identify sample wells that possess this artefact, thus indicating that compounds in those wells may have an erroneous activity assignment in AID 731.
This assay is based on the efficient FRET between a GST-PPARg ligand binding domain (LBD) fusion protein and a FLAG-tagged SRC3 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. A high ratio in sample well measured immediately after addition of compound suggests TR-FRET assay format artefact.
Prior to the start of the assay 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 1536-well assay plates. 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, 2 nM of GST-tagged PPARg-LBD [aa 204-477] and 35 nM of FLAG-tagged SRC-3 protein [aa 601-762]. Next, the microplates were centrifuged for 30s at 300g. The assay was started by dispensing 40 nL of GW1929 (8 uM final nominal concentration), test compounds (8 uM final nominal concentration), or DMSO alone (0.8% final concentration) into the appropriate wells. Immediately after centrifugation fluorescence 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). Values measured from both wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = I671 nm / I617 nm x 10,000
I671nm represents the measured fluorescence emission at 671 nm and I617nm represents the measured fluorescence emission at 617nm.
A mathematical algorithm was used to determine nominally fluorescent compounds.
Two values were calculated: (1) the average Ratio of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter. Any compound that exhibited greater fluorescence than the cutoff parameter was flagged as a possible TR-FRET artefact.
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. 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. A possible artefact of this assay can include, but are not limited to: fluorescent compounds, compounds that initiate aggregation of the TR-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 14 to 100 and inactive compounds have range of activity score from 0 to 14.
** Test Concentration.
Data Table (Concise)