TR-FRET-based biochemical dose response competitive binding lanthascreen assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)
Name: TR-FRET-based biochemical dose response competitive binding lanthascreen assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg). ..more
BioActive Compounds: 11
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patrick Griffin, TSRI
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: MH079861-01
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: PPARG_AG_TRFRET_0384_3XIC50 LANTHASCREEN DRUN
Name: TR-FRET-based biochemical dose response competitive binding lanthascreen assay for partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg).
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 identified, PPAR gamma (NR1C3) is implicated in several important disorders such as atherosclerosis, diabetes, obesity and cancer, providing strong justification for the search for 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, the different physiological pathways triggered by PPARg activation have to be decoupled. Recently, new classes of PPARg ligands, the so called selective PPARg modulators (SPPARgMs), have been developed. These compounds respond as partial agonists in a GAL-4 luciferase assay and are assumed to display a different binding mode in the PPARg subunit compared to the full agonist, glitazones (8). Selective recruitment of transcriptional coactivators by partial agonists has also been demonstrated, suggesting that different PPARg binding mode leading to a distinct coactivator recruitment profile may explain the change in gene expression patterns compared to those of full agonists (glitazones). Further, due to their improved pharmacodynamic properties, there is substantial interest and need to develop insulin-sensitizing PPARg modulators with minimal classical activation of PPARg and reduced side effects, while maintaining robust antidiabetic efficacy (9-11).
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.
8. Berger J, Leibowitz MD, Doebber TW, Elbrecht A, Zhang B, Zhou G, Biswas C, Cullinan CA, Hayes NS, Li Y, Tanen M, Ventre J, Wu MS, Berger GD, Mosley R, Marquis R, Santini C, Sahoo SP, Tolman RL, Smith RG, Moller DE. Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects. J Biol Chem. 1999 Mar 5;274(10):6718-25.
9. Berger JP, Petro AE, Macnaul KL, Kelly LJ, Zhang BB, Richards K, Elbrecht A, Johnson BA, Zhou G, Doebber TW, Biswas C, Parikh M, Sharma N, Tanen MR, Thompson GM, Ventre J, Adams AD, Mosley R, Surwit RS, Moller DE.Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator. Mol Endocrinol. 2003 Apr;17(4):662-76.
10. Minoura H, Takeshita S, Ita M, Hirosumi J, Mabuchi M, Kawamura I, Nakajima S, Nakayama O, Kayakiri H, Oku T, Ohkubo-Suzuki A, Fukagawa M, Kojo H, Hanioka K, Yamasaki N, Imoto T, Kobayashi Y, Mutoh S.
Eur J Pharmacol. 2004 Jun 28;494(2-3):273-81. Pharmacological characteristics of a novel nonthiazolidinedione insulin sensitizer, FK614.
11. Vidovic D, Busby SA, Griffin PR, SchC. A combined ligand- and structure-based virtual screening protocol identifies submicromolar PPARg partial agonists. ChemMedChem. 2011 Jan 3;6(1):94-103.
PPAR gamma, PPARg, PPARG1, PPARG2, PPAR, peroxisome proliferator-activated receptor gamma, partial agonist, agonist, competition, inhibit, polarscreen, Invitrogen, binding, assay provider, CBI, center based initiative, center-based, biochemical, fluorescence, lantha, lanthascreen, FRET, TRFRET, TR-FRET, competitive, selective, nuclear receptor, tumor, cancer, dose response, triplicate, 384, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to confirm compounds that can directly bind to PPARg through competition with a fluorescently labeled high affinity PPARg compound. The fluorescent ligand when bound to the PPARg LBD protein is in close proximity to the Tb-anti PPARg antibody bound to the N-terminal His tag on the PPARg LBD. In the absence of test compound, this provides a robust TR-FRET signal which is the ratio of the fluorescein emission at 520 nm and the Tb emission at 490 nm. When test compound displaces the fluorescently labeled control compound, it causes a loss of the TR-FRET signal which is proportional to how much of the compound is displaced. This assay allows for the separation of compounds positive in the cell-based luminescence assays that are working through direct binding to PPARg versus compounds modulating PPARg transactivation activity through indirect mechanisms. In addition, it provides a more sensitive measurement of compound binding to PPARg than the Polarscreen PPARg Competitor assay based on head to head comparisons with positive controls such as Rosiglitazone. Therefore, positive hits from the Polarscreen PPARg competitor assay were also evaluated in this assay along with analogs to our probe SID 91762765.
This assay is a commercially available assay from Invitrogen (http://products.invitrogen.com/ivgn/product/PV4894) and was conducted per manufacturers instructions. Compounds that were actives from the Polarscreen PPARg competitor assay were tested in this assay in dose response using concentrations in the range 5 uM to 0.002 uM.
For every dose, FRET ratio was determined as follows:
FRET_ratio = Signal_Test_Compound_520nm / Signal_Test_Compound_490nm
Test_Compound is defined as wells containing test compound.
FRET ratio was then plotted versus compound concentration and IC50s were calculated using GraphPad Prism. As a positive control, Rosiglitazone was tested in the same dose response experiment and calculated IC50 values were compared to values in literature as an assessment of assay robustness.
PubChem Activity Outcome and Score:
Compounds with a calculated IC50 less than or equal to 5 uM were considered active in this assay.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-78, and for inactive compounds 42-0.
List of Reagents:
Lanthascreen TR-FRET PPARg Competitive Binding Assay, (Invitrogen, Part: PV4894)
384-well plates (Greiner 384, small volume, Black Part: 784076)
This assay was performed by the assay provider. As a control for assay robustness, dose-response experiments using Rosiglitazone were performed with every set of test compounds in the range 10 uM - 0.001 uM. Calculated IC50 values for Rosiglitazone samples were compared to published values and were consistently 20 +/- 5 nM which is slightly better than the published EC50 of 47 nM.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)