Late stage assay provider results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay to identify transactivation inhibitor of PPRE (PPARG response element)
Name: Late stage assay provider results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay to identify transactivation inhibitor of PPRE (PPARG response element). ..more
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: MH084512
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: PPARG_NON-AG_LUMI_0384_3XFOLD-CHANGE TRANSACTIVATION DRUN PPRE
Name: Late stage assay provider results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): Luminescence-based cell-based dose response assay to identify transactivation inhibitor of PPRE (PPARG response element).
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). The objective of this project is to identify compounds that bind to PPARgamma and do not induce PPARg transactivation (12).
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.B7
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, Schurer SC. A combined ligand- and structure-based virtual screening protocol identifies submicromolar PPARg partial agonists. ChemMedChem. 2011 Jan 3;6(1):94-103.
12. Choi JH, Banks AS, Estall JL, Kajimura S, Bostrom P, Laznik D, Ruas JL, Chalmers MJ, Kamenecka TM, Bluher M, Griffin PR, Spiegelman BM. Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5. Nature. 2010 Jul 22;466(7305):451-6.
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The purpose of this assay is to determine dose response curves for compounds identified as possible PPAR gamma partial agonist probe candidates. In this assay, Cos-1 cells co-transfected with a full length PPARg construct in a pSport6 vector backbone (pS6-hPPARg) and three copies of a PPARg response element (3x-PPRE)-Aromatase-luciferase reporter construct, are incubated for 20 hours with test compound. As designed, a compound that activates PPARg activity will bind and activate the pS6-PPARg construct, thereby stimulating PPARg-mediated activation of the 3xPPRE-Aromatase-luciferase reporter, leading to an increase in well luminescence. Compounds are tested in triplicate using an 12-point titration series.
Luciferase reporter assays were conducted using a pSport-hPPARg full length construct and 3x PPRE luciferase reporter cotransfected into Cos-1 cells. Reverse transfections were performed in bulk using 3x10^6 cells in 10 cm plates, 7 ug of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and replated in 384 well plates at a density of 10,000 cells/well. Following 4 hour incubation, 10nL of DMSO or test compound (at 10 mM stock concentration) was dispensed from the compound plates to the 20 uL sample of cells. That gives a final nominal concentration of 5 uM for the single point assay. Cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated cells.
For every dose, %TA (transactivation) in activation over low control was determined as follows:
%_TA = Test_Compound_(Given dose) / Median_Low_Control
Low_Control is defined as wells containing DMSO.
Test_Compound is defined as wells containing test compound.
%TA over low control was then plotted versus compound concentration and calculated using GraphPad Prism.
PubChem Activity Outcome and Score:
Compounds with a %TA value equal to or less than 5% at all doses 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 100-2, and 0-0 for inactive compounds.
List of Reagents:
Cells (Cos-1 cells (CRL-1650-ATCC))
Plasmids (pSport6-hPPARg-full-length), (PPRE-X3-TK-luc (Addgene))
Britelite Plus (PerkinElmer, part 6016767)
DMEM (Mediatech Inc, Part 10 013 CV)
Fugene 6 (Roche Applied Science, part 11814443001).
384-well plates (Perkin Elmer 384 well Culture Plates, part 6007680)
* Activity Concentration. ** Test Concentration.
Data Table (Concise)