Luminescence-based cell-based primary high throughput screening assay to identify partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg)
Name: Luminescence-based cell-based primary high throughput screening assay to identify partial agonists of the peroxisome proliferator-activated receptor gamma (PPARg). ..more
BioActive Compounds: 60
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_LUMI_0384_2XRLU PRUN
Name: Luminescence-based cell-based primary high throughput screening assay to identify 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.
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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, transcriptional assay, assay provider, CBI, center based initiative, center-based, luciferase, aromatase, sport6, COS, cell-based, RLU, luminescence, selective, nuclear receptor, tumor, cancer, primary screen, 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 identify compounds that can increase the activity of PPARg. In this assay, Cos-1 cells co-transfected with a full length PPARgamma 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 duplicate at a final nominal concentration of 5 uM.
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 3E6 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, 10 nL 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.
The percent activation for each compound compared to full agonist and partial agonist control was calculated as follows:
The percent activation for each compound compared to full agonist control was calculated as follows:
%_Activation_of_Full_Agonist_Control = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_Full_Agonist_Control - Median_Low_Control ) )
The percent activation for each compound compared to partial agonist control was calculated as follows:
%_Activation_of_Partial_Agonist_Control = 100 * ( ( Test_Compound Median_Low_Control ) / ( Median_Partial_Agonist_Control Median_Low_Control ) )
Low_Control is defined as wells containing DMSO.
Test_Compound is defined as wells containing test compound.
Full_Agonist_Control is defined as wells containing Rosiglitazone.
Partial_Agonist_Control is defined as wells containing MRL-24.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Three values were calculated: (1) the average percent activation of all compounds tested,(2) three times their standard deviation and (3) percent activation values of the partial agonist control and of the full agonist control.
PubChem Activity Outcome and Score:
A compound is active if the % activation of the partial agonist control is greater than 85% and the % activation of the full agonist control is less than 65% percent. A compound is considered inactive if either one or both of these statements is false.
The reported PubChem Activity Score has been normalized to 100% observed average relative luminescence units (RLU).
The PubChem Activity Score range for active compounds is 100-60, and for inactive compounds 59-12.
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)
This assay was performed by the assay provider. The compound library selected for the primary screen was a subset of the MLSMR library of 340K compounds that were selected based on a 3D virtual screen looking for compounds predicted to bind to PPARg in a partial agonist binding mode (see reference 11). All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. 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 requested for testing.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)