Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): chemiluminescence-based biochemical western blot assay for inhibitors of phosphorylation of PPARG protein levels
Name: Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): chemiluminescence-based biochemical western blot assay for inhibitors of phosphorylation of PPARG protein levels. ..more
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: U54 MH084512
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: PHOSPHO-PPARG_AG_WESTERN-BLOT-KINASE-ASSAY MDRUN (CDK5) NON-AG
Name: Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): chemiluminescence-based biochemical western blot assay for inhibitors of phosphorylation of PPARG protein levels.
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 PPARG 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 PPARG 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 PPARg and do not induce PPARg transactivation (non-agonists) (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 PPARG 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 PPARGs 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, Schurer SC. A combined ligand- and structure-based virtual screening protocol identifies subuM 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 PPARg by Cdk5. Nature. 2010 Jul 22;466(7305):451-6.
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The purpose of this assay is to determine whether powder samples of compounds identified as possible PPARG non- agonist probe candidates can inhibit cdk5-mediated PPARG phosphorylation at S273 position. This biochemical assay monitors the transfer of inorganic phosphorous from ATP to the PPARG target protein by the cdk5 enzyme. This assay employs recombinant proteins incubated in the presence of ATP and test compounds, followed by western blot analysis of the reaction products using a phospho-PPARG antibody and standard western blotting techniques. Compounds were tested at a nominal concentration of 2 uM.
In vitro CDK kinase assay was performed using 0.5 ug of purified PPARG protein that was incubated with active CDK kinase (0.5 ug) in assay buffer (25 mM Tris-HCl pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2). Active Cdk5/p35 was purchased from Millipore (Cat 14-477). The assay mixture was incubated with test compounds for 30 minutes followed by the addition of 20 uM ATP and further incubation for 15 min at 30 C. Laemmli buffer was added to stop the reaction and subjected to electrophoresis followed by transfer on nitrocellulose membrane and developed with antibody anti-phospho-PPARG selective for S273. The blots were developed with antibody anti-phospho-PPARG selective for S273 position. Low_Control is defined as wells containing DMSO.
PubChem Activity Outcome and Score:
Compounds that reduced phosphorylation > or equal to 10% at 2 uM were considered active in this assay. (ie, Activity cutoff is > 10% inhibition)
The PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for actives and 0 for inactives.
The PubChem Activity Score range for active compounds is 100-50. There are no inactive compounds.
List of Reagents:
Purified PPARg protein (prepared in the assay provider's lab)
Active Cdk5/p35 Millipore
ATP (assay provider)
Laemmli buffer (prepared in the assay provider's lab)
Novex(R) 4-20% Tris-Glycine Gels (Invitrogen, Cat. EC60285BOX)
Nitrocellulose memberane (iBlot(R) Transfer Stack, Mini Invitrogen, Cat. IB3010-02)
Primary antibody: anti-phospho-PPARG selective for S273 position (custom, assay provider's lab)
Chemiluminescent developer for rabbit antibody (Pierce Fast Western Blot Kit, SuperSignal West Femto Substrate 35081).
This assay was run by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to cytotoxic compounds and compounds that modulate protein synthesis or processing. 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)