Sequence: peroxisome proliferator-activated receptor gamma isoform 2 [Homo sapiens] Protein Family: The ligand binding domain of peroxisome proliferator-activated receptors Gene:PPARG     Related Protein 3D Structures

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Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Konstantin Petrukhin, Columbia University
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS061718-01 Fast Track
Grant Proposal PI: Konstantin Petrukhin, Columbia University
External Assay ID: PPARG-NCOR2_IAG_HTRF_1536_3XIC50 DCSRUN

Name: Counterscreen for NR2E3 inverse agonists: TR-FRET-based biochemical high throughput dose response assay to identify inverse agonists of the interaction between peroxisome proliferator-activated receptor gamma (PPARg) and nuclear receptor co-repressor 2 (NCOR2).

Description:

Nuclear receptors are small molecule- and hormone-regulated transcription factors with discrete DNA-binding and ligand-binding domains, and are essential during development and for maintenance of proper cell function in adults. Small pharmacological compounds that bind to the cleft of the ligand-binding domain could alter receptor conformation and subsequently modify transcription of target genes. Such ligands (agonists and antagonists) have been designed for 23 nuclear receptors among the 48 identified in the human genome (1-3). NR2E3 is an orphan nuclear receptor expressed exclusively in rod and cone photoreceptor cells of the retina (4-7). In its unliganded state, NR2E3 acts as a transcriptional repressor (4, 8, 9) due to interaction with co-repressors such as retinal RetCOR (10), NCOR (11) or SMRT (11). Defects in this gene are a cause of several retinopathies (12-15). Studies showing that mice with a spontaneous deletion in the Nr2e3 gene develop late-onset, progressive retinal degeneration (7), suggest that this nuclear receptor is essential for photoreceptor development and survival. The identification of selective NR2E3 agonists or inverse agonists would provide useful tools for the understanding of the biological role of NR2E3 in retinal diseases.

References:

1. Evans, R.M., The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol, 2005. 19(6): p. 1429-38.
2. Kliewer, S.A., Lehmann, J.M., and Willson, T.M., Orphan nuclear receptors: shifting endocrinology into reverse. Science, 1999. 284(5415): p. 757-60.
3. Li, Y., Lambert, M.H., and Xu, H.E., Activation of nuclear receptors: a perspective from structural genomics. Structure, 2003. 11(7): p. 741-6.
4. Chen, J., Rattner, A., and Nathans, J., The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes. J Neurosci, 2005. 25(1): p. 118-29.
5. Cheng, H., Khanna, H., Oh, E.C., Hicks, D., Mitton, K.P., and Swaroop, A., Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors. Hum Mol Genet, 2004. 13(15): p. 1563-75.
6. Haider, N.B., Naggert, J.K., and Nishina, P.M., Excess cone cell proliferation due to lack of a functional NR2E3 causes retinal dysplasia and degeneration in rd7/rd7 mice. Hum Mol Genet, 2001. 10(16): p. 1619-26.
7. Akhmedov, N.B., Piriev, N.I., Chang, B., Rapoport, A.L., Hawes, N.L., Nishina, P.M., Nusinowitz, S., Heckenlively, J.R., Roderick, T.H., Kozak, C.A., Danciger, M., Davisson, M.T., and Farber, D.B., A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse. Proc Natl Acad Sci U S A, 2000. 97(10): p. 5551-6.
8. Gerber, S., Rozet, J.M., Takezawa, S.I., dos Santos, L.C., Lopes, L., Gribouval, O., Penet, C., Perrault, I., Ducroq, D., Souied, E., Jeanpierre, M., Romana, S., Frezal, J., Ferraz, F., Yu-Umesono, R., Munnich, A., and Kaplan, J., The photoreceptor cell-specific nuclear receptor gene (PNR) accounts for retinitis pigmentosa in the Crypto-Jews from Portugal (Marranos), survivors from the Spanish Inquisition. Hum Genet, 2000. 107(3): p. 276-84.
9. Kobayashi, M., Hara, K., Yu, R.T., and Yasuda, K., Expression and functional analysis of Nr2e3, a photoreceptor-specific nuclear receptor, suggest common mechanisms in retinal development between avians and mammals. Dev Genes Evol, 2008. 218(8): p. 439-44.
10. Takezawa, S., Yokoyama, A., Okada, M., Fujiki, R., Iriyama, A., Yanagi, Y., Ito, H., Takada, I., Kishimoto, M., Miyajima, A., Takeyama, K., Umesono, K., Kitagawa, H., and Kato, S., A cell cycle-dependent co-repressor mediates photoreceptor cell-specific nuclear receptor function. EMBO J, 2007. 26(3): p. 764-74.
11. Kapitskaya, M., Cunningham, M.E., Lacson, R., Kornienko, O., Bednar, B., and Petrukhin, K., Development of the high throughput screening assay for identification of agonists of an orphan nuclear receptor. Assay Drug Dev Technol, 2006. 4(3): p. 253-62.
12. Bernal, S., Solans, T., Gamundi, M.J., Hernan, I., de Jorge, L., Carballo, M., Navarro, R., Tizzano, E., Ayuso, C., and Baiget, M., Analysis of the involvement of the NR2E3 gene in autosomal recessive retinal dystrophies. Clin Genet, 2008. 73(4): p. 360-6.
13. Coppieters, F., Leroy, B.P., Beysen, D., Hellemans, J., De Bosscher, K., Haegeman, G., Robberecht, K., Wuyts, W., Coucke, P.J., and De Baere, E., Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa. Am J Hum Genet, 2007. 81(1): p. 147-57.
14. Gire, A.I., Sullivan, L.S., Bowne, S.J., Birch, D.G., Hughbanks-Wheaton, D., Heckenlively, J.R., and Daiger, S.P., The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa. Mol Vis, 2007. 13: p. 1970-5.
15. Sharon, D., Sandberg, M.A., Caruso, R.C., Berson, E.L., and Dryja, T.P., Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration. Arch Ophthalmol, 2003. 121(9): p. 1316-23.


Keywords:

late stage, SAR, purchased, synthesized, inverse, inverse agonists, nuclear receptor subfamily 2, group E, member 3, NR2E3; RetCOR, corepressor, photoreceptor-specific nuclear receptor; PNR, blindness, age-related macular degeneration, AMD, PPAR, PPARg, PPAR gamma, peroxisome proliferator-activated receptor gamma, NCOR2, nuclear receptor co-repressor 2, counterscreen, orphan nuclear receptor, fluorescence, TR-FRET, HTRF, agonist, activator, dose response, HTS, 1536, Scripps, Scripps Florida, Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this biochemical assay is to determine whether compounds presenting an inverse agonist profile as active in a previous set of experiments entitled, TR-FRET-based primary biochemical high throughput screening assay to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3) (AID 2300), were nonselective due to activation of the interaction between PPARgamma-NCOR2. This assay determines dose response curves for tested compounds. In this HTRF-format assay GST-PPARg and its interaction partner, a biotinylated corepressor NCOR2 are incubated in the presence of test compounds, Eu(K)-anti GST antibody, and Streptavidin-D2. Interaction between the NCOR2 and PPARg partners brings the fluorophores together, leading to well FRET. As designed, test compounds that act as PPARg inverse agonists will lead to strengthening of PPARg and corepressor NCOR2 interaction, thereby enhancing interaction of the fluorescent tags, leading to increased well FRET. Compounds are tested in triplicate 10-point 1:3 dilution series starting at a nominal test concentration of 39.8 uM.

Protocol Summary:

Prior to the start of the assay 5 uL of Assay Buffer (10 mM Tris-HCL, pH 7.5, 0.05% NP-40 alternative, 6% glycerol, 100 mM potassium fluoride, 1 mM dithiothreitiol and 0.05% w/v bovine serum albumin) were dispensed into columns 1 and 2 of 1536-well assay plates. Next, 5 uL of 1.05X Assay Mixture containing 7.35 nM GST-tagged PPARg and 315 nM biotinylated NCOR2 in Assay Buffer were dispensed into the remaining 46 columns. The compounds were then pinned into each assay plate. Next, 1 uL of 6X Detection Mix containing 4.5 nM Eu(K)-anti-GST and 42 nM Streptavidin-D2 in Assay Buffer was dispensed into all wells. After dispensing, the final concentrations of the reagents were: 7.0 nM GST-PPARg, 300 nM Biotin-NCOR2, 0.75 nM Eu(K)-anti-GST and 42 nM Streptavidin-D2. The plates were then incubated for 5 hours at 4 C and well FRET was measured. After excitation at 340 nm, well fluorescence was monitored at 617 nm (Eu(K)) and 671 nm (D2) with the ViewLux microplate reader (Perkin Elmer). For each well, a fluorescence ratio was calculated according to the following mathematical expression:

Ratio = I671nm / I617nm x 10,000

Where:

I671nm represents the measured fluorescence emission at 671 nm. I617nm represents the measured fluorescence emission at 617 nm.

The percent inhibition for each compound was calculated using as follows:

% Inhibition = -( 100 * ( 1 - ( ( Ratio_Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) )

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing no protein.
Low_Control is defined as wells containing 0.6% DMSO.

Please note: the positive control used here was the same control utilized in the agonist mode of the assay. No other pharmacological positive controls were available at the time of the screen.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 39.8 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 39.8 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-70, and for inactive compounds 68-0.

List of Reagents:

GST-PPAR gamma (supplied by Assay Provider)
Biotinylated NCOR2 (supplied by Assay Provider)
Eu(K)-antiGST (Cisbio, 61GSTKLB)
Streptavidin-D2 (Cisbio, 61OSADAB)
Tris-HCl, pH 7.5, 1 M solution (Invitrogen, 15567-027)
BSA, 30% solution (Sigma, A8327-50ML)
Biotin (Sigma, B4501-1G)
Rosiglitazone (control) (vendor, part)
Glycerol (Invitrogen, 15514-011)
NP-40 alternative, 10% solution (Calbiochem, 492018)
DTT, 1M solution (Fluka, 43816)
Potassium fluoride powder (Fluka, 60238)
1536-well plates (Greiner, part 789173)

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 campaigns specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay, the positive control Rosiglitazone had an average IC50 of 50 nM. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. 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 unable to supply all samples requested.

Grant Number: 1 R21 NS061718-01