TR-FRET-based biochemical high throughput dose response assay to identify NR2E3 inverse agonists
Name: TR-FRET-based biochemical high throughput dose response assay to identify NR2E3 inverse agonists. ..more
BioActive Compounds: 83
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: NR2E3_IAG_HTRF_1536_3XIC50 DRUN
Name: TR-FRET-based biochemical high throughput dose response assay to identify NR2E3 inverse agonists.
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.
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.
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, dose response, titration, triplicate, orphan nuclear receptor, fluorescence, TR-FRET, agonist, activator, HTS, 1536, Scripps, Scripps Florida, Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this biochemical assay is to determine dose response curves for 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). This assay assesses the ability of compounds to disrupt NR2E3 transcriptional activity through the enhancement of binding to its corepressor, RetCOR. In this assay, GST-NR2E3 and its interaction partner, a biotinylated corepressor RetCOR, are incubated together in the presence of test compounds, Eu(K)-anti GST antibody, and Streptavidin-D2. Interaction between the RetCOR and NR2E3 partners brings the fluorophore-tagged antibodies together, leading to FRET between the fluorophores. As designed, test compounds that act as NR2E3 inverse agonists will lead to strengthening of RetCOR and NR2E3 interaction, thereby increasing the interaction of the fluorescent tags, leading to increased well FRET. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 39.8 uM.
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 1.42 nM GST-tagged NR2E3 and 7.35 biotinylated RetCOR in Assay Buffer were dispensed into the remaining 46 columns. The compounds were then pinned into triplicate assay plates. Column 3 of each plate contained 10 uM biotin as high control. Next, 1 uL of 6X Detection Mix containing 4.5 nM Eu(K)-anti-GST and 252 nM Streptavidin-D2 in Assay Buffer was dispensed into all wells. After dispensing, final concentrations of the different reagents were: 0.75 nM Eu(K)-anti GST, 42 nM Streptavidin-D2, 1.35nM GST-tagged NR2E3 and 7 nM biotinylated RetCOR. 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
I671nm represents the measured fluorescence emission at 671 nm.
I617nm represents the measured fluorescence emission at 617 nm.
The percent response 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 ) ) )
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing biotin.
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 activation 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% activation 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% activation, 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 100-61, and for inactive compounds 60-0.
List of Reagents:
GST-NR2E3 (supplied by Assay Provider)
Biotinylated RetCoR (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)
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. The IC50 of biotin in this assay was 50 nM. 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, 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 provide all compounds selected for testing.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)