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BioAssay: AID 2379

TR-FRET-based biochemical high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)

Name: TR-FRET-based biochemical high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3). ..more
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 Tested Compounds
 Tested Compounds
All(363)
 
 
Active(140)
 
 
Inactive(223)
 
 
 Tested Substances
 Tested Substances
All(363)
 
 
Active(140)
 
 
Inactive(223)
 
 
AID: 2379
Data Source: The Scripps Research Institute Molecular Screening Center (NR2E3_AG_TR-FRET_1536_3X%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-18
Modify Date: 2010-06-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 140
Related Experiments
Show more
AIDNameTypeComment
2300TR-FRET-based primary biochemical high throughput screening assay to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3).Screeningdepositor-specified cross reference: Primary screen (NR2E3 agonists)
2325Summary of probe development efforts to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3).Summarydepositor-specified cross reference: Summary (NR2E3 agonists)
2758TR-FRET-based biochemical high throughput dose response assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Confirmatorydepositor-specified cross reference
2759Counterscreen for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3): TR-FRET-based biochemical high throughput dose response assay to identify agonists of the interaction between peroxisome proliferator-activated receptor gamma (PPARg) and nuclear receptor co-repressor 2 (NCOR2)Confirmatorydepositor-specified cross reference
463256TR-FRET-based biochemical high throughput dose response assay to identify NR2E3 inverse agonistsConfirmatorydepositor-specified cross reference
463257Counterscreen 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)Confirmatorydepositor-specified cross reference
504787Counterscreen for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3): TR-FRET-based biochemical high throughput assay to identify agonists of the interaction between peroxisome proliferator-activated receptor gamma (PPARg) and nuclear receptor co-repressor 2 (NCOR2)Screeningdepositor-specified cross reference
624378Luminescence-based cell-based high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Screeningdepositor-specified cross reference
624379Counterscreen for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3): Luminescence-based cell-based high throughput screening assay to identify agonists of the Herpes Virus Virion Protein 16 (VP16)Screeningdepositor-specified cross reference
651846Late Stage Counterscreen for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3): Luminescence-based cell-based high throughput dose response screening assay to identify agonists of the Herpes Virus Virion Protein 16 (VP16)Confirmatorydepositor-specified cross reference
651849Late stage Luminescence-based cell-based high throughput dose response assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Confirmatorydepositor-specified cross reference
602229Luminescence-based cell-based high throughput primary screening assay to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Screeningsame project related to Summary assay
624394Luminescence-based cell-based high throughput dose response assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Confirmatorysame project related to Summary assay
624395Counterscreen for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3):Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)Confirmatorysame project related to Summary assay
Description:
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_AG_TR-FRET_1536_3X%INH

Name: TR-FRET-based biochemical high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3).

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 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:

nuclear receptor subfamily 2, group E, member 3, NR2E3; RetCOR, corepressor, photoreceptor-specific nuclear receptor; PNR, blindness, age-related macular degeneration, AMD, confirmation, 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.
Protocol
Assay Overview:
The purpose of this biochemical assay is to confirm activity of compounds identified 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 activate NR2E3 through disruption of binding to its corepressor, RetCOR. The assay employs GST-NR2E3 and its interaction partner, a biotinylated corepressor RetCOR. These reagents are added together in the presence of test compounds and 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 agonists will lead to release of RetCOR from NR2E3, thereby preventing interaction of the fluorescent tags, leading to reduced well FRET. Compounds are tested in triplicate at a final nominal concentration of 3.98 micromolar.
Protocol Summary:
Prior to the start of the assay 5 microliters 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 microliters 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 microliter 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 degrees Celsius 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 671nm and;
I617nm represents the measured fluorescence emission at 617nm.
The percent inhibition for each compound was calculated using as follows:
% Inhibition = 100 x ( 1 - ( ( Ratio_TestCompound - Median_Ratio_HighControl ) / ( Median_Ratio_LowControl - Median_Ratio_HighControl ) )
Where:
Test_Compound is defined as wells containing test compound.
Positive_Control is defined as wells containing biotin.
Negative_Control is defined as wells containing 0.6% DMSO.
The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score for active compounds is 100-20, for inactive 20-0.
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)
Comment
Due to the increasing size of the MLPCN compound library, 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 campaign's 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 not able to provide all compounds selected for testing in this AID.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (3.98μM**)Normalized percent activation at a compound concentration of 3.98 micromolar.Float%
2Standard DeviationStandard deviation of the confirmation assay derived from the normalized percent activation of the triplicate data for each compound.Float

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS061718-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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