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BioAssay: AID 463256

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
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 Tested Compounds
 Tested Compounds
All(114)
 
 
Active(83)
 
 
Inactive(31)
 
 
 Tested Substances
 Tested Substances
All(114)
 
 
Active(83)
 
 
Inactive(31)
 
 
AID: 463256
Data Source: The Scripps Research Institute Molecular Screening Center (NR2E3_IAG_HTRF_1536_3XIC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-09-23
Hold-until Date: 2010-10-01
Modify Date: 2010-10-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 83
Related Experiments
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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 in singlicate)
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)
2379TR-FRET-based biochemical high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Screeningdepositor-specified cross reference: Confirmation (NR2E3 agonists in quadruplicate)
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: Dose response (NR2E3 agonists in quadruplicate)
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: Dose response counterscreen (PPARg and NCOR2 interaction agonists in triplicate)
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
602229Luminescence-based cell-based high throughput primary screening assay to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Screeningdepositor-specified cross reference
624394Luminescence-based cell-based high throughput dose response assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Confirmatorydepositor-specified cross reference
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)Confirmatorydepositor-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
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)Confirmatorysame project related to Summary assay
624378Luminescence-based cell-based high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)Screeningsame project related to Summary assay
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)Screeningsame 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_IAG_HTRF_1536_3XIC50 DRUN

Name: TR-FRET-based biochemical high throughput dose response assay to identify NR2E3 inverse agonists.

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, 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.
Protocol
Assay Overview:
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.
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 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
Where:
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 ) ) )
Where:
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)
Comment
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
From PubChem:
Assay Format: Biochemical
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 0.002 uM [1] (0.002μM**)Value of % inhibition at 0.002 uM inhibitor concentration; replicate one.Float%
12Inhibition at 0.002 uM [2] (0.002μM**)Value of % inhibition at 0.002 uM inhibitor concentration; replicate two.Float%
13Inhibition at 0.002 uM [3] (0.002μM**)Value of % inhibition at 0.002 uM inhibitor concentration; replicate three.Float%
14Inhibition at 0.006 uM [1] (0.006μM**)Value of % inhibition at 0.006 uM inhibitor concentration; replicate one.Float%
15Inhibition at 0.006 uM [2] (0.006μM**)Value of % inhibition at 0.006 uM inhibitor concentration; replicate two.Float%
16Inhibition at 0.006 uM [3] (0.006μM**)Value of % inhibition at 0.006 uM inhibitor concentration; replicate three.Float%
17Inhibition at 0.018 uM [1] (0.018μM**)Value of % inhibition at 0.018 uM inhibitor concentration; replicate one.Float%
18Inhibition at 0.018 uM [2] (0.018μM**)Value of % inhibition at 0.018 uM inhibitor concentration; replicate two.Float%
19Inhibition at 0.018 uM [3] (0.018μM**)Value of % inhibition at 0.018 uM inhibitor concentration; replicate three.Float%
20Inhibition at 0.055 uM [1] (0.055μM**)Value of % inhibition at 0.055 uM inhibitor concentration; replicate one.Float%
21Inhibition at 0.055 uM [2] (0.055μM**)Value of % inhibition at 0.055 uM inhibitor concentration; replicate two.Float%
22Inhibition at 0.055 uM [3] (0.055μM**)Value of % inhibition at 0.055 uM inhibitor concentration; replicate three.Float%
23Inhibition at 0.164 uM [1] (0.164μM**)Value of % inhibition at 0.164 uM inhibitor concentration; replicate one.Float%
24Inhibition at 0.164 uM [2] (0.164μM**)Value of % inhibition at 0.164 uM inhibitor concentration; replicate two.Float%
25Inhibition at 0.164 uM [3] (0.164μM**)Value of % inhibition at 0.164 uM inhibitor concentration; replicate three.Float%
26Inhibition at 0.492 uM [1] (0.492μM**)Value of % inhibition at 0.492 uM inhibitor concentration; replicate one.Float%
27Inhibition at 0.492 uM [2] (0.492μM**)Value of % inhibition at 0.492 uM inhibitor concentration; replicate two.Float%
28Inhibition at 0.492 uM [3] (0.492μM**)Value of % inhibition at 0.492 uM inhibitor concentration; replicate three.Float%
29Inhibition at 1.48 uM [1] (1.48μM**)Value of % inhibition at 1.48 uM inhibitor concentration; replicate one.Float%
30Inhibition at 1.48 uM [2] (1.48μM**)Value of % inhibition at 1.48 uM inhibitor concentration; replicate two.Float%
31Inhibition at 1.48 uM [3] (1.48μM**)Value of % inhibition at 1.48 uM inhibitor concentration; replicate three.Float%
32Inhibition at 4.42 uM [1] (4.42μM**)Value of % inhibition at 4.42 uM inhibitor concentration; replicate one.Float%
33Inhibition at 4.42 uM [2] (4.42μM**)Value of % inhibition at 4.42 uM inhibitor concentration; replicate two.Float%
34Inhibition at 4.42 uM [3] (4.42μM**)Value of % inhibition at 4.42 uM inhibitor concentration; replicate three.Float%
35Inhibition at 13.3 uM [1] (13.3μM**)Value of % inhibition at 13.3 uM inhibitor concentration; replicate one.Float%
36Inhibition at 13.3 uM [2] (13.3μM**)Value of % inhibition at 13.3 uM inhibitor concentration; replicate two.Float%
37Inhibition at 13.3 uM [3] (13.3μM**)Value of % inhibition at 13.3 uM inhibitor concentration; replicate three.Float%
38Inhibition at 39.8 uM [1] (39.8μM**)Value of % inhibition at 39.8 uM inhibitor concentration; replicate one.Float%
39Inhibition at 39.8 uM [2] (39.8μM**)Value of % inhibition at 39.8 uM inhibitor concentration; replicate two.Float%
40Inhibition at 39.8 uM [3] (39.8μM**)Value of % inhibition at 39.8 uM inhibitor concentration; replicate three.Float%

* Activity Concentration. ** 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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