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BioAssay: AID 2758

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

Name: TR-FRET-based biochemical high throughput dose response assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3). ..more
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 Tested Compounds
 Tested Compounds
All(125)
 
 
Active(32)
 
 
Inactive(93)
 
 
 Tested Substances
 Tested Substances
All(125)
 
 
Active(32)
 
 
Inactive(93)
 
 
AID: 2758
Data Source: The Scripps Research Institute Molecular Screening Center (NR2E3_AG_TR-FRET_1536_3XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-04-08
Modify Date: 2010-05-18

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 32
Depositor Specified Assays
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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).screeningScreening (NR2E3 agonists, 1X)
2325Summary of probe development efforts to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3).summarySummary
2379TR-FRET-based biochemical high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)screeningScreening (NR2E3 agonists, 3X)
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)confirmatory
651849Late stage Luminescence-based cell-based high throughput dose response assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)confirmatory
463256TR-FRET-based biochemical high throughput dose response assay to identify NR2E3 inverse agonistsconfirmatory
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)confirmatory
624378Luminescence-based cell-based high throughput confirmation assay for agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)screening
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)screening
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_3XIC50

Name: TR-FRET-based biochemical high throughput dose response 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, dose response, titration, quadruplicate, 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 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), and that confirmed activity in a previous set of experiments entitled, "TR-FRET-based biochemical high throughput confirmation assay to identify agonists of nuclear receptor subfamily 2, group E, member 3 (NR2E3)" (AID 2379). This assay assesses the ability of compounds to activate NR2E3 through disruption 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 agonists will lead to release of RetCOR from NR2E3, thereby preventing interaction of the fluorescent tags, leading to reduced well FRET. Compounds were tested in quadruplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 39.8 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 quadruplicate assay plates. Column 3 of each plate contained 10 μM 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.

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 micromolar) did not result in greater than 50% activation, the IC50 was determined manually as greater than 39.8 uM. 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.

PubChem Activity Outcome and Score:

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 100-75, for inactive 63-0.

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 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.
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 concentration.Float
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 inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 2.0 nM (0.002μM**)Value of %inhibition at 2.0 nanomolar inhibitor concentration; average of quadruplicate measurement.Float%
12Inhibition at 6.1 nM (0.0061μM**)Value of %inhibition at 6.1 nanomolar inhibitor concentration; average of quadruplicate measurement.Float%
13Inhibition at 18.2 nM (0.0182μM**)Value of %inhibition at 18.2 nanomolar inhibitor concentration; average of quadruplicate measurement.Float%
14Inhibition at 54.7 nM (0.0547μM**)Value of %inhibition at 54.7 nanomolar inhibitor concentration; average of quadruplicate measurement.Float%
15Inhibition at 164.0 nM (0.164μM**)Value of %inhibition at 164.0 nanomolar inhibitor concentration; average of quadruplicate measurement.Float%
16Inhibition at 491.9 nM (0.4919μM**)Value of %inhibition at 491.9 nanomolar inhibitor concentration; average of quadruplicate measurement.Float%
17Inhibition at 1.5 uM (1.5μM**)Value of %inhibition at 1.5 micromolar inhibitor concentration; average of quadruplicate measurement.Float%
18Inhibition at 4.4 uM (4.4μM**)Value of %inhibition at 4.4 micromolar inhibitor concentration; average of quadruplicate measurement.Float%
19Inhibition at 13.3 uM (13.3μM**)Value of %inhibition at 13.3 micromolar inhibitor concentration; average of quadruplicate measurement.Float%
20Inhibition at 39.8 uM (39.8μM**)Value of %inhibition at 39.8 micromolar inhibitor concentration; average of quadruplicate measurement.Float%

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

Data Table (Concise)
Classification
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