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: VP16_INH_LUMI_1536_3XIC50 DCSRUN

Name: Counterscreen 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).

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:

dose response, counterscreen, triplicate, VP16, Herpes Virus Virion Protein 16, CHO, cell-based, lumi, luminescence, alternate, nuclear receptor subfamily 2, group E, member 3, NR2E3; RetCOR, corepressor, photoreceptor-specific nuclear receptor; PNR, blindness, age-related macular degeneration, AMD, orphan nuclear receptor, agonist, activator, HTS, 1536, Scripps, Scripps Florida, Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this cell-based assay is to determine whether powder samples of compounds identified as potential NR2E3 agonists or inverse agonists are nonselective or promiscuous transcriptional modulators. This assay determines dose response VP16 inhibition curves for compounds.

In this counterscreen, the strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) is fused to the yeast GAL4 DNA Binding Domain (DBD) in place of the original NCOR moeiety used in the NR2E3 assay. Cells are co-transfected with the pGL4.31 luciferase reporter plasmid containing UAS repeats. As designed, compounds that repress VP16 transcriptional activity and/or prevent GAL4 binding to the UAS sequence will lead to a reduced expression of the pGL4.31 luciferase reporter gene, resulting in decreased well luminescence. These compounds are likely to be nonselective inhibitors or cytotoxic. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 40 uM.

Protocol Summary:

The CHO-S cell line was routinely cultured in 850 sq cm smooth surface, vented cap roller bottles at 37 C, 95% relative humidity (RH) and shaken at 135-155 rpm. The growth media consisted of Freestyle CHO Expression Medium supplemented with 8 mM L-Glutamine and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

CHO-S cells were suspended in roller bottles at a density of 0.6 million cells/mL in CHO Expression Medium supplemented with 8 mM L-Glutamine. The following day, cells were diluted to a density of 1 million cells/mL and transfected one of two ways: one population was transfected with 0.112 ug/mL of pGL4.31 reporter plasmid and 0.54 ug/mL of VP16 plasmid, complexed with 1.2 uL/mL of Freestyle Max Reagent in 38.4 uL/mL of OptiPRO SFM according to the Freestyle Max Reagent manufacturer's protocol (+VP16 cells). The second poplulation was transfected with 0.112 ug/mL of pGL4.31 reporter plasmid complexed with 1.2 uL/mL of Freestyle Max Reagent in 38.4 uL/mL of OptiPRO SFM according to the Freestyle Max Reagent manufacturer's protocol (-VP16 cells). Sixteen hours after transfection, 3750 cells in 5 uL of media were seeded into each well of 1536 well microtiter plates. The -VP16 cell population was dispensed to column 1 and 2 and was used as a high control mimicking inhibition. The +VP16 cell population was dispensed over the rest of the plate, with the exception of column 48 that received only media. Next, 20 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 24 hours at 37 C, 5% CO2, and 95 % RH. Luciferase levels were measured by adding 5 uL of One-Glo Luciferase Assay (prepared according to the manufacturer's protocol) to each well; followed by 10 minute incubation at room temperature. Then, Well Luminescence was read on the ViewLux plate reader. The percent inhibition for each compound was calculated as follows:

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

Where:

High_Control is defined as wells containing DMSO and -VP16 cells.
Test_Compound is defined as wells containing test compounds, DMSO and +VP16 cells.
Low_Control is defined as wells containing DMSO and +VP16 cells.

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. 40 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 40 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 of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-75, for inactive 73-0.

List of Reagents:

Freestyle CHO-S Cells (Invitrogen, part R800-07)
Freestyle CHO-S Expression Medium (Invitrogen, part 12651-022)
Freestyle Max Reagent (Invitrogen, part 16447-100)
OptiPRO SFM (Invitrogen, part 12309-050)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
L-Glutamine-200mM (Invitrogen, part 25030-081)
BD Falcon Roller Bottles-Smooth Surface (Bd Vacutainer Labware Medical, part 353154 )
pGL4.31 (Promega, part C935)
pVP16 (Assay provider)
1536-well plates (Corning, part 7298)

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. 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, and compounds that modulate well luminescence. 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.

Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]

Assay: CurveFit [1]: Mask: Excluded Points

Grant Number: 1 R21 NS061718-01