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BioAssay: AID 651615

Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)

Name: Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16). ..more
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 Tested Compounds
 Tested Compounds
All(2338)
 
 
Active(986)
 
 
Inactive(1352)
 
 
 Tested Substances
 Tested Substances
All(2342)
 
 
Active(989)
 
 
Inactive(1353)
 
 
AID: 651615
Data Source: The Scripps Research Institute Molecular Screening Center (VP16_INH_LUMI_1536_3X%INH CSRUN for LRH1 IAG)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-03

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 986
Depositor Specified Assays
Show more
AIDNameTypeComment
602396Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)screeningPrimary screen (LRH1 inverse agonists in singlicate)
602418Summary of the probe development efforts to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)summarySummary (LRH1 inverse agonists)
651968Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inverse agonists of the Steroidogenic Factor 1 Nuclear Receptor (SF1; NR5A1)confirmatory
651969Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify nonselective inhibitors of the Steroidogenic acute regulatory protein (StAR) promoter or luminescence assay artifactsconfirmatory
651970Luminescence-based cell-based high throughput dose response assay for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)confirmatory
651973Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)confirmatory
651974Late stage counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)confirmatory
651975Late stage counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify nonselective inhibitors of the Steroidogenic acute regulatory protein (StAR) promoter or luminescence assay artifactsconfirmatory
651976Late stage luminescence-based cell-based high throughput dose response assay for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)confirmatory
651977Late stage counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inverse agonists of the Steroidogenic Factor 1 Nuclear Receptor (SF1; NR5A1)confirmatory
686941Late stage assay provider counterscreen for P450 inhibition. LC-MS/MS-based assay to determine inhibitory activity of compounds against purified recombinantly expressed CYP3A4 and CYP3A5other
686944Late stage assay provider counterscreen for P450 inhibition based on the formation of Vincristine M1 formation in Genotyped HLMother
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patrick Griffin, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: CA134873
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: VP16_INH_LUMI_1536_3X%INH CSRUN for LRH1 IAG

Name: Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16).

Description:

The goal of this project is to identify modulators (inverse agonists) of the orphan nuclear receptor LRH-1, which has been implicated in cancer by enhancing proliferation and cell cycle progression and metabolic disorders through its regulation of genes involved cholesterol and bile acid homeostasis.

NR5A2 or Liver receptor homologue-1 (LRH-1) is a member of the NR5A, or Ftz-F1, subfamily V nuclear receptors for which there are four members (1). Murine LRH-1 was originally identified due to its sequence homology to the Drosophila Fushi tarazu factor-1 but orthologs have been subsequently identified in several other species including rat, chicken, horse, zebrafish and human (2-7). LRH-1, and its closest family member steroidogenic factor-1 (SF-1, NR5A1), bind to identical DNA consensus sequences (response elements or REs) and both have the ability to bind phospholipids in their ligand binding domains (LBDs) (8-10). However, LRH-1 and SF-1 are expressed in different tissues and thus are considered likely to have non-overlapping, non-redundant functions. SF-1 expression is confined to steroidogenic tissues and adrenals where it regulates development, differentiation, steroidogenesis and sexual determination (5, 7, 11). LRH-1 is highly expressed in tissues of endodermal origin and its expression is essential for normal liver, intestine, and pancreas function. LRH-1 has also been shown to be expressed in the ovary and adipose tissue.

In a recent report, Chand and colleagues investigated the mechanism of action of LRH-1 in invasive breast cancer cells. They found that LRH-1 promotes motility and cell invasiveness in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells and similar effects were observed in non-tumorigenic mammary epithelial cells. Interestingly, both remodeling of the actin cytoskeleton and E-cadherin processing were observed when LRH-1 was over-expressed. These findings implicate LRH-1 in promotion of migration and invasion in breast cancer independent of estrogen sensitivity. Together these findings provided strong evidence that LRH-1 plays a significant role in tumor formation both in vitro and in vivo. Therefore, the identification of potent and selective LRH-1 inverse agonists may provide new approaches for the treatment of cancer.

References:

1. Fayard, E., J. Auwerx, and K. Schoonjans, LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol, 2004. 14(5): p. 250-60.
2. Galarneau, L., J.F. Pare, D. Allard, D. Hamel, L. Levesque, J.D. Tugwood, S. Green, and L. Belanger, The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family. Mol Cell Biol, 1996. 16(7): p. 3853-65.
3. Kudo, T. and S. Sutou, Molecular cloning of chicken FTZ-F1-related orphan receptors. Gene, 1997. 197(1-2): p. 261-8.
4. Boerboom, D., N. Pilon, R. Behdjani, D.W. Silversides, and J. Sirois, Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology, 2000. 141(12): p. 4647-56.
5. Broadus, J., J.R. McCabe, B. Endrizzi, C.S. Thummel, and C.T. Woodard, The Drosophila beta FTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone. Mol Cell, 1999. 3(2): p. 143-9.
6. Ellinger-Ziegelbauer, H., A.K. Hihi, V. Laudet, H. Keller, W. Wahli, and C. Dreyer, FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis. Mol Cell Biol, 1994. 14(4): p. 2786-97.
7. Lavorgna, G., H. Ueda, J. Clos, and C. Wu, FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu. Science, 1991. 252(5007): p. 848-51.
8. Li, Y., M. Choi, G. Cavey, J. Daugherty, K. Suino, A. Kovach, N.C. Bingham, S.A. Kliewer, and H.E. Xu, Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1. Mol Cell, 2005. 17(4): p. 491-502.
9. Solomon, I.H., J.M. Hager, R. Safi, D.P. McDonnell, M.R. Redinbo, and E.A. Ortlund, Crystal structure of the human LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity. J Mol Biol, 2005. 354(5): p. 1091-102.
10. Krylova, I.N., E.P. Sablin, J. Moore, R.X. Xu, G.M. Waitt, J.A. MacKay, D. Juzumiene, J.M. Bynum, K. Madauss, V. Montana, L. Lebedeva, M. Suzawa, J.D. Williams, S.P. Williams, R.K. Guy, J.W. Thornton, R.J. Fletterick, T.M. Willson, and H.A. Ingraham, Structural Analyses Reveal Phosphatidyl Inositols as Ligands for the NR5 Orphan Receptors SF-1 and LRH-1. Cell, 2005. 120(3): p. 343-355.
11. Luo, X., Y. Ikeda, and K.L. Parker, A cell-specific nuclear receptor isa essential for adrenal and gonadal development and sexual differentiation. Cell, 1994. 77(4): p. 481-90.

Keywords:

CSRUN, counterscreen, VP16, VP16, Herpes Virus Virion Protein 16, triplicate, HTS, high throughput, 1536, Nuclear receptor, NR, CYP7A promoter-binding factor; alpha-1-fetoprotein transcription factor; b1-binding factor, hepatocyte transcription factor which activates enhancer II of hepatitis B virus; fetoprotein-alpha 1 (AFP) transcription factor; hepatocytic transcription factor; liver receptor homolog 1; liver receptor homolog-1; nuclear receptor NR5A2; nuclear receptor subfamily 5 group A member 2, LRH1, liver, activator, inverse agonist, transcriptional assay, luciferase, luminescence, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine whether compounds identified as active in a set of previous experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)" (AID 602396) are nonselective or promiscuous transcriptional modulators.

This assay determines VP16 inhibition activity 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). 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 are tested in triplicate at a nomimal test concentration of 3.6 uM.

Protocol Summary:

Seven million HEK293 cells were seeded in T-175 flasks 23 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v Anti-Anti. Flasks were then incubated for 48 hours at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, cells were harvested using TrypLE, resuspended in fresh media at a density of 1 million cells per mL and seeded into new T-175 flasks (23 mL per flask). After being allowed to attach for one hour at 37 C, 5% CO2 and 95% RH, cells were transfected with 1 mL of preincubated mix of serum-free OptiMEM containing 23 ug of the pGL4.31 reporter plasmid, 100 ng of the VP16-Gal4 expression vector and 80 uL of transfection reagents. Twenty four hours post transfection, cells were harvested using 5 mL of TrypLE and resuspended at a concentration of 750,000 cells per mL in phenol-red free DMEM media supplemented as described above.

The assay was started by dispensing 5 uL of cell suspension into each well of a white, solid-bottom 1536-well plate using a flying reagent dispenser (i.e. 3,750 cells per well). The first two columns received cells transfected with the reporter plasmid only (no cotransfection with the VP16-Gal4 expressing vector) as a control for background luminescence (no VP16 cells). Cells were then treated with 18 nL/well of test compounds, DMSO as a negative control (final concentration 0.36%) using a PinTool transfer unit (GNF). Plates were then placed in the incubator at 37 C, 5% CO2 and 95%RH. Twenty four hours later, plates were removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase was detected by adding 5 uL per well of ONE-Glo luciferase detection reagent. After a 15 minutes incubation time, light emission was measured with the ViewLux reader (PerkinElmer). The percent inhibition of each test 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.

The average percent inhibition and standard deviation of each compound tested were calculated.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the counterscreen. Two values were calculated: (1) the average percent inhibition of test low control wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-30, and for inactive compounds 30-0.

List of Reagents:

HEK-293 cells (ATCC, part CRL-1573)
DMEM media (Invitrogen, part 11965)
Fetal Bovine Serum (Hyclone, part SH30088.03)
Anti-Anti (Gibco, part 15240)
TrypLE (Invitrogen, part 12604)
T-175 flasks (Falcon, part 353112)
pGL4.31 (Promega)
VP16-Gal4 expression vector (Assay Provider)
TransIT 293 transfection reagent (Mirus Corporation, part MIR-2700)
ONE-Glo luciferase reagent (Promega, part E6130)
White, solid-bottom 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. 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 not able to supply all compounds selected for testing in this assay.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 3.6 uM (3.6μM**)Average percent inhibition of the counterscreen at a compound concentration of 3.6 uM.Float%
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the triplicate data for each compound.Float
3Inhibition at 3.6 uM [1] (3.6μM**)Percent inhibition of the counterscreen at a compound concentration of 3.6 uM; replicate 1.Float%
4Inhibition at 3.6 uM [2] (3.6μM**)Percent inhibition of the counterscreen at a compound concentration of 3.6 uM; replicate 2.Float%
5Inhibition at 3.6 uM [3] (3.6μM**)Percent inhibition of the counterscreen at a compound concentration of 3.6 uM; replicate 3.Float%

** Test Concentration.
Additional Information
Grant Number: CA134873

Data Table (Concise)
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