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BioAssay: AID 488780

Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based dose response counterscreen assay to identify SF1 inhibitors

Name: Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based dose response counterscreen assay to identify SF1 inhibitors. ..more
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 Tested Compounds
 Tested Compounds
All(16)
 
 
Inactive(16)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Inactive(16)
 
 
AID: 488780
Data Source: The Scripps Research Institute Molecular Screening Center (SF1_INH_LUMI_0384_3XIC50 MDCSRUN Round 0)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-10-13
Hold-until Date: 2011-10-01
Modify Date: 2011-10-01

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
488781Summary of the probe development efforts to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2)Summary depositor-specified cross reference
504928Late stage assay provider results from the probe development effort to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): absorbance-based cell-based assay to identify cytotoxic compounds in various cell typesOther1 depositor-specified cross reference
504933Late stage assay provider results from the probe development effort to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): luminescence-based cell-based assay to identify inhibitors of Star (Steroidogenic acute regulatory protein)Confirmatory2 depositor-specified cross reference
504934Late stage assay provider results from the probe development effort to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): luminescence-based high throughput cell-based assay to identify modulators of human nuclear receptorsOther1 depositor-specified cross reference
485348Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based primary assay to identify LRH1 inhibitors (3X%INH)Other same project related to Summary assay
488769Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): fluorescence-based cell-based quantitative PCR assay to identify inhibitors of LRH-1 target gene expressionOther same project related to Summary assay
488775Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based counterscreen assay to identify inhibitors of the human herpes virus VP16 transcriptional activator protein (VP16)Other same project related to Summary assay
488779Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based counterscreen assay to identify SF-1 inhibitorsOther same project related to Summary assay
488782Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based dose response assay to identify LRH1 inhibitors (Cyp19 aromatase-luciferase reporter)Confirmatory same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patrick Griffin, TSRI
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: U54 MH084512
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: SF1_INH_LUMI_0384_3XIC50 MDCSRUN Round 0

Name: Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based dose response counterscreen assay to identify SF1 inhibitors.

Description:

NR5A2 or Liver receptor homologue-1 (LRH1) is a member of the NR5A, or Ftz-F1, subfamily V nuclear receptors for which there are four members (1). Murine LRH1 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). LRH1, 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, LRH1 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). LRH1 is highly expressed in tissues of endodermal origin and its expression is essential for normal liver, intestine, and pancreas function. LRH1 has also been shown to be expressed in the ovary and adipose tissue.

In a very recent report, Chand and colleagues investigated the mechanism of action of LRH1 in invasive breast cancer cells. They found that LRH1 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 LRH1 was over-expressed. These findings implicate LRH1 in promotion of migration and invasion in breast cancer independent of estrogen sensitivity. Together these findings provided strong evidence that LRH1 plays a significant role in tumor formation both in vitro and in vivo. Therefore, the identification of potent and selective LRH1 inverse agonists may provide new approaches for the treatment of cancer (12).

References:

1. Fayard, E., J. Auwerx, and K. Schoonjans, LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends in Cell Biology, 2004. 14(5): p. 250-260.
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 is essential for adrenal and gonadal development and sexual differentiation. Cell, 1994. 77(4): p. 481-90.
12. Chand, A., K.A. Herridge, E.W. Thompson, and C. Clyne, The orphan nuclear receptor LRH1 promotes breast cancer motility and invasion. Endocr Relat Cancer, 2010.

Keywords:

Late stage, late stage AID, assay provider, purchased, synthesized, dose response, counterscreen, SF1, steroidogenic factor 1, nuclear receptor, library, liver receptor homolog 1; liver receptor homolog-1; nuclear receptor NR5A2; nuclear receptor subfamily 5 group A member 2, LRH1, liver, inhibitor, inverse agonist, transcriptional assay, assay provider, center based initiative, center-based, luciferase, luminescence, selective, 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 powder samples of possible LRH1 inverse agonist probe candidates are nonselective due to inhibition of another nuclear receptor, SF-1. This assay also serves to determine SF-1 inhibitory dose response curves for compounds In this assay, HEK293T cells, co-transfected with a full length SF-1 construct in a pSport6 vector backbone (pS6-SF-1) and a 5xSFRE -luciferase reporter construct, are incubated for 20 hours with test compound. As designed, a compound that inhibits SF-1 activity will prevent activation of the pS6 SF-1 construct, thereby preventing SF-1-mediated activation of the 5xSFRE-luciferase reporter, leading to a decrease in well luminescence. Compounds are tested in triplicate using a 9-point dose response series starting at a nominal concentration of 10 uM.

Protocol Summary:

Luciferase reporter assays were conducted using a pSport6 full-length SF-1 construct and 5xSFRE luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 3E6 cells in 10 cm plates,7 ug of total DNA and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and replated in 384 well plates at a density of 10,000 cells/well. Following 4 hour incubation, cells were treated with DMSO/compounds for 20 hours. The luciferase levels were measured by addition of BriteLite Plus (Perkin Elmer). Data was normalized to luciferase signal from DMSO treated 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 GraphPad Prism software. 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. 10 uM) did not result in greater than 50% inhibition, the IC50 value was determined manually as greater than 10 uM.

PubChem Activity Outcome and Score:

Compounds with a IC50 value greater than 5 uM were considered inactive. Compounds with a IC50 value equal to or less than 5 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 inactive compounds is 100-0. There are no active compounds.

List of Reagents:

384 well plates (PerkinElmer, part 6007688)
Britelite Plus (PerkinElmer, part 6016767)
DMEM (Mediatech Inc, Part 10 013 CV)
Fugene 6 (Roche Applied Science, part 11814443001)
Comment
This assay was performed by the assay provider. 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. 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, or 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (average IC50) shown in micromolar.FloatμM
2Standard DeviationThe standard deviation of the value of the IC50.Float
3Hill 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
4RsquareThis 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
5Fold Change at 10 uM [1] (10μM**)Value of fold change at 10 micromolar inhibitor concentration; replicate one.Float
6Fold Change at 3 uM [1] (3μM**)Value of fold change at 3 micromolar inhibitor concentration; replicate one.Float
7Fold Change at 1 uM [1] (1μM**)Value of fold change at 1 micromolar inhibitor concentration; replicate one.Float
8Fold Change at 0.333 uM [1] (0.333μM**)Value of fold change at 0.333 micromolar inhibitor concentration; replicate one.Float
9Fold Change at 0.111 uM [1] (0.111μM**)Value of fold change at 0.111 micromolar inhibitor concentration; replicate one.Float
10Fold Change at 0.037 uM [1] (0.037μM**)Value of fold change at 0.037 micromolar inhibitor concentration; replicate one.Float
11Fold Change at 0.012 uM [1] (0.012μM**)Value of fold change at 0.012 micromolar inhibitor concentration; replicate one.Float
12Fold Change at 0.004 uM [1] (0.004μM**)Value of fold change at 0.004 micromolar inhibitor concentration; replicate one.Float
13Fold Change at 0.001 uM [1] (0.001μM**)Value of fold change at 0.001 micromolar inhibitor concentration; replicate one.Float
14Fold Change at 10 uM [2] (10μM**)Value of fold change at 10 micromolar inhibitor concentration; replicate two.Float
15Fold Change at 3 uM [2] (3μM**)Value of fold change at 3 micromolar inhibitor concentration; replicate two.Float
16Fold Change at 1 uM [2] (1μM**)Value of fold change at 1 micromolar inhibitor concentration; replicate two.Float
17Fold Change at 0.333 uM [2] (0.333μM**)Value of fold change at 0.333 micromolar inhibitor concentration; replicate two.Float
18Fold Change at 0.111 uM [2] (0.111μM**)Value of fold change at 0.111 micromolar inhibitor concentration; replicate two.Float
19Fold Change at 0.037 uM [2] (0.037μM**)Value of fold change at 0.037 micromolar inhibitor concentration; replicate two.Float
20Fold Change at 0.012 uM [2] (0.012μM**)Value of fold change at 0.012 micromolar inhibitor concentration; replicate two.Float
21Fold Change at 0.004 uM [2] (0.004μM**)Value of fold change at 0.004 micromolar inhibitor concentration; replicate two.Float
22Fold Change at 0.001 uM [2] (0.001μM**)Value of fold change at 0.001 micromolar inhibitor concentration; replicate two.Float
23Fold Change at 10 uM [3] (10μM**)Value of fold change at 10 micromolar inhibitor concentration; replicate three.Float
24Fold Change at 3 uM [3] (3μM**)Value of fold change at 3 micromolar inhibitor concentration; replicate three.Float
25Fold Change at 1 uM [3] (1μM**)Value of fold change at 1 micromolar inhibitor concentration; replicate three.Float
26Fold Change at 0.333 uM [3] (0.333μM**)Value of fold change at 0.333 micromolar inhibitor concentration; replicate three.Float
27Fold Change at 0.111 uM [3] (0.111μM**)Value of fold change at 0.111 micromolar inhibitor concentration; replicate three.Float
28Fold Change at 0.037 uM [3] (0.037μM**)Value of fold change at 0.037 micromolar inhibitor concentration; replicate three.Float
29Fold Change at 0.012 uM [3] (0.012μM**)Value of fold change at 0.012 micromolar inhibitor concentration; replicate three.Float
30Fold Change at 0.004 uM [3] (0.004μM**)Value of fold change at 0.004 micromolar inhibitor concentration; replicate three.Float
31Fold Change at 0.001 uM [3] (0.001μM**)Value of fold change at 0.001 micromolar inhibitor concentration; replicate three.Float

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: U54 MH084512

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