Bookmark and Share
BioAssay: AID 488782

Center 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)

Name: Center 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). ..more
_
   
 Tested Compounds
 Tested Compounds
All(16)
 
 
Active(13)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Active(13)
 
 
Inactive(3)
 
 
AID: 488782
Data Source: The Scripps Research Institute Molecular Screening Center (LRH1_IAG_LUMI_0384_3XIC50 MDRUN 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 ):           Active    All
Target
BioActive Compounds: 13
Depositor Specified Assays
AIDNameTypeComment
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)confirmatory
488781Summary of the probe development efforts to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2)summary
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 receptorsother
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 typesother
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: LRH1_IAG_LUMI_0384_3XIC50 MDRUN Round 0

Name: Center 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).

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.
13. Clyne CD, Speed CJ, Zhou J, Simpson ER, Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem. 2002 Jun 7;277(23):20591-7. Epub 2002 Apr 1.

Keywords:

Late stage, late stage AID, assay provider, purchased, synthesized, dose response, 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 dose response curves for powder samples of possible LRH1 inverse agonist probe candidates can inhibit the activity of LRH1. This assay employs a luciferase reporter for the aromatase gene, Cyp19. Tissue specific expression of Cyp19 is regulated by hormones (specifically estrogens) and is increased in response to breast tumor derived factors. LRH1 has been shown to bind to the Cyp19 promoter and regulates its expression in adipose tissue (13). In this assay, HEK293T cells, co-transfected with a full length LRH1 construct in a pSport6 vector backbone (pS6-LRH1) and a Cyp 19 aromatase-luciferase reporter construct, are incubated for 20 hours with test compound. As designed, a compound that inhibits LRH1 activity will prevent activation of the pS6 LRH1 construct, thereby preventing LRH1-mediated activation of the Cyp19 aromatase-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 LRH1 construct and Cyp19 aromatase 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.

PubChem Activity Outcome and Score:

Compounds with a IC50 value greater than 5 uM were considered inactive. Compounds with an 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 active compounds is 100-1, and for inactive compounds 0-0.

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
Show more
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)
Classification
PageFrom: