AID	Name	Type	Comment
485348	Center 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	Primary screen (LRH1 inhibitors in triplicate)
488769	Center 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 expression	other	Screen (LRH1 target gene expression inhibitors in triplicate)
488775	Center 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	Counterscreen (VP16 inhibitors in triplicate)
488779	Center Based Initiative to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2): Luminescence-based counterscreen assay to identify SF-1 inhibitors	other	Counterscreen (SF-1 inhibitors in triplicate)
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	confirmatory	Dose response counterscreen (SF-1 inhibitors in triplicate)
488781	Summary of the probe development efforts to identify novel inverse agonists of the liver receptor homolog-1 (LRH1; NR5A2)	summary	LRH1 IAG Summary
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)	confirmatory	Dose response (LRH1 inhibitors in triplicate)

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: STAR_INH_LUMI_0384_4XDCSRUN Round 0 LRH1 IAG

Name: Late 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).

Description:

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 (12).

In a very 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 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. 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, counterscreen, STAR, STARD1, steroidogenic acute regulatory protein, ML180, ML179, mitochondria, steroid, hormone, lumi, luminescence, luciferase, reporter, luc, dose response, titration, IC50, liver receptor homolog 1, liver receptor homolog-1; nuclear receptor NR5A2; nuclear receptor subfamily 5 group A member 2, LRH1, liver, inhibitor, inverse agonist, IAG, 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.

Assay Overview:

The purpose of this assay is to determine whether powder samples of possible LRH1 inverse agonist probe candidates can inhibit the activity of LRH1, as measure by inhibition of promoter activity of the Steroidogenic acute regulatory protein (Star). In this assay, HEK293T cells, co-transfected with a full length LRH1 construct in a pSport6 vector backbone (pS6-LRH1) and a Star-luciferase reporter construct are incubated for 20 hours with test compound. StAR is a transport protein that regulates cholesterol transfer within the mitochondria, which is the rate-limiting step in the production of steroid hormones. It is primarily present in steroid-producing cells, including theca cells and luteal cells in the ovary, Leydig cells in the testis and cell types in the adrenal cortex. Compounds were tested in triplicate at a nominal concentration of 10 uM, and 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 StAR luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 3E6 cells in 10 cm plates, 9 mg of total DNA and X-tremeGENE 9 DNA Transfection Reagent in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells from were counted and re-plated in 384 well plates at a density of 8,000 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 Reagent. Data was normalized to luciferase signal from DMSO treated cells.

The maximal response (inhibition) for each replicate well of each compound was calculated as follows:

Maximal_Response = Cells_treated_with_Test_Compound / Cells_treated_with_Vehicle_(DMSO)

PubChem Activity Outcome and Score:

For dose response assays, 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. Compounds with an IC50 value greater than 5 uM were considered inactive. Compounds with an IC50 value equal to or less than 5 uM were considered active.

Probe compounds were given a score of 100, actives a score of 50 and inactives a score of 0.

The PubChem Activity Score range for active compounds is 100-100. There were no inactive compounds.


List of Reagents:

Fugene6 transfection reagent (Roche Applied Sciences)
BriteLite reagent (Perkin Elmer)
pGL4.31 construct (Promega)
384-well plates (Greiner, part 789176)

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.

Grant Number: U54 MH084512