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: CELL-VIABILITY_INH_MTT_096_Fold-Change-DRUN 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): absorbance-based cell-based assay to identify cytotoxic compounds in various cell types.

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, MTT, ML180, ML179, absorbance, cytotoxicity, viability, survival, growth, proliferation, spectrophotometer, duplicate, dose response, titration, IC50, CC50, 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, 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.

Cell Lines

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description
			Active	Inactive
1		Raji Cell Line	1
2		EuMyc Lymphoma Cell Line		1
3		70Z/3 Cell Line	1
4		9E10 Cell Line		1
5		Jurkat Cell Line	1
6		MCF7 Cell Line	1
7		T47D Cell Line	1
8		MD-MDA-231 Cell Line	1
9		HS578 Cell Line	1
10		SKBR Cell Line	1

---

§ Panel component ID.

Assay Overview:

The purpose of this assay is to determine cytotoxicity/viability fold change or cytotoxicity/viability dose response curves for powder samples of possible LRH1 inverse agonist probes candidates, using a variety of tumor cell lines. This assay is based upon the reduction of the yellow tetrazolium salt (MTT) in metabolically active cells to form insoluble purple-blue formazan crystals, which are solubilized by the addition of a detergent. MTT reduction occurs inside cells via the action of mitochondrial dehydrogenases. Formazan production is directly proportional to cell number, and metabolically inactive cells produce low levels of formazan. All values were normalized to DMSO negative control. As designed, a compound that inhibits cell growth or proliferation or is directly cytotoxic, will lead to decreased production of purple formazan crystals, thereby reducing well absorbance. Some compounds were tested in duplicate using a 5-point dilution series starting at a nominal concentration of 10 uM. For some assays, doses tested were 10.0, 3.3, 1.1, 0.37, 0.123, 0.041, 0.0137, and 0.0045 uM. Control treatment was incubation of cells in media alone. Values were normalized to DMSO.

Protocol Summary:

A selection of some of the possible cell types tested in these assays are listed here: Human B lymphocyte (Lymphoma) Raji cells, Mouse B lymphocyte EuMyc Lymphoma cells, Mouse Pre-B lymphocyte (cancer) 70Z/3 cells, 9E10 cells, Mouse T lymphocyte (Leukemia) Jurkat cells, Human Breast Cancer MCF7 cells, Human Breast Cancer T47D cells, Human Breast Cancer MD-MDA-231 cells, Human breast (non-cancer) HS578 cells, Human Breast Cancer SKBR cells. The following describes the culture conditions for each cell line, seeded in 50 ul per well of a 96-well plate (ME indicates mercaptoethanol)

Raji: 1.5x10^4 cells per well, cultured for 2 days in 10%FBS RPMI1640, 2 mM L-Glutamine, Penstrep.
SCR lymphoma ex vivo: 1.5x10^4 cells per well, cultured for 2 days in 10% FBS RPMI1640, 2mM L-Glutamine, Penstrep, 0.5 ng/ml IL7.
ATG7 null lymphoma : 1.5x10^4 cells per well, cultured for 2 days in 10% FBS RPMI1640, 2mM L-Glutamine, Penstrep, 0.5 ng/ml IL7.
U266: 3.0x10^4 cells per well, cultured for 4 days in10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep.
NCI H929: 3.0x10^4 cells per well, cultured for 4 days in 10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep, 5 0nM 2ME.
RPMI8226: 1.5x10^4 cells per well, cultured for 3 days in 10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep.
70Z/3: 1.5x10^4 cells per well, cultured for 3 days in 10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep, 50 nM 2ME.
Jurkat: 3.0x10^4 cells per well, cultured for 3 days in 10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep.
9E10:1.5x10^4 cells per well, cultured for 3 days in 10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep.
MDA231: 0.5x10^4 cells per well, cultured for 3 days in10% FBS DMEM with 25 mM Glucose, 2 mM L-Glutamine, Penstrep.
HS578: 1.0x10^4 cells per well, cultured for 4 days in 10% FBS DMEM with 25 mM Glucose, 2 mM L-Glutamine, Penstrep.
MCF7: 0.5x10^4 cells per well, cultured for 3 days in 10% FBS DMEM with 25 mM Glucose, 2 mM L-Glutamine, Penstrep.
T47D: 0.5x10^4 cells per well, cultured for 3 days in 10% FBS DMEM with 25 mM Glucose, 2 mM L-Glutamine, Penstrep.
SKBR: 1.0x10^4 cells per well, cultured for 4 days in 10% FBS DMEM with 25 mM Glucose, 2 mM L-Glutamine, Penstrep.
PC3: 0.5x10^4 cells per well, cultured for 3 days in 10% FBS RPMI1640, 2 mM L-Glutamine, Penstrep.

Cells were seeded in 96-well plate based on the cell concentrations indicated above, in a volume of 50 ul per well. Cells were cultured in 37 C, 5% CO2 for 3-5 days, as indicated. MTT (Chemicon International) was then added to all well, 10 ul per well, and the cells were allowed to continue culturing for 4 hours. Next was addition of isopropanol/0.04N HCL, 100ul per well, and the well contents were mixed by pipetting up and down to dissolve the formazan into a homogeneous blue solution. Finally, the plates were read at 570 nm and 630 nm. Use dual wavelength of 570 nm subtracted from reference 630 nm.

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

%_Inhibition = Cells_treated_with_Test_Compound / Cells_treated_with_Vehicle_(DMSO)

For selected test compounds, fold inhibition was plotted against compound concentration. Fold change values were calculated from GraphPad Prism software.

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

Compounds with a fold change value greater than 0.6 were considered inactive. Compounds with a fold change value less than 0.6 were considered active.

Active compounds were given a score of 100 and inactives were given a score of 0.

Raji Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

EuMyc Lymphoma Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

70Z/3 Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

9E10 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

Jurkat Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

MCF7 Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

T47D Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

MD-MDA-231 Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

HS578 Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

SKBR Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

Overall Outcome and Score:

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

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

List of Reagents:

MTT (Chemicon International)

This assay was performed by a collaborator of 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 absorbance. 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