Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)
Name: Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2). ..more
BioActive Compounds: 458
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: LRH1_INH_LUMI_1536_1X%INH PRUN
Name: Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2).
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.
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.
Primary, 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.
The purpose of this assay is to identify compounds that decrease LRH-1 activity. This assay monitors LRH-1 activity, as measured by inhibition of promoter activity of the Steroidogenic acute regulatory protein (Star). In this assay, HEK293T cells, co-transfected with a full length LRH-1 construct in a pSport6 vector backbone (pS6-LRH-1) and a Star-luciferase reporter construct are incubated for 24 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 are tested in singlicate at a nominal concentration of 3.6 uM.
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 StAR-Luc reporter plasmid, 11.5 ug of the LRH-1 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 LRH-1 expressing vector) as a control for background luminescence (no LRH-1 cells). Cells were then treated with 18 nL/well of test compounds, DMSO as a negative control (final concentration 0.36%) or SR-1848 as a positive control (36 uM final for full inhibition and 1.2 uM for 50% inhibition) 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 for each compound was calculated as follows:
%_Inhibition = ( 1 - ( Median_Positive_Control - Test_Compound ) / ( Median_Positive_Control - Median_Negative_Control ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing cells transfected with the reporter only (no LRH-1 expressed).
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, 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 100-68, and for inactive compounds 68-0.
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)
StAR-Luc plasmid (Assay Provider)
LRH-1 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)
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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: inducible reporter: luciferase induction
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: microplate: 1536 well plate
BAO: bioassay specification: assay measurement throughput quality: single concentration single measurement
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: primary
BAO: bioassay specification: bioassay type: functional: reporter gene
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of gene expression
BAO: meta target: molecular target: protein target: receptor: nuclear receptor
BAO: version: 1.4b1090
Assay Format: Cell-based
Assay Cell Type: MDA-MB-231
** Test Concentration.
Data Table (Concise)