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
Name: 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. ..more
BioActive Compounds: 2
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: HAPTOGLOBIN_SAA1-SAA4_INH_FLUOR_0384_3XQPCR Round 0
Name: 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.
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 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.
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.
Late stage, late stage AID, assay provider, purchased, synthesized, counterscreen, QPCR, qRT-PCR, real time PCR, quantitative, PCR, polymerase chain reaction, gene expression, TaqMan, Haptoglobin, HP, binding peptide; haptoglobin alpha(1S)-beta; haptoglobin alpha(2FS)-beta; haptoglobin, alpha polypeptide; haptoglobin, beta polypeptide; SAA1, serum amyloid A1, SAA; PIG4; SAA2; TP53I4; MGC111216; SAA1, apolipoprotein; SAA4, serum amyloid A4, constitutive, constitutively expressed serum amyloid A protein; serum amyloid A-4 protein, C-SAA, CSAA; percent, 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.
§ Panel component ID.
The purpose of this assay is to determine whether powder samples of possible LRH1 inverse agonist probe candidates are able to block the expression of pro-inflammatory target genes haptoglobin, SAA1, and SAA4. LRH-1 activation has been shown to prevent the cytokine induced stimulation of certain proinflammatory genes from the liver that are activated in the acute phase response. The genes are Haptoglobin, SAA1 and SAA4. In these assays huh7 cells endogenously expressing LRH-1 were treated with 10 uM compound or DMSO vehicle for 18 hours. Following compound incubation, 3nM IL1b and IL6 inflammatory cytokines were added to cells and incubated for a further 3 hours followed by isolation of RNA, conversion to cDNA, and Taqman-based QPCR. As designed, a compound that inhibits LRH1 activity will reduce target gene expression following addition of cytokines, leading to decreased production of the PCR amplicon, thereby reducing fluorescence, and increasing Ct.
huh7 cells endogenously expressing LRH-1 were plated in 6 well plates at a density of 200,000 cells/well and after 18 hour incubation, treated with 10 uM compound or DMSO vehicle for additional 18 hours. Following compound incubation, 3 nM IL1b and IL6 inflammatory cytokines were added to cells and incubated for a further 3 hours. Then cells were lysed and RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA). DNA was generated using the Taqman reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using LightCycler RNA Amplification Kit HybProbe master mix (Roche) with Taqman MGB Probe 6FAM--MGBNFQ on a model LightCycler480 real time PCR system (Roche). Data are expressed as the mean percent inhibition plus or minus SD of 3 replicates normalized to 100 ug total RNA.
The percent inhibition was normalized based on measurement of total RNA.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Any compound that exhibited a percent inhibition greater than the hit cutoff calculated ( > 25 % Inhibition) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
Haptoglobin Score: The PubChem Activity Score range for active compounds is 100-98. There are no inactive compounds.
SAA1 Score: The PubChem Activity Score range for active compounds is 100-97. There are no inactive compounds.
SAA4 Score: The PubChem Activity Score range for active compounds is 100-99. There are no inactive compounds.
Overall Outcome and Score:
The overall outcome was active if the compound was active in at least one panel, inactive otherwise.
The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive 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).
QiaShredder (Qiagen, 79656)
RNeasy mini kit (Qiagen, 74104)
LightCycler RNA Amplification Kit HybProbe (Roche, 12015145001)
Forward primer (Lifetech Applied Biosystems)
Reverse primer (Lifetech Applied Biosystems)
MGBProbe (Lifetech Applied Biosystems, 4304971, 6FAM)
LightCycler480 multiwell plate 96 (Roche, 04729692001)
LightCycler480 sealing foil (Roche, 04729757001)
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.
Categorized Comment - additional comments and annotations
** Test Concentration. § Panel component ID.