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)
Name: 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). ..more
BioActive Compounds: 9
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: VP16_INH_LUMI_0384_3X%INH Round 0
Name: 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).
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.
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, VP16, 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, fold-change, fold change, selective, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether powder samples of possible LRH1 inverse agonist probe candidates are nonselective due to inhibition of VP16 In this counterscreen assay the nuclear receptor plasmid was replaced by the GAL4DBD-VP16LBD plasmid, which expresses the strong transactivation domain of the herpes simplex virus Virion Protein 16 (VP16) fused to the GAL4 DBD. Cells are co-transfected with the 5xGAL4 response element (UAS) luciferase reporter to monitor GAL4DBD-VP16LBD activity, followed by incubation with test compounds for 18-24 hours. As designed, compounds that inhibit VP16 activity will decrease pGAL4DBD-VP16LBD activity, leading to reduced activation of the pG5-luc and decreased well luminescence. These compounds are likely to be nonselective inhibitors or cytotoxic. Compounds are tested in triplicate at a nominal concentration of 5 uM.
Luciferase reporter assays were conducted using a pBind GAL4DBD-VP16LBD construct and UAS luciferase reporter cotransfected into HEK293T cells. Reverse transfections were performed in bulk using 4E6 cells in 10 cm plates, 9 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.
The fold change inhibition for each compound was calculated as follows:
[ Fold_Change_Inhibition ] = [ Cells_treated_with_Test_Compound ] / [ Cells_treated_with_Vehicle_(DMSO) ]
The average fold change of each compound tested is reported.
PubChem Activity Outcome and Score:
Any compound that exhibited a fold change inhibition greater than the hit cutoff calculated ( > 10 % 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.
The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 43-0.
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)
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.
Data Table (Concise)