Late stage assay provider luminescence-based cell-based dose response assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1)
Name: Late stage assay provider luminescence-based cell-based dose response assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1). ..more
BioActive Compounds: 2
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Enzo Lalli, CNRS
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03 DA030558-01
Grant Proposal PI: Enzo Lalli, CNRS
External Assay ID: DAX1_INH_LUMI_0096_3XIC50 MDRUN (run by assay provider)
Name: Late stage assay provider luminescence-based cell-based dose response assay for inhibitors of the orphan nuclear receptor subfamily 0, group B, member 1 (DAX1; NR0B1).
Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding (DBD) and ligand-binding domains (LBD). Small pharmacological compounds able to bind to the cleft of the ligand-binding domain could alter its conformation and subsequently modify transcription of target genes. Such ligand agonists and/or antagonists have already been successfully designed for 23 nuclear receptors among the 48 previously identified in the human genome (1-3). Of interest, DAX-1 (NR0B1; dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1) has been shown to act as a robust transcriptional repressor, inhibiting genes involved in steroidogenesis through interaction with corepressors and regulating the pluripotency of stem cells (4-8). The human DAX-1 gene encodes a protein whose C terminus is similar to the LBD of nuclear hormone receptors, while its N terminus is composed of three cysteine-rich 70 amino acids with little similarity to known proteins (4, 7). Mutations in Dax-1 have been shown to cause the X-linked form of adrenal hypoplasia congenita (AHC), associated with hypogonadotropic hypogonadism (HHG). AHC-HHG-associated mutations share an altered DAX-1 C-terminal domain (5, 9), resulting in loss of transcriptional repression activity (5, 7, 9). This finding suggests that the impairment of the DAX-1 transcriptional activity is directly linked to the pathogenesis of AHC-HHG. In addition, Dax-1 has also been shown to be highly expressed in pediatric Ewing tumors (10). As a result, the identification of selective inhibitors of Dax-1 will serve as useful tools to elucidate the developmental and tumorigenic roles of Dax-1, and its maintenance of stem cell pluripotency.
1, Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol 19:1429-1438, 2005.
2. Kliewer SA, Lehmann JM, and Willson TM. Orphan nuclear receptors: shifting endocrinology into reverse. Science 284: 757-760, 1999.
3. Li Y, Lambert MH, and Xu HE. Activation of nuclear receptors: a perspective from structural genomics. Structure (Camb) 11: 741-746., 2003.
4. Lalli, E., M. H. Melner, D. M. Stocco, and P. Sassone-Corsi. 1998. DAX-1 blocks steroid production at multiple levels. Endocrinology 139:4237-4243. 22. Lalli, E., and P. Sassone-Corsi. 1999. DAX-1 and the adrenal cortex. Curr. Opin. Endocrinol. Diabetes 6:185-190.
5. Ito, M., R. Yu, and J. L. Jameson. 1997. DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol. Cell. Biol. 17:1476-1483.
6. Zazopoulos, E., E. Lalli, D. M. Stocco, and P. Sassone-Corsi. 1997. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311-315.
7. Lalli, E., B. Bardoni, E. Zazopoulos, J.-M. Wurtz, T. M. Strom, D. Moras, and P. Sassone-Corsi. 1997. A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita. Mol. Endocrinol. 11:1950-1960.
8. Lalli E, Alonso J. Targeting DAX-1 in embryonic stem cells and cancer. Expert Opin Ther Targets. 2010 Feb;14(2):169-77.
9. Zanaria, E., F. Muscatelli, B. Bardoni, T. M. Strom, S. Guioli, W. Guo, E. Lalli, C. Moser, A. P. Walker, E. R. B. McCabe, T. Meitinger, A. P. Monaco, P. Sassone-Corsi, and G. Camerino. 1994. An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635-641.
10. Mendiola M, Carrillo J, Garcia E, Lalli E, Hernandez T, de Alava E, Tirode F, Delattre O, Garcia-Miguel P, Lopez-Barea F, Pestana A, Alonso J. The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors. Int J Cancer. 2006 Mar 15;118(6):1381-9.
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The purpose of this assay is to determine dose response curves for powder samples of compounds identified as Dax-1 inhibitor probe candidates. This assay monitors the ability of compounds to inhibit the activity of the DAX-1 nuclear receptor (NR0B1), a robust transcriptional repressor. This assay employs HEK293 cells co-transfected with a GAL4 DBD-DAX1 C-terminal construct (pG4D 207-470) and a luciferase reporter under control of the thymidine kinase minimal promoter preceded by yeast Gal4 binding sites (pGAL4-tk-luc). In this assay, transfected cells are incubated with test compounds, followed by measurement of well luminescence. DAX1 activity represses expression of the luciferase reporter plasmid. As designed, a DAX1 inhibitor will prevent or reduce DAX1-mediated transcriptional repression, leading to increased expression of the luciferase reporter gene, and increased well luminescence. Compounds are tested in triplicate at a single concentration of 100 uM and using a 3-point, 1:2 dilution series, starting at a nominal test concentration of 50 uM.
HEK293 cells were routinely cultured in T-75 flasks containing 15 mL of DMEM (4.5 g/L glucose) medium supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin-streptomycin at 37 C, 5% CO2 and 95% relative humidity (RH). When cells reached about 70% confluency, they were transfected with 1.5 mL of serum-free OptiMEM containing 7.8 ug of the pGal4-tk-luc reporter plasmid, 7.8 ug of the Gal4 DBD-DAX1 fusion expression vector pG4D 207-470, and 31.25 uL of TransIT-293 transfection reagent. In the absence of a pharmacological positive control, DAX-1 inhibition was mimicked by transfecting cells with the empty vector pG4M polyII instead of the pG4D 207-470 vector. Four hours post transfection, cells were harvested using 3 mL of trypsin-EDTA solution and resuspended at a concentration of 400,000 cells per mL in the wells of culture-treated white 96-well plates (50 uL per well), diluted in DMEM supplemented as above. Transfected cells were then incubated at 37 C, 5% CO2 and 95% RH for 16 hours before luciferase assay. 50 uL of each dilution of the test compounds or DMSO control to reach the appropriate final concentration were also added to the wells. Transfection experiments were performed in duplicate and repeated three times for each tested drug concentration.
Luciferase assays were performed using the Luciferase Assay System as described by the manufacturer except that 50 uL of Luciferase Assay Reagent were injected in each well using a GloMax luminometer equipped with an automatic injector (Promega). Luminescence in each well was integrated for 10 seconds with a 2-second delay after Luciferase Assay Reagent injection. The relative luciferase activity of each test compound was calculated as follows:
Relative_Activity = (average luminescence of test compound wells) / (average luminescence of DMSO wells)
PubChem Activity Outcome and Score:
Compounds with a relative activity equal to or greater than 1.25 at any dose were considered active. Compounds with a relative activity less than 1.25 at all doses were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed relative activity at 100 uM.
The PubChem Activity Score range for active compounds is 100-78, and for inactive compounds 28-0.
List of Reagents:
pGAL4-tk-luc reporter plasmid (supplied by Assay Provider)
pG4D 207-470 DAX-1 expression plasmid (supplied by Assay Provider)
pG4M polyII empty plasmid (supplied by Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Invitrogen, part 10106-169)
Penicillin-Streptomycin 100X Liquid Solution (Invitrogen, part 15140-130)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
Trypsin-EDTA solution (Invitrogen, part 25300-104)
Luciferase Assay System (Promega, part E1500)
White, clear bottom 96-well plates (Corning part 3903)
This assay was performed by the assay provider's lab. 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)