Primary Cell-based High Throughput Screening assay for inhibitors of the nuclear receptor Steroidogenic Factor 1 (SF-1)
Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding and ligand-binding domains. ..more
BioActive Compounds: 359
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Orphagen Pharmaceuticals, San Diego, CA
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1X01-MH077624-01
Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding and ligand-binding domains.
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].
The nuclear receptor SF-1 (steroidogenic factor-1) belongs to the class of "unexplored" orphan nuclear receptors that have been poorly investigated at a pharmacological level.
SF-1 is expressed in the pituitary, testes, ovaries, and adrenal gland and regulates steroid hormone production at many levels, including direct regulation of expression of major P450 enzymes involved in steroid hormone synthesis .
To explore the potential of SF-1 as novel drug target, small molecule ligands have to be identified. To this end, we screened the MLSCN library via a cell-based assay developed by Orphagen Pharmaceuticals (San Diego, CA).
Ligands for SF-1 may have clinical applications as modulators of adrenal steroid synthesis. In particular, a properly designed antagonist to SF-1 is predicted to have therapeutic utility in the treatment of metastatic prostate cancer though suppression of both adrenal androgen and gonadal testosterone synthesis.
Another potential benefit of this effort could be the identification of SF-1 ligands that could become a novel class of centrally acting small molecules that regulate energy metabolism and obesity. Indeed, SF-1 is also a potential therapeutic target for the regulation of the ventromedial hypothalamus (VMH), known to process peripheral metabolic input and regulate energy homeostasis .
 Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol 19:1429-1438, 2005.
 Kliewer SA, Lehmann JM, and Willson TM. Orphan nuclear receptors: shifting endocrinology into reverse. Science 284: 757-760, 1999.
 Li Y, Lambert MH, and Xu HE. Activation of nuclear receptors: a perspective from structural genomics. Structure (Camb) 11: 741-746., 2003.
 Hammer GD and Ingraham HA. Steroidogenic Factor-1: Its role in endocrine organ development and differentiation. Frontiers in Neuroendocrinology 20: 199-223, 1999.
 Majdic G, Young M, Gomez-Sanchez E, Anderson P, Szczepaniak LS, Dobbins RL, McGarry JD, and Parker KL. Knockout mice lacking steroidogenic factor 1 are a novel genetic model of hypothalamic obesity. Endocrinology 143: 607-614., 2002.
steroidogenic factor, SF-1, nuclear receptor, NR5A1, FTZ1, FTZF1, ELP, AD4BP, transcriptional assay, cancer, obesity, CHO-K1, luciferase, luminescence
The transcriptional cell-based assay utilizes a fusion of the DNA-binding domain of the yeast transcriptional factor Gal4 with the ligand-binding domain of target receptor SF-1 (encoded by the pFA-hSF-1 plasmid, Orphagen Pharmaceuticals) to regulate a luciferase reporter containing 5xGal4 response elements at its promoter region (pG5-luc, Stratagene). Both pFA-hSF-1 and pG5-luc plasmids are transiently co-transfected in CHO-K1 (Chinese Hamster Ovary) cells. The presence in this cell line of required co-activators allows the expression of luciferase driven by activated SF-1 nuclear receptors. Compounds that inhibit the basal transcription of luciferase are detected through the suppression of light emission using the SteadyLite luciferase detection kit (Perkin Elmer). Such compounds hence constitute potential inhibitors of the SF-1 nuclear receptor.
The primary HTS assay was conducted in 1536-well format. All compounds were tested once at a 10 micromolar final concentration.
Six million CHO-K1 cells were seeded in T-175 flasks (Corning part 431080) containing 20 milliliters of F12 media (Invitrogen part 31765-092) supplemented with 10% v/v fetal bovine serum (Gemini part 900-108) and 1% v/v penicillin-streptomycin-neomycin mix (Invitrogen part 15640-055). Flasks were then incubated for 20 hours at 37 degrees Celsius, 5%CO2 and 95% relative humidity.
The following day, CHO-K1 cells were transiently transfected with the pG5-luc reporter plasmid (Stratagene) and the SF-1/Gal4 fusion expressing plasmid (pFA-hSF-1, Orphagen Pharmaceuticals). Transfection was performed using the TransIT-CHO transfection kit (Mirus part 2176) according to the manufacturer's protocol.
Flasks were designated +SF1 or -SF1. +SF1 flasks received 1.2 milliliters of F12 media containing 54 microliters of TransIT CHO reagent (Mirus), 9 microliters of CHO Mojo reagent (Mirus), 9 ug of pG5-luc (Stratagene), 8.75 ug of empty pcDNA3.1 (Invitrogen), and 250 ng of pFA-hSF-1 plasmid (Orphagen Pharmaceuticals).
-SF1 designated flasks received exactly the same transfection reagents and DNA excepting plasmid pFA-hSF-1.
Flasks were then placed back in the incubator at 37 degrees Celsius, 5%CO2 and 95% relative humidity.
Four hours after transfection, cells were trypsinized and suspended to a concentration of 800,000 cells per milliliter in F12 media (Invitrogen part 31765-092) supplemented with 10% v/v heat inactivated fetal bovine serum (Gemini part 900-108) and 1% v/v penicillin-streptomycin-neomycin mix (Invitrogen part 15640-055).
The assay began by dispensing 5 microliters of cell suspension to each well (i.e. 4,000 cells/well) of a white solid-bottom 1536-well plate. Cells from flasks designated -SF1 were seeded in the first two columns of the 1536-well plate (mock-transfected positive control) and the remaining 46 columns were filled with +SF1 cells.
One hour after seeding, +SF1 cells were treated with 50 nL/well of test compounds (i.e. 10 micromolar) or DMSO (negative control) and -SF1 cells with 50nL/well of DMSO (positive control). Plates were then placed in the incubator at 37 degrees Celsius, 5% CO2 and 95% relative humidity.
Twenty hours later, plates were equilibrated to room temperature for 20 minutes. A luciferase assay was performed by adding 5 microliters per well of the SteadyLite HTS reagent (Perkin Elmer part#6016989). After a 15 minutes incubation time, light emission was measured with the ViewLux reader (Perkin Elmer).
The percent inhibition of each compound has been calculated as follow:
%inhibition = (1-(median_positive_control - test_compound)/(median_positive_control - median_negative_control)*100
with positive control : -SF1 cells treated with 1% DMSO
and negative control : +SF1 cells treated with 1% DMSO
A mathematical algorithm was used to determine nominally active compounds.
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% of the highest observed primary inhibition. Negative % inhibition values are reported as activity score zero.
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to:
toxic compounds, compounds that inhibit luciferase activity, compounds that non-specifically inhibit or activate transcriptional activity.
Data Table (Concise)