Dose-response cell-based assay for activators 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. 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. ..more
BioActive Compounds: 75
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
External Assay ID: SF1_AG_LUMI_1536_EC50
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 . Ligands for SF-1 may have clinical applications as modulators of adrenal steroid synthesis, and also as modulators of energy metabolism and obesity through the regulation of the ventromedial hypothalamus (VMH).
To explore the function of SF-1, small molecule probes have to be identified. To this end, we screened the MLSCN library via a cell-based assay developed by Orphagen Pharmaceuticals (San Diego, CA).
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,
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, agonist
Twelve hundred and twenty nine compounds have been identified as hits during a previously described set of experiments entitled "Primary Cell-based High Throughput Screening assay for activators of the nuclear receptor Steroidogenic Factor 1 (SF-1)". Further information on the primary screen can be found by searching on this website for PubChem AID=522.
Among those hits, a subset of compounds showing selectivity towards SF-1 was selected by applying the following criteria:
1) SF1% >70% AND S/A>4
OR(2) 70>SF1 % >50 AND S/A>6
OR(3) SF1% >50 AND RORa% <0
where "SF1%" represents the percentage of activation measured in the SF-1 primary screening (AID=522), "RORa%" the percentage of activation measured in the RORa primary screening (AID=560) and "S/A" the ratio SF1% / RORa%.
One hundred and seven compounds selected using criteria stated above were assessed in dose-response experiments in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 100 micromolar.
As with the primary screen, the dose-response 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 increase the basal transcription of luciferase are detected through the increase of light emission using the SteadyLite luciferase detection kit (Perkin Elmer). Such compounds hence constitute potential activators of the SF-1 nuclear receptor.
This assay was conducted in triplicate in 1536-well format.
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 54uL of TransIT CHO reagent (Mirus), 9 uL 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, background) 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 or DMSO (negative control) and -SF1 cells with
50nL/well of DMSO (positive control). Each compound dilution was assayed in triplicate, for a nominal total of 30 data points per
dose-response. 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 activation of each compound has been calculated as follows:
%activation = 100 x (( Median_RLU_negative_control - RLU_compound ) / ( Median_RLU_negative_control - Median_RLU_background ))
with background: -SF1 cells treated with 1% DMSO
and negative control: +SF1 cells treated with 1% DMSO
For each compound, percentage activations were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported EC50 values were generated from fitted curves by solving for X-intercept at a Y-value corresponding to half of the maximum %activation reached.
In cases where compounds showed toxicity at a defined concentration (characteristic pattern of activation followed by a dramatic
inhibition), datapoints were invalidated to allow curve fitting and the corresponding result annotated as "possibly cytotoxic".
Compounds with EC50 of greater than 10 micromolar were considered inactive; compounds with EC50 of equal to or less than 10 micromolar
were considered active.
The activity score was calculated based on pIC50 values for compounds from the reported EC50 value generated from the fitted curves, as mentioned above.
Since no SF-1 activators have been reported in the literature at the time this screening effort was performed, the assay described here
has not been optimized in reference to a positive control activator. Instead, the %activation values reported here have been normalized
to "-SF1 cells" treated with 1% DMSO. Results should be reviewed with this caveat in mind.
All data reported were normalized on a per-plate basis.
Possible artifacts of this assay can include, but are not limited to:
compounds that induce cell proliferation, compounds that increase luciferase activity, compounds that non-specifically activate
Categorized Comment - additional comments and annotations
Data Table (Concise)