Primary Cell-based High Throughput Screening assay for inhibitors of the Retinoic Acid Receptor-related orphan receptor A (RORA)
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: 278
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.
RORA is one of three related orphan nuclear receptors, including RORB and RORC, known as "Retinoic Acid Receptor-related orphan receptors".
RORA has unusual potential as a therapeutic target for "metabolic syndrome". This refers to a convergence of pathogenic factors, including insulin resistance, dyslipidemia, hypertension, and a proinflammatory state, that greatly elevate the risk of diabetes and atherosclerosis. RORA has been shown to be implicated in several key aspects of this pathogenesis. For instance, the staggerer mouse, which carries a homozygous germline inactivation of RORA, shows low body weight, high food consumption, elevated angiogenesis in response to ischemia, susceptibility to atherosclerosis, and an abnormal serum lipid profile. A combination of genetic and cellular studies also showed that RORA regulates lipoprotein levels and very likely has an impact on circadian rhythm and metabolism in peripheral tissue such as the liver.
Taken together, those observations highlight the need to identify specific ligands of RORA that could help understand its therapeutic potential and provide good chemical starting points for further drug development.
RAR-related orphan receptor A, RORA, nuclear receptor, RZRA, ROR1, ROR2, ROR3, NR1F1, transcriptional assay, CHO-K1, luciferase, luminescence, Scripps, primary, inhibition
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 RORA (encoded by the pFA-hRORA plasmid, Orphagen Pharmaceuticals) to regulate a luciferase reporter containing 5xGal4 response elements at its promoter region (pG5-luc, Stratagene). Both pFA-hRORA 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 RORA 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 RORA 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 RORA/Gal4 fusion expressing plasmid (pFA-hRORA, Orphagen Pharmaceuticals). Transfection was performed using the TransIT-CHO transfection kit (Mirus part 2176) according to the manufacturer's protocol.
Flasks were designated +RORA or -RORA. +RORA 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 125 ng of pFA-hRORA plasmid (Orphagen Pharmaceuticals).
-RORA designated flasks received exactly the same transfection reagents and DNA excepting plasmid pFA-hRORA.
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 -RORA were seeded in the first two columns of the 1536-well plate (mock-transfected positive control) and the remaining 46 columns were filled with +RORA cells.
One hour after seeding, +RORA cells were treated with 50 nL/well of test compounds (i.e. 10 micromolar) or DMSO (negative control) and -RORA 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 follows:
%inhibition = (1-(median_positive_control - test_compound)/(median_positive_control - median_negative_control)*100
with positive control : -RORA cells treated with 1% DMSO
and negative control : +RORA 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 inhibit transcriptional activity.
Data Table (Concise)