AID	Name	Type	Comment
522	Primary Cell-based High Throughput Screening assay for activators of the nuclear receptor Steroidogenic Factor 1 (SF-1)	screening
560	Primary Cell-based High Throughput Screening assay for activators of the Retinoic Acid Receptor-related orphan receptor A (RORA)	screening
1954	Summary of probe development efforts to identify activators of the Retinoic Acid Receptor-related orphan receptor A (RORA).	summary

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 (1-X01-MH077624-01)
Network: Molecular Library Screening Center Network (MLSCN)

External Assay ID: SF1_AG_LUMI_1536_CS_EC50

Name:

Counterscreen for activators of the Retinoic Acid Receptor-related orphan receptor A (RORA): A cell-based dose-response assay for inhibition of the Steroidogenic Factor 1 (SF-1)

Description:

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].

RORA is one of three related orphan nuclear receptors, including RORB and RORC, known as "Retinoic Acid Receptor-related orphan receptors" [4].

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 [5]. 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 [6-8], elevated angiogenesis in
response to ischemia [9], susceptibility to atherosclerosis [8], and an abnormal serum lipid profile [10]. 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.

References:

[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]Jetten AM, Kurebayashi S, and Ueda E. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog Nucleic Acid Res Mol Biol 69: 205-247, 2001.
[5]Grundy SM, Brewer HB, Jr., Cleeman JI, Smith SC, Jr., and Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Arterioscler Thromb Vasc Biol 24: e13-18, 2004.
[6]Bertin R, Guastavino JM, and Portet R. Effects of cold acclimation on the energetic metabolism of the staggerer mutant mouse. Physiol Behav 47: 377-380, 1990
[7]Guastavino JM, Bertin R, and Portet R. Effects of the rearing temperature on the temporal feeding pattern of the staggerer mutant mouse. Physiol Behav 49: 405-409, 1991
[8]Mamontova A, Seguret-Mace S, Esposito B, Chaniale C, Bouly M, Delhaye-Bouchaud N, Luc G, Staels B, Duverger N, Mariani J, and Tedgui A. Severe atherosclerosis and hypoalphalipoproteinemia in the staggerer mouse, a mutant of the nuclear receptor RORalpha. Circulation 98: 2738-2743., 1998
[9]Besnard S, Silvestre J-S, Duriez M, Bakouche J, Lemaigre-Dubreuil Y, Mariani J, Levy BI, and Tedgui A. Increased ischemia-induced angiogenesis in the staggerer mouse, a mutant of the nuclear receptor RORa. Circ Res 89: 1209-1215, 2001.
[10]Raspe E, Duez H, Gervois P, Fievet C, Fruchart J-C, Besnard S, Mariani J, Tedgui A, and Staels B. Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha. J Biol Chem 276: 2865-2871, 2001.

Keywords:

RAR-related orphan receptor A, RORA, nuclear receptor, RZRA, ROR1, ROR2, ROR3, NR1F1, transcriptional assay, CHO-K1, luciferase, luminescence, Scripps, primary, activation

Assay Overview:

Nine hundred and seventy 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 Retinoic Acid Receptor-related orphan receptor A (RORA)". Further information on the primary screen can be found by searching on this website for PubChem AID=560.

Among those hits, a subset of compounds showing selectivity towards RORA was selected by applying the following criteria:

(1) RORA% >70% AND S/A>4
OR(2) 70>RORA % >50 AND S/A>6
OR(3) RORA% >50 AND SF1% <0

where "RORA%" represents the percentage of activation measured in the RORA primary screening (AID=560), "SF1%" the percentage of activation measured in the SF1 primary screening (AID=522) and "S/A" the ratio RORA% / SF1%.

Sixty three 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.

This counterscreen employs the same format as the assay used to select potential RORA activators; except that here the biological target is SF1. A compound that appears as an activator in both the RORA and SF1 assays would be less desirable for follow-up, since the compound may be a non-selective activator or format-specific artifact. On the other hand, compounds found inactive in this assay and confirmed active in RORA dose-response experiments are likely to be selective activators of RORA.

This dose-response counterscreen 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.

Protocol Summary:

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 transcriptional activity.