Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patrick Griffin, TSRI
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: U54 MH084512
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: ROR_INH_RAD_0384_IC50 MDRUN Round 1

Name: Late stage assay provider results from the probe development effort to identify selective inverse agonists of the Retinoic acid receptor-related Orphan Receptors (RORA): radioligand binding assay for RORa using [3H]25-hydroxycholesterol to determine whether probe candidates bind directly to RORa.

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). RORalpha (RORa ; RORA; NR1F1) is one of three related orphan nuclear receptors, including RORbeta (RORB ; RORB; NR1F2) and RORgamma (RORg; RORC; NR1F3), known as "Retinoic Acid Receptor-related orphan receptors" (4).

RORA has unusual potential as a therapeutic target for the "metabolic syndrome" which results in pathologies such as insulin resistance, dyslipidemia, hypertension, and a pro-inflammatory state, that greatly elevates the risk of diabetes and atherosclerosis (5).The related RORC demonstrates significant expression in metabolic tissues such as liver, adipose, and skeletal muscle (6). These two receptors are implicated in several key aspects of this metabolic pathogenesis. For instance, the staggerer mouse, which carries a homozygous germline inactivation of RORA, shows low body weight, high food consumption (7-9), elevated angiogenesis in response to ischemia (10), susceptibility to atherosclerosis (9), and an abnormal serum lipid profile (11). RORG null mice exhibit normal plasma cholesterol levels, but when bred with the RORA staggerer mice, the resulting RORalpha/gamma knockout exhibits hypoglycemia not found in the single mutant animals. These studies reveal the functional redundancy of RORa and RORg in regulating blood glucose levels and highlight the need for RORalpha/gamma ligands that can bind to these receptors and modulate their transcriptional activity (12, 13, 14).

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. Medvedev A, Yan ZH, Hirose T, Giguere V, Jetten AM. Cloning of a cDNA encoding the murine orphan receptor RZR/ROR gamma and characterization of its response element. Gene. 1996 Nov 28;181(1-2):199-206.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, Shan B. Role of LXRs in control of lipogenesis. Genes Dev. 2000 Nov 15;14(22):2831-8.
13. Kumar N, Solt LA, Conkright JJ, Wang Y, Istrate MA, Busby SA, Garcia-Ordonez R, Burris TP, Griffin PR. The benzenesulfoamide T0901317 is a novel RORalpha/gamma Inverse Agonist. Mol Pharm. Feb;77(2):228-36. Epub 2009 Nov 3.
14. Kumar N, Kojetin DJ, Solt LA, Kumar KG, Nuhant P, Duckett DR, Cameron MD, Butler AA, Roush WR, Griffin PR, Burris TP.Identification of SR3335 (ML-176): A Synthetic RORalpha Selective Inverse Agonist. ACS Chem Biol. 2011 Mar 18;6(3):218-22. Epub 2010 Dec 6.

Keywords:

Late stage, late stage AID, assay provider, purchased, synthesized, RAR-related orphan receptor A, ROR alpha, RORa, RORA, nuclear receptor, low throughput assay, RZRA, ROR1, ROR2, ROR3, NR1F1, inhibitor, inverse agonist, radioligand, RLB, radioactivity, cholesterol, competitive, ML176, assay provider, center based initiative, center-based, luciferase, luminescence, selective, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this assay is to determine whether a powder sample of a possible RORA inverse agonist probe candidate can bind directly to the RORA target receptor. In this assay, HepG2 cells are incubated for 20 hours with test compound and radiolabeled 25-hydroxycholesterol . Some cells were pre-treated with siRNAs against RORs RORA & RORG) to mimic the ROR inhibition effect of the compound. As designed, a compound that binds to RORA will displace radiolabeled 25-hydroxycholesterol. The compound was tested at a nominal concentration of 10 uM.

Protocol Summary:

This protocol has been previously described (see PMID 21090593). For ligand binding studies, RORa ligand binding domain (amino acids 304-556) was PCR-amplified and cloned into a pGEX-2T (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) encoding an N-terminal GST-Tag according to the manufacturer's instructions. The protein was induced with 1 mM isopropyl B-d-thiogalactoside in BL21 gold (DE3) cells (Invitrogen, Carlsbad, CA) and purified by affinity chromatography with Protino GST/4B column (Macherey-Nagel, Bethlehem, PA) followed by size-exclusion chromatography with HiLoad 26/60 Superdex 200 column (GE Healthcare). The protein was eluted, concentrated, and stored in 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM dithiothreitol, and 10% glycerol.

Forty-five or 90 ng of purified GST-RORalpha or GST-RORgamma was incubated with various concentrations of [ 3H]25-hydroxycholesterol in assay buffer (50 mM HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mM NaCl, and 5 mM MgCl2) to determine the Kd value. Nonspecific binding was defined in the absence of protein and excess of nonradioactive 25-hydroxycholesterol and was shown to be identical. The assays were terminated by rapid filtration through presoaked Whatman GF/B filters (0.5% polyethylenimine in phosphate-buffered saline) in Multiscreen plates (Millipore, Billerica, MA) and were washed (3 x 0.1 ml) with ice-cold assay buffer. The radioligand binding results were analyzed using Prism software (GraphPad Software, Inc., San Diego, CA). For the competition assay, various concentrations of compound were incubated with receptor in the presence of 3 nM [3H]25-hydroxycholesterol. The results of these competition radioligand binding assay illustrate the ability of SR3335 (SID 85261497; ML176) to displace radiolabeled 25-hydroxycholesterol from RORalpha LBD. The Ki was calculated as 220 nM using the Cheng-Prusoff equation. Importantly, this probe candidate did not compete well for binding when the LBD of RORa was utilized, demonstrating the RORA selectivity of this probe. See PMID 21090593 for details.

The fold-change inhibition for each compound was calculated as follows:

Fold_Change = Cells_treated_with_Test_Compound / Cells_treated_with_Vehicle_(DMSO)

The Ki of each compound tested was calculated. All Ki values were determined by non-linear regression (hyperbolic equation) analysis using the mixed inhibition model which allows for simultaneous determination of mechanism of inhibition. Mechanism of inhibition was determined using the "alpha" parameter derived from a mixed-model inhibition by GraphPad Prism.

PubChem Activity Outcome and Score:

Any compound with a Ki value > 500 nM is considered inactive. Any compound with a Ki < 500 nM is considered active.

Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

List of Reagents:

Radioligand 25-[26,27-3H]hydroxycholesterol was from PerkinElmer Life and Analytical Sciences (Waltham, MA).
384 well plates (PerkinElmer, part 6007688)

This assay was performed by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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 the binding vessel, or compounds that modulate receptor binding affinities. 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.

Grant Number: U54 MH084512