Kinase experiment:

The ALPHA screen assays are performed. Assays are performed in triplicate in white opaque 384-well plates under green light conditions (<100 lux) at room temperature. The final assay volume is 20 μL. All dilutions are made in assay buffer (100 mM NaCl, 25 mM Hepes, 0.1% (w/v) BSA, pH 7.4). The final DMSO concentration is 0.25% (v/v). A mix of 12 μL of GST-RORγ-LBD (10 nM), beads (12.5 μg/mL of each donor and acceptor), and 4 μL of increasing concentrations (210 nM-50 μM) of compound SR1078 are added to the wells, the plates are sealed and incubated for 1h. After this preincubation step, 4 μL of Biotin-TRAP220-2 peptide (50 nM) is added, the plates are sealed and further incubated for 2h. The plates are read on PerkinElmer Envision 2104 and data analyzed using GraphPad Prism software[1].

Cell experiment:

HEK293 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum at 37℃ under 5% CO2. HepG2 cells are maintained and routinely propagated in minimum essential medium supplemented with 10% (v/v) fetal bovine serum at 37℃ under 5% CO2. In experiments where lipids and sterols are depleted, cells are maintained on charcoal treated serum (10% (v/v) fetal bovine serum) and treated with 7.5 μM lovastatin and 100 μM mevalonic acid. 24 h prior to transfection, HepG2 or HEK293 cells are plated in 96-well plates at a density of 15×103 cells/well. Transfections are performed using LipofectamineTM 2000. 16 h post-transfection, the cells are treated with vehicle or SR1078. 24 h post-treatment, the luciferase activity is measured using the Dual-GloTM luciferase assay system. The experiments are repeated at least three times[1].

Animal experiment:

Mice[1] Plasma levels of SR1078 are evaluated in C57BL6 mice (n=3 per time point) administered by i.p. injection. After 1, 2, 4, and 8h blood is taken. In the 2h time point liver is taken for target gene analysis. Plasma is generated using standard centrifugation techniques, and the plasma and tissues are frozen at –80℃. Plasma and tissues are mixed with acetonitrile (1:5 (v/v) or 1:5 (w/v), respectively), sonicated with a probe tip sonicator, and analyzed for drug levels by liquid chromatography/tandem mass spectrometry.

SR1078 is an agonist of retinoic acid receptor-related orphan receptor (ROR) RORα and RORγ with IC50 values of both 1-3 μM [1].

RORα and RORγ are two members of nuclear receptor superfamily, which possesses a highly conserved DNA-binding and a ligand-binding domain. These two receptors are considered to play important roles in the regulation of metabolism and the function of immune system [1].

SR1078 is a selective RORα and RORγ agonist, which activate RORα and RORγ directed transcription via inducing conformational changes [1].

SR1078 selectively targets on RORα and RORγ as an agonist but not on LXR and FXR [1]. It was shown that SR1078 was able to stimulate ROR activity and subsequent ROR-directed mRNA expression. When HepG2 cells were treated with SR1078 for 24h, the ROR directed reporter gene FG21 mRNA level was increased by 3-fold, while another reporter gene G6Pase was increased by 2-fold [1].

In mouse model, i.p. injection of 10 mg/kg SR1078 for 1 hr resulted in 3.6 μM plasma concentration., and after 8 hr the concentration still sustained at 800 nM. i.p. injection of 10 mg/kg SR1078 for 2 hr resulted in significantly increased level of mRNA of reporter gene G6Pase and FG21 [1].

Reference:
[1] Wang Y et al. , Identification of a Synthetic Agonist for the Orphan Nuclear Receptors RORα and RORγ, SR1078. ACS Chem Biol., 2010, 5(11): 1029-1034.