SR1078

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°C under 5% CO2. HepG2 cells are maintained and routinely propagated in minimum essential medium supplemented with 10% (v/v) fetal bovine serum at 37°C 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°C. 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.

References:

[1]. Wang Y, et al. Identification of SR1078, a synthetic agonist for the orphan nuclear receptors RORα and RORγ. ACS Chem Biol. 2010 Nov 19;5(11):1029-34.