| Assay Method Information | |
| | RORgamma Gal4 Reporter Gene Assay (FRET) |
| Description: | Determination of a ligand mediated Gal4 promoter driven transactivation to quantify ligand binding to RORγ was performed as follows: DNA encoding three different RORγ protein fragments was cloned into vector pCMV-BD (Stratagene). Expression was under control of a CMV promoter and as fusion to the DNA-binding domain of the yeast protein GAL4. The amino acid boundaries of the three proteins and the respective database entries are listed in Table 2. Other vectors used were pFR-Luc (Stratagene) as regulated reporter plasmid. pFR-Luc contains a synthetic promoter with five tandem repeats of the yeast GAL4 binding sites that control expression of the Photinus pyralis (American firefly) luciferase gene. In order to improve experimental accuracy the plasmid pRL-CMV was cotransfected. pRL-CMV contains the constitutive CMV promoter, controlling the expression of the Renilla reniformis luciferase. All Gal4 reporter gene assays were done in 293T cells (DSMZ (German Collection of Microorganisms and Cell Cultures), Braunschweig, Germany, ACC635) grown in Minimum Essential Medium (MEM) with Phenol Red. The medium is supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 1% Glutamax and 100 units Penicilin/Streptavidin per mL at 37° C. in 5% CO2.For the assay, 5×105 cells were plated per well in 96 well plates in 100 μL per well, incubated over night at 37° C. in 5% CO2. The following day, medium was discarded and the cells were transiently transfected using 20 μL per well of a OptiMEM PEI-based transfection-reagent (Sigma-Aldrich, 408727) including the three plasmids described above. About 4 h after addition of the transfection solution, fresh Minimal Essential Medium (MEM, same composition as used for plating cells, but without serum) was added. Then compound stocks, prediluted in MEM (same composition as used for plating cells) were added (final vehicle concentration not exceeding 0.1%).Cells were incubated for additional 16 h before firefly (FF) and renilla (REN) luciferase activities were measured sequentially in the same cell extract using a Dual-Light-Luciferase-Assay system (Dyer et al., Anal. Biochem. 2000, 282:158). All experiments were done at least in triplicates.Applying the Gal4 reporter gene assay as described above, the Examples of the present invention usually have an inhibition activity (IC50 FF resp. IC50 RENnorm) ranging from below 10 nM to about 20 μM, and typically, from about 10 nM to about 1 μM. The RORγ modulating compounds of the invention desirably have an inhibition in the Gal4 reporter gene assay ranging from below 10 nM to about 1 μM. Table 3 list the pIC50-value of typical examples of compounds of the invention that have an RORγ activity in the Gal4 reporter gene assay for firefly (FF) and renilla normalised (RENnorm) luciferase measurements (nt=not tested). It is understood that the data illustrated below may have reasonable variation depending on the specific conditions and procedures used by the person conducting the test. The efficacy was determined in comparison to the RORγt inhibitor T0901317 (equals 100%) and the pIC50-value is underlined, when the efficacy of the compound is below 50% of the reference. |
| Affinity data for this assay | |
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