| Assay Method Information | |
| | Radioligand Binding Assay for EP4 Receptor |
| Description: | The bindings, to the radioligand EP4, of a control compound and the compounds prepared by Examples 1 to 43 were tested using recombinant human EP4 receptor membrane protein (prepared from 293 cells overexpressing human EP4 receptor). The compounds to be tested and PGE2 were formulated as 10 mM stocks in a DMSO solvent, and then 8 concentration points of 4× working solution were serially diluted using a buffer (50 mM HBSS, 0.1% BSA, 500 mM NaCl). 1 μL of the compound working solution, DMSO, and the PGE2 working solution were added to the assay plate, respectively; 100 μL of a EP4 receptor membrane protein (20 μg/well) and 100 μL of a radioligand [3H]-PGE2 (PerkinElmer, Cat: NET428250UC, Lot: 2469552) (final concentration of 1.5 nM) were added and incubated at sealed room temperature for 1 hour. Unifilter-96 GF/C filter plate (Perkin Elmer) was soaked with 0.5% BSA, 50 μL per well, for at least 30 min at room temperature. After binding was complete, the reaction mixture was harvested through a GF/C plate using a Perkin Elmer Filtermate Harvester, followed by washing the filter plate and drying the filter plate for 1 hour at 50° C. After drying, the bottom of the filter plate wells was sealed using a Perkin Elmer Unifilter-96 sealing tape, and 50 μL of MicroScint™-20 cocktail (Perkin Elmer) was added to seal the top of the filter plate. |
| Affinity data for this assay | |
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