| Assay Method Information | |
| | Selectivity Assay with Human NK-2 |
| Description: | The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC50/(1+[L]/KD) where [L] is the concentration of free radioligand and KD is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).The compounds of the invention, which were tested in the above NK-1 and NK-2 described assays, demonstrated a low affinity at the human NK-1 and human NK-2 receptors: more than 200 fold shift of the Ki compared to the human NK-3 receptor (table 3). Thus, compounds according to the invention have been shown to be selective over NK-1 and NK-2 receptors. |
| Affinity data for this assay | |
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