Abstract
Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y-1, Y-2, and Y-5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y-4 receptor (hY(4)R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K-4] hPP has high ...
Abstract
Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y-1, Y-2, and Y-5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y-4 receptor (hY(4)R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K-4] hPP has high affinity (K-d 5.6 nM) to the hY(4)R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K-4] hPP to hY(4)R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino) methyl] propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pK(i) 4.17), a lead to be optimized in terms of potency and selectivity.