Determination of Affinity and Activity of Ligands at the Human Neuropeptide Y Y4 Receptor by Flow Cytometry and Aequorin Luminescence

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y₁, Y₂, and Y₅ receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y₄ receptor (hY₄R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K⁴]hPP has high affinity (K_d 5.6 nM) to the hY₄R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K⁴]hPP to hY₄R was visualized by confocal microscopy. The hY₄R, 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.