Illuminating Neuropeptide Y Y(4) Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y(4) receptor (Y(4)R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y(4)R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y(4)R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y(4)R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y(4)R ligands derived from recently reported cyclic hexapeptides showing picomolar Y(4)R binding affinity. With pK(i) values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y(4)R affinity. Y(4)R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y(4)R (equilibrium pK(d): 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y(4)R competition binding studies and imaging techniques such as single-receptor molecule tracking.