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 p K 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 p K 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.