Illuminating Neuropeptide Y Y₄ Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as...

The neuropeptide Y (NPY) Y₄ receptor (Y₄R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y₄R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y₄R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y₄R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y₄R ligands derived from recently reported cyclic hexapeptides showing picomolar Y₄R binding affinity. With pK_i values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y₄R affinity. Y₄R 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 Y₄R (equilibrium pK_d: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y₄R competition binding studies and imaging techniques such as single-receptor molecule tracking.