Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools

Gleixner J, Kopanchuk S, Grätz L, Tahk MJ, Laasfeld T, Veikšina S, Höring C, Gattor AO, Humphrys LJ, Müller C, Archipowa N, Köckenberger J, Heinrich M, Kutta RJ, Rinken A, Keller M (2024)

Publication Type: Journal article

Publication year: 2024

Journal

ACS Pharmacology & Translational Science American Chemical Society

DOI: 10.1021/acsptsci.4c00013

Abstract

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 pKi 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 pKd: 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.

Authors with CRIS profile

Johannes Köckenberger Lehrstuhl für Pharmazeutische Chemie Markus Heinrich Professur für Pharmazeutische Chemie

Additional Organisation(s)

FAU Forschungszentrum Neue Wirkstoffe (FAU NeW)

Involved external institutions

University of Tartu EE Estonia (EE) Universität Regensburg DE Germany (DE)

How to cite

APA:

Gleixner, J., Kopanchuk, S., Grätz, L., Tahk, M.J., Laasfeld, T., Veikšina, S.,... Keller, M. (2024). Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools. ACS Pharmacology & Translational Science. https://doi.org/10.1021/acsptsci.4c00013

MLA:

Gleixner, Jakob, et al. "Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools." ACS Pharmacology & Translational Science (2024).

BibTeX: Download