Fluorescence Labeling of Neurotensin(8-13) via Arginine Residues Gives Molecular Tools with High Receptor Affinity

ACS Med Chem Lett. 2019 Nov 19;11(1):16-22. doi: 10.1021/acsmedchemlett.9b00462. eCollection 2020 Jan 9.


Fluorescence-labeled receptor ligands have emerged as valuable molecular tools, being indispensable for studying receptor-ligand interactions by fluorescence-based techniques such as high-content imaging, fluorescence microscopy, and fluorescence polarization. Through application of a new labeling strategy for peptides, a series of fluorescent neurotensin(8-13) derivatives was synthesized by attaching red-emitting fluorophores (indolinium- and pyridinium-type cyanine dyes) to carbamoylated arginine residues in neurotensin(8-13) analogues, yielding fluorescent probes with high NTS1R affinity (pK i values: 8.15-9.12) and potency (pEC50 values (Ca2+ mobilization): 8.23-9.43). Selected fluorescent ligands were investigated by flow cytometry and high-content imaging (saturation binding, kinetic studies, and competition binding) as well as by confocal microscopy using intact CHO-hNTS1R cells. The study demonstrates the applicability of the fluorescent probes as molecular tools to obtain, for example, information about the localization of receptors in cells and to determine binding affinities of nonlabeled ligands.