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

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 (pKi 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.