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

Abstract

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 NTS 1 R affinity (pK i values: 8.15−9.12) and potency (pEC 50 values (Ca 2+ 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-hNTS 1 R 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.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

“…Synthesis of the red-emitting dibenzodiazepinone-type MR ligands (structures also shown in Figure 2), containing a pyridinium-type (10,16) or an indolinium-type (12, 14, 17, 18, 20, 22, 23, 27, 28, 30) cyanine fluorophore, and reference compound 25. Reagents and conditions: (a) triethylamine, DMF, rt, 2 h, 21% (10), 34% (16); (b) DIPEA, DMF, rt, 1-2 h, 40% (12), 37% (14), 31% (17), 30% (18), 30% (20), 34% (22), 41% (23), 36% (27), 26% (28), 34% (30).…”

“…Fluorescence arbitrary values from high-content imaging fluorescence images were obtained using a previously described procedure. 13,27…”

“…For this reason, the pharmacophore (ligand) and the fluorophore are often held apart by a linker or spacer moiety. [6][7][8] There are numerous reports on fluorescent probes for GPCRs, for instance for neuropeptide Y, [9][10][11][12][13][14] histamine, [15][16][17][18][19] opioid, [20][21][22] dopamine, 23,24 neurotensin, [25][26][27][28] and adenosine 29 receptors.…”

Screening and quantification of the encapsulation of dyes in supramolecular particles

“…In the study of colloidal particles, like multilamellar vesicles, 18 this technique is primarily used to determine morphology and size. The technique can be used to study the affinity constant of molecules towards cell receptors, 19,20 the internalization of microparticles in cells 21 and the diffusion of small molecules in multilayer particles. 22 There are, however, relatively few studies of FC used for the measurement of affinity constants of particles.…”

Fluorescent H2 Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications

“…Replacing the propionic acid amide of the high affinity radioligand [ 3 H]­UR-DE257 ( 11 ) ( K d value: 31 nM), a squaramide derived from BMY 2536 ( 4 ), by red-emitting fluorophores was the starting point for the development of high affinity fluorescent hH 2 R ligands. As the physicochemical properties of the fluorescent labels can considerably effect, e.g., nonspecific binding or internalization, we chose two different types of red-emitting fluorophores with various electrical charge: the positively charged pyrilium dye (Py-5, 12 ) and differently charged cyanine dyes (positive: S0223 ( 13a ), neutral: S0436 ( 14a ), or negative: S0386 ( 15a ), free acids) (Scheme ).…”