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 (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.

Neurotensin (NT), a 13 amino acid neuropeptide (cf. Figure ) which was first isolated and purified from bovine hypothalamus,[1] is mainly found in the gastrointestinal tract and the central nervous system.[2,3] Three transmembrane receptors were identified to mediate the physiological actions of NT: the NTS1R and NTS2R, both family A G-protein coupled receptors (GPCRs), and the NTS3R (sortilin), a member of the Vps10p-domain receptor family.[2,4−6] As the NTS1R is overexpressed in various malignant tumors such as pancreatic adenocarcinoma, breast cancer, and colorectal carcinoma, it represents a potential target for tumor radiodiagnosis and endoradiotherapy,[7−10] an approach requiring labeled, hydrophilic, stable, and potent NTS1R ligands. Noteworthily, the carboxy-terminal hexapeptide of NT (NT(8–13), 1; Figure ) is equipotent with NT at NTS1 and NTS2 receptors.[11]

Figure 1

Structures of the tridecapeptide neurotensin, NT(8–13) (1, blue), the radiolabeled NT(8–13) derivative [3H]2,[19] and the fluorescent NT(8–13) derivative 3.[19]Granier et al.[20]Einsiedel et al.[21]Keller et al.[19]

Generally, the determination of dissociation constants of receptor ligands is fundamental in terms of studying ligand receptor interactions. For this purpose, well characterized labeled receptor ligands, used as tools for competition binding studies, are indispensable. Classical competition binding assays are based on radiolabeled ligands exhibiting high receptor affinity. During the past few decades, fluorescent receptor ligands have gained increasing importance as molecular tools,[12−18] representing an attractive alternative to radioligands, e.g. with respect to safety issues and costs.

Moreover, in contrast to radioligand binding assays, fluorescent ligands enable the measurement of bound ligand under homogeneous conditions both at equilibrium and in kinetic analyses.[22−27]

To date, a few fluorescently labeled NTS1R ligands have been reported, representing derivatives of NT,[28] NT(2–13),[29] or NT(8–13).[25,30] These fluorescent peptides have in common that the fluorophore was attached to the peptide via the N-terminus, which can result in a considerable decrease in receptor affinity.[30] Recently, a new labeling strategy for arginine-containing peptides, based on the bioisosteric replacement of arginine by an amino-functionalized, Nω-carbamoylated arginine, was introduced.[19] This proof-of-concept study included derivatives of 1, for example the radioligand [3H]2 and the cyanine dye-conjugated peptide 3 (Figure ). In the previous study, fluorescent ligand 3 was characterized in terms of NTS1R affinity by competition binding with [3H]2,[19] but its suitability as molecular tool for fluorescence-based techniques was not explored.

In this study, we conjugated two types of red-emitting fluorophore core structures (indolinium- and pyridinium-type cyanine dyes) to analogues of 1 containing an Nω-carbamoylated arginine in position 8 or 9. The used dyes (indolinium, pyridinium) are excitable with a red (635 nm) and a 488 nm argon laser, respectively, being standard equipment in many instruments. As the physicochemical properties of fluorescent dyes are a crucial factor effecting, e.g. solubility and unspecific interactions of the respective fluorescent ligands in biological systems,[31] we applied three differently substituted indolinium-type dyes (5, 8, 10; cf. Scheme and Figure ), accounting for a negative net charge, a positive charge, or no net charge of the fluorophore. All fluorescence-labeled peptides were investigated with respect to NTS1R affinity in radioligand competition binding assays. Selected fluorescent probes (including the previously reported compound 3(19)) were characterized by flow cytometry, high-content imaging, and confocal microscopy.

Scheme 1

Synthesis of the Fluorescent NT(8–13) Derivatives 6, 9, 11, 12, 14, 16, and 17 as well as the Fluorescent “Dummy Ligands” 19 and 20

Reagents and conditions: (a) DIPEA, DMF, NMP, rt, 30 min, 42% (6), 27% (11), 38% (12), 24% (16), 42% (17); (b) DIPEA, KHCO3, DMF, H2O, rt, 30 min, 26%; (c) triethylamine, DMF, NMP, rt, 2 h, 25%; (d) DIPEA, DMF, rt, 15 min, 27% (19), 64% (20).

Figure 2

Structures of the synthesized and investigated fluorescent NT(8–13) derivatives 3,[19]6, 9, 11, 12, 14, 16, and 17 as well as structures of the fluorescent “dummy ligands” 19, 20, and 21.[19]

The indolinium-type cyanine dye-labeled NT(8–13) derivatives 6, 9, 11, 12, 16, and 17 were prepared by treatment of the amino-functionalized precursor peptides 4, 7, or 15,[19] containing an Nω-carbamoylated arginine either in position 8 (4, 7) or in position 9 (15) with the succinimidyl esters of the respective dyes (5, 8, or 10) (Scheme ). The pyridinium dye-labeled peptide 14 was obtained by treatment of 7 with the pyrylium derivative 13(32) in the presence of triethylamine (Scheme ). The reference compounds 19 and 20 (fluorescent “dummy ligands”) were prepared from propylamine (18) and succinimidyl esters 10 and 5, respectively (Scheme ).

The stability of the fluorescently labeled NT(8–13) derivatives 6, 11, 14, and 17 was investigated by incubating these peptides in PBS, pH 7.4, at 22 °C for up to 48 h followed by RP-HPLC analysis. Whereas indolinium-type cyanine dye-labeled fluorescent probes (6, 11, 17) exhibited excellent stabilities (Figures S1, S2, and S4, Supporting Information), fluorescent probe 14, containing a pyridinium-type dye, showed minor decomposition after incubation times >24 h (Figure S3, Supporting Information).

Fluorescence quantum yields were estimated (reference: cresyl violet perchlorate) for the indolinium-type fluorescent probes 3,[19]6, and 11 as well as for the pyridinium-type fluorescent peptide 14 in PBS, pH 7.4, and in PBS supplemented with 1% BSA (Table S1, Figures S5–S8, Supporting Information). For all investigated compounds, fluorescence quantum yields, determined in PBS supplemented with 1% BSA, were higher compared to the quantum yields determined in neat PBS (Table S1, Supporting Information).

NTS1R binding data of the fluorescent peptides 6, 9, 11, 12, 14, 16, and 17 were determined by competition binding with the NT(8–13) derivative [3H]2[19] (for structure, see Figure ) at intact HT-29 colon carcinoma cells endogenously expressing the hNTS1R[33] but no NTS2R.[19] In addition, NTS1R binding data of 3, 6, 9, 11, 12, and 14 were determined by competition binding with [3H]2 at whole Chinese hamster ovary cells stably transfected with the hNTS1R (CHO-hNTS1R cells[21]). The pKi values of 3, 6, 9, 11, 12, and 14, obtained from these competition binding assays, were in excellent agreement with the pKi values determined at HT-29 cells (Table ).

Table 1

NTS1R Binding Data (pKi) of 1, 3, 6, 9, 11, 12, 14, 16, and 17, NTS2R Binding Data (pKi) of 1, 3, 6, 9, 12, and 17, pKd Values of 3, 6, 9, and 12 Determined by Saturation Binding (hNTS1R), and hNTS1R Potencies (pEC50) of 1, 3, 6, and 12 from Ca2+ Assays

	hNTS1R							hNTS2R
compound	pKia	pKib	pKd flow cytometryc	pKd high-content imagingd		pEC50		pKig
				60 min/no wash	75 min/with wash	1e	2f
1	9.89 ± 0.01h	n.d.	n.a.	n.a.	n.a.	8.92 ± 0.12	10.16 ± 0.14	9.13 ± 0.07
3	8.38 ± 0.02h	8.15 ± 0.03	8.44 ± 0.01	7.82 ± 0.16	8.09 ± 0.04	8.99 ± 0.07	n.d.	8.92 ± 0.15
6	8.86 ± 0.05	8.88 ± 0.12	8.41 ± 0.1	8.20 ± 0.07	8.42 ± 0.21	8.23 ± 0.07	9.38 ± 0.22	8.40 ± 0.05
9	8.66 ± 0.01	8.61 ± 0.08	n.d.	7.99 ± 0.14	8.61 ± 0.29	n.d.	n.d.	9.02 ± 0.06
11	8.37 ± 0.07	8.60 ± 0.05	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
12	8.70 ± 0.05	8.81 ± 0.03	n.d.	7.59 ± 0.10	8.17 ± 0.22	n.d.	9.43 ± 0.23	8.46 ± 0.14
14	9.12 ± 0.03	8.89 ± 0.09	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
16	8.57 ± 0.002	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	8.14 ± 0.02
17	8.45 ± 0.02	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.

Determined by radioligand competition binding with [3H]2 at HT-29 colon carcinoma cells (Kd ([3H]2) = 0.55 nM); mean values ± SEM from two (16), three (3, 6, 9, 11, 12, 17), or four (14) independent experiments (performed in triplicate).

Determined by radioligand competition binding with [3H]2 at CHO-hNTS1R cells (Kd ([3H]2) = 0.29 nM); mean values ± SEM from three independent experiments (performed in triplicate).

Determined by flow cytometric saturation binding at CHO-hNTS1R cells using PBS as binding buffer; mean values ± SEM from two (3) or four (6) independent experiments (performed in duplicate).

Determined by high-content imaging saturation binding at CHO-hNTS1R cells (binding buffer: Leibovitz’s L15 medium, incubation period: 60 or 75 min); “no wash” indicates that no washing step was performed before the measurement, “with wash” indicates that one washing step was performed shortly before the measurement; mean values ± SEM from four (3, 6, 9) or five (12) (60 min incubation), and three (9), four (3) or five (6, 12) (75 min incubation) independent experiments (performed in triplicate).

Determined in a Fura-2 Ca2+ assay at human HT-29 cells; mean values ± SEM from two (1) or three (3, 6) independent experiments (performed in singlet).

Determined in a Fluo-4 Ca2+ assay at CHO-hNTS1R cells; mean values ± SEM from eight independent experiments (performed in triplicate).

Determined by competition binding with [3H]2 at HEK-hNTS2R cell homogenates (Kd ([3H]2) = 0.79 nM); mean values ± SEM from three (3, 6, 9), four (16), five (12), or seven (1) independent experiments (performed in triplicate).

Data were previously reported as Ki value by Keller et al. and were reanalyzed to give the pKi value.[19] n.d.: not determined; n.a.: not applicable

As reported for 3,[19] the fluorescent NT(8–13) derivatives 6, 9, 11, 12, 14, 16, and 17 exhibited high NTS1R affinities with pKi values of 8.15–9.12 (Table ; competition binding curves shown in Figure S9, Supporting Information). This demonstrated that the recently introduced concept of peptide labeling via the nonclassical bioisosteric replacement of arginine by a functionalized Nω-carbamoylated arginine can be successfully applied to either arginine in 1 (Arg8: 3, 6, 9, 11, 12 and 14, Arg9: 16 and 17; cf. Figure ), even if bulky moieties such as fluorescent dyes are attached. Moreover, these results showed that the type of fluorophore (different core structures and charges; cf. Figure ) had little impact on receptor binding of the fluorescent peptides. In addition to hNTS1R affinities, hNTS2R binding data were determined for peptides 1, 3, 6, 9, 12 and 16 by competition binding at homogenates of HEK-293 cells, transiently transfected with the hNTS2R, using [3H]2 as radiolabeled probe (Kd (hNTS2R): 0.79 nM). Whereas hNTS2R affinities of 3 and 9 proved to be slightly higher compared to their NTS1R affinities, hNTS2R binding of 6, 12 and 16 was marginally lower than hNTS1R binding (Table ; competition binding curves shown in Figure S10, Supporting Information). This revealed that Nω-carbamoylation and fluorescence labeling at Arg8 or Arg9 of the weakly NTS1R-selective parent compound 1 did not induce selectivity for either NT receptor subtype, being consistent with the bioisosteric character of the carbamoylated arginine.

Agonist activities at the NTS1 receptor were studied for compounds 1, 3, 6, and 12 in a Fura-2 (1, 3, 6) or in a Fluo-4 (1, 6, 12) Ca2+ assay using HT-29 cells and CHO-hNTS1R cells, respectively (Figure S12A and S12C, Supporting Information; pEC50 values shown in Table ; representative time courses of Fluo-4 fluorescence shown in Figure S13, Supporting Information). The pEC50 values of compounds 3, 6, and 12, exhibiting maximal effects (efficacies) comparable to that of 1 (cf. upper curve plateaus in Figure S12A and S12C, Supporting Information) were in good agreement with the respective pKi values obtained from competition binding experiments with [3H]2 (Table ). It should be mentioned that CHO-hNTS1R cells, used for the Fluo-4 assay, show a considerably higher NTS1R expression compared to HT-29 cells,[19] used for the Fura-2 assay, presumably resulting in higher Fluo-4 assay potencies compared to Fura-2 assay potencies due to a receptor reserve in CHO-hNTS1R cells (see pEC50 values of compounds 1 and 6, Table ). The use of 6 and 12 for studying NTS1R antagonism of the NTS1R antagonists SR142948A (22) and SR48692 (23) (structures see Figure S11, Supporting Information) is described in the Supporting Information.

The fluorescent NT(8–13) derivatives 3 and 6 were studied by flow cytometric saturation binding at CHO-hNTS1R (3, 6) and HT-29 cells (6) (for representative isotherms, see Figure S14, Supporting Information). The obtained pKd values of 3 and 6 were in excellent agreement with the respective pKi values from competition binding studies with [3H]2 (Table ). Moreover, high-content imaging saturation binding studies were performed with 3, 6, 9, and 12 at CHO-hNTS1R cells (pKd values see Table , for saturation binding curves, see Figure S15, Supporting Information). A more detailed description of saturation binding studies as well as flow cytometric and high-content imaging competition binding studies (6, 12) and flow cytometric kinetic investigations (3, 6) (NTS1R) are provided in the Supporting Information.

In addition to flow cytometric and high-content imaging binding studies, binding of 3 (indolinium-type fluorophore with positive net charge), 6 (indolinium-type fluorophore with negative net charge), 11 (indolinium-type fluorophore without net charge), and 14 (pyridinium-type fluorophore with positive net charge) to CHO-hNTS1R cells was investigated by confocal microscopy at 22–25 °C. For all fluorescent probes (3, 6, 11, 14), fluorescence appeared to be mainly plasma membrane-associated until approximately 10 min of incubation, followed by a continuous increase in intracellular fluorescence, appearing to be located in vesicles (Figure (3, 6, 11, 14) and Movies 1, 3, and 7 (3, 6, 11)). Unspecific binding of 3, 6, 11, and 14 was very low (Figure ). The observed internalization of NTS1 receptors bound to 3, 6, 11, or 14 was consistent with previous reports on the internalization of NTS1 receptors upon agonist binding.[6,25,30,34] As a representative example, internalization of ligand 6 was also investigated at physiological temperature (37 °C), including a nuclear counterstain (Hoechst dye 33342). These experiments also revealed a fast cellular and vesicular uptake of fluorescent ligand (Figure S21, Supporting Information), which was more pronounced compared to the studies at lower temperature (Figure ). The dissociation kinetics of 3, 6, and 11 studied by confocal microscopy at CHO-hNTS1R cells, revealing recycling of NTS1Rs to the plasma membrane, are described in the Supporting Information.

Figure 3

Binding of the cyanine dye labeled fluorescent peptides 3 (fluorophore with positive net charge) (panel A), 6 (fluorophore with negative net charge) (panel B), and 11 (fluorophore with no net charge) (panel C) as well as the pyridinium dye labeled ligand 14 (fluorophore with positive net charge) (panel D) to live CHO-hNTS1R cells at 22–25 °C at the given concentrations, investigated by confocal microscopy (3, 6, and 11: excitation 633 nm, emission LP 650, 14: excitation 488 nm, emission LP 560). Images (representative of at least two experiments) were acquired with a LSM 510 confocal microscope after incubation times of 5, 15, 30, and 45 min. Unspecific binding was determined in the presence of 1 (100-fold excess to the fluorescent peptide).

Interestingly, incubation of CHO-hNTS1R cells with the fluorescent dummy ligands 19 (indolinium-type fluorophore without net charge), 20 (indolinium-type fluorophore with negative net charge), and 21 (indolinium-type fluorophore with positive net charge) revealed a strong cellular uptake of the positively charged compound 21 at a low concentration of 30 nM but no uptake of the neutral and negatively charged compounds 19 and 20, respectively, applied at a concentration of 200 nM (Figure S22, Supporting Information). This demonstrated that the physicochemical properties of fluorescent dyes should be taken into consideration for the design of fluorescence-labeled biologically active compounds.

In conclusion, we demonstrate that fluorescence labeling of NT(8–13) via carbamoylated arginine residues represents a useful alternative to N-terminal conjugation of fluorescent dyes to NT peptide analogues. The presented fluorescent NTS1R ligands represent useful molecular tools to study NTS1R expression and internalization in cells and determine NTS1R binding affinities of nonlabeled compounds by competition binding, which is useful e.g. for the screening of potential NTS1R ligands and compound profiling in drug development programs. As the fluorescent probes also exhibit high NTS2R affinity, they represent potential molecular tools for fluorescence-based ligand binding studies at the NTS2R. However, due to the missing selectivity, studies of cellular systems or tissues expressing both NT receptor subtypes would be challenging as they require highly selective NTS1R or NTS2R ligands for selective receptor subtype blocking. Regarding unspecific interactions (e.g., adsorption to plastic), fluorescent probes containing a fluorophore with a negative net charge or without net charge proved to be superior to compounds containing a positively charged fluorophore. In conjunction with a recently reported study on the stabilization of the NT(8–13) backbone against enzymatic degradation,[35] the present study can potentially be exploited for the design and preparation of fluorescent and radiolabeled molecular tools useful for the imaging of NTS1R positive tumors.