Neurotensin (NT), a 13 amino acid neuropeptide 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 NTS 1 R and NTS 2 R, both family A G-protein coupled receptors (GPCRs), and the NTS 3 R (sortilin), a member of the Vps10p-domain receptor family. (2,4−6) As the NTS 1 R 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 NTS 1 R ligands. Noteworthily, the carboxy-terminal hexapeptide of NT (NT(8–13), 1) is equipotent with NT at NTS 1 and NTS 2 receptors. (11)
Figure 1. Structures of the tridecapeptide neurotensin, NT(8–13) (1, blue), the radiolabeled NT(8–13) derivative [3 H]2, (19) and the fluorescent NT(8–13) derivative 3. (19)a Granier et al. (20)b Einsiedel et al. (21)c 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 NTS 1 R 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, N ω-carbamoylated arginine, was introduced. (19) This proof-of-concept study included derivatives of 1, for example the radioligand [3 H]2 and the cyanine dye-conjugated peptide 3. In the previous study, fluorescent ligand 3 was characterized in terms of NTS 1 R affinity by competition binding with [3 H]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 N ω-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) 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 NTS 1 R 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 20a
a Reagents and conditions: (a) DIPEA, DMF, NMP, rt, 30 min, 42% (6), 27% (11), 38% (12), 24% (16), 42% (17); (b) DIPEA, KHCO 3, DMF, H 2 O, 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 N ω-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). The pyridinium dye-labeled peptide 14 was obtained by treatment of 7 with the pyrylium derivative 13 (32) in the presence of triethylamine. The reference compounds 19 and 20 (fluorescent “dummy ligands”) were prepared from propylamine (18) and succinimidyl esters 10 and 5, respectively.
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, fluorescent probe 14, containing a pyridinium-type dye, showed minor decomposition after incubation times >24 h.
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. 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.
NTS 1 R 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 [3 H]2 (19) at intact HT-29 colon carcinoma cells endogenously expressing the hNTS 1 R (33) but no NTS 2 R. (19) In addition, NTS 1 R binding data of 3, 6, 9, 11, 12, and 14 were determined by competition binding with [3 H]2 at whole Chinese hamster ovary cells stably transfected with the hNTS 1 R (CHO-hNTS 1 R cells (21)). The p K i values of 3, 6, 9, 11, 12, and 14, obtained from these competition binding assays, were in excellent agreement with the p K i values determined at HT-29 cells.
a Determined by radioligand competition binding with [3 H]2 at HT-29 colon carcinoma cells (K d ([3 H]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). b Determined by radioligand competition binding with [3 H]2 at CHO-hNTS 1 R cells (K d ([3 H]2) = 0.29 nM); mean values ± SEM from three independent experiments (performed in triplicate). c Determined by flow cytometric saturation binding at CHO-hNTS 1 R cells using PBS as binding buffer; mean values ± SEM from two (3) or four (6) independent experiments (performed in duplicate). d Determined by high-content imaging saturation binding at CHO-hNTS 1 R 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). e Determined in a Fura-2 Ca 2+ assay at human HT-29 cells; mean values ± SEM from two (1) or three (3, 6) independent experiments (performed in singlet). f Determined in a Fluo-4 Ca 2+ assay at CHO-hNTS 1 R cells; mean values ± SEM from eight independent experiments (performed in triplicate). g Determined by competition binding with [3 H]2 at HEK-hNTS 2 R cell homogenates (K d ([3 H]2) = 0.79 nM); mean values ± SEM from three (3, 6, 9), four (16), five (12), or seven (1) independent experiments (performed in triplicate). h Data were previously reported as K i value by Keller et al. and were reanalyzed to give the p K i 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 NTS 1 R affinities with p K i values of 8.15–9.12. This demonstrated that the recently introduced concept of peptide labeling via the nonclassical bioisosteric replacement of arginine by a functionalized N ω-carbamoylated arginine can be successfully applied to either arginine in 1 (Arg 8: 3, 6, 9, 11, 12 and 14, Arg 9: 16 and 17) even if bulky moieties such as fluorescent dyes are attached. Moreover, these results showed that the type of fluorophore (different core structures and charges) had little impact on receptor binding of the fluorescent peptides. In addition to hNTS 1 R affinities, hNTS 2 R 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 hNTS 2 R, using [3 H]2 as radiolabeled probe (K d (hNTS 2 R): 0.79 nM). Whereas hNTS 2 R affinities of 3 and 9 proved to be slightly higher compared to their NTS 1 R affinities, hNTS 2 R binding of 6, 12 and 16 was marginally lower than hNTS 1 R binding.
