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 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 1) 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 1) 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.^19aGranier et al.^20bEinsiedel et al.^21cKeller 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, N^ω-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 1). 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 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; cf. Scheme 1 and Figure 2), 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 3,¹⁹6, 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) (Scheme 1). The pyridinium dye-labeled peptide 14 was obtained by treatment of 7 with the pyrylium derivative 13(32) in the presence of triethylamine (Scheme 1). The reference compounds 19 and 20 (fluorescent “dummy ligands”) were prepared from propylamine (18) and succinimidyl esters 10 and 5, respectively (Scheme 1).

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 3,¹⁹6, 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]2¹⁹ (for structure, see Figure 1) at intact HT-29 colon carcinoma cells endogenously expressing the hNTS₁R³³ 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 cells²¹). The pK_i values of 3, 6, 9, 11, 12, and 14, obtained from these competition binding assays, were in excellent agreement with the pK_i values determined at HT-29 cells (Table 1).

Table 1. NTS₁R Binding Data (pK_i) of 1, 3, 6, 9, 11, 12, 14, 16, and 17, NTS₂R Binding Data (pK_i) of 1, 3, 6, 9, 12, and 17, pK_d 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.

^aDetermined by radioligand competition binding with [³H]2 at HT-29 colon carcinoma cells (K_d ([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).

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

^cDetermined 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).

^dDetermined 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).

^eDetermined 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).

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

^gDetermined by competition binding with [³H]2 at HEK-hNTS₂R cell homogenates (K_d ([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).

^hData were previously reported as K_i value by Keller et al. and were reanalyzed to give the pK_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 NTS₁R affinities with pK_i values of 8.15–9.12 (Table 1; 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 N^ω-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 2), 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 2) 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 (K_d (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 1; competition binding curves shown in Figure S10, Supporting Information). This revealed that N^ω-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 1; 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 pK_i values obtained from competition binding experiments with [³H]2 (Table 1). 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 1). 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 pK_d values of 3 and 6 were in excellent agreement with the respective pK_i values from competition binding studies with [³H]2 (Table 1). Moreover, high-content imaging saturation binding studies were performed with 3, 6, 9, and 12 at CHO-hNTS₁R cells (pK_d values see Table 1, 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 (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 3). 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 3). 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.

Glossary

Abbreviations

CHO cells

Chinese hamster ovary cells

DIPEA

diisopropylethylamine

GPCR

G-protein coupled receptor

K_d

dissociation constant obtained from a saturation binding experiment

PBS

phosphate buffered saline

pK_d

negative logarithm of the K_d (in M)

pK_i

negative logarithm of the dissociation constant K_i (in M) obtained from a competition binding experiment

TFA

trifluoroacetic acid

Vps10p

vacuolar protein sorting 10 protein.

Biographies

Max Keller is a medicinal chemist by training and received his doctoral degree at the University of Regensburg under the supervision of Prof. Armin Buschauer. Currently, he is an Assistant Professor at the Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg. His work focuses on the development of molecular tools to study ligand receptor interactions by radiochemical and fluorescence-based methods. Maxʼs main research interest is the design, synthesis, and characterization of selective and labeled ligands of G-protein coupled receptors.

Nick Holliday obtained his Ph.D. and performed postdoctoral work at Kingʼs College London, prior to establishing his own research group at the University of Nottingham (UK), where he is now Associate Professor of Pharmacology. His work focuses on developing various bioluminescence and fluorescence imaging methodologies to understand the binding and signaling of G protein coupled receptors and other membrane proteins and the molecular properties of their effector complexes.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00462.

• Figures S1–S10; investigation of NTS₁R antagonism of SR142948A and SR48692 by the use of fluorescent ligands 6 and 12 (including Figures S11–S13); Tables S1–S3; saturation binding studies with 3, 6, 11, and 12 and competition binding studies with 6 and 12 (including Figures S14–S18); association and dissociation kinetics of 3 and 6 at CHO-hNTS₁R cells studied by flow cytometry (including Figures S19 and S20); Figures S21 and S22; association and dissociation kinetics of 3, 6 and 11 at CHO-hNTS₁R cells studied by confocal microscopy (including Figures S23–S30); experimental procedures; data processing; RP-HPLC chromatograms of compounds 6, 9, 11, 12, 14, 16, and 17 (PDF)

• Molecular formula strings (XLSX)

• Movie 1: Compound 3 association; Figure S23 (AVI)

• Movie 2: Compound 3 association and dissociation; Figure S24 (AVI)

• Movie 3: Compound 6 association; Figure S25 (AVI)

• Movie 4: Compound 6 association and dissociation; Figure S26 (AVI)

• Movie 5: Compound 6 association and dissociation; Figure S27 (AVI)

• Movie 6: Compound 6 dissociation; Figure S28 (AVI)

• Movie 7: Compound 11 association; Figure S29 (AVI)

• Movie 8: Compound 11 association and dissociation; Figure S30 (AVI)

• Movie 9: Addition of 3 nM 1 to CHO-hNTS1R cells (AVI)

• Movie 10: Vehicle control for addition of 3 nM 1 to CHO-hNTS1R cells (AVI)

Author Present Address

^§ A.P.: Ramboll Environment & Health GmbH, Werinherstraße 79, 81541 Munich, Germany.

Author Contributions

M.K. and A.P. performed the synthesis. M.K., S.A.M., V.H.Y., J.C., L.S., T.L., H.H., N.H., and P.G. performed functional and binding studies. M.K. initiated and planned the project. M.K., N.D.H., and G.B. supervised the research. M.K., N.D.H., and G.B. wrote the manuscript. All authors have given approval to the final version of the manuscript.

This work was funded by the Research Grants KE 1857/1-1 and 1-2 of the Deutsche Forschungsgemeinschaft (DFG)) and additionally supported by the Graduate Training Program GRK 1910 of the DFG and the International Doctoral Program “Receptor Dynamics” Elite Network of Bavaria.

The authors declare no competing financial interest.