Double Methotrexate-Modified Neuropeptide Y Analogues Express

Article pubs.acs.org/jmc

Double Methotrexate-Modified Neuropeptide Y Analogues Express Increased Toxicity and Overcome Drug Resistance in Breast Cancer Cells David Böhme, Jan Krieghoff, and Annette G. Beck-Sickinger* Universität Leipzig, Institute of Biochemistry, Brüderstraße 34, 04103 Leipzig, Germany S Supporting Information *

ABSTRACT: Bioconjugates containing the neuropeptide Y (NPY) analogue [F7,P34]-NPY as targeting moiety are able to deliver toxic agents specifically to breast cancer cells that overexpress the human Y1-receptor (hY1R). To increase their activity, multiple toxophores can be attached to one peptide. Herein, synthesis and characterization of [F7,P34]-NPY conjugates containing two methotrexate (MTX) molecules are presented. First, carboxytetramethylrhodamine was linked to [F7,P34]-NPY by amide or enzymatic linkage. The conjugate containing the enzymatic cleavage site showed high extracellular stability and fast intracellular release. Then, MTX was introduced at positions four and 22 of [F7,P34]-NPY, connected by enzymatic or amide linkage. The toxicity of the analogues on breast cancer cells was hY1R-mediated and dependent on the used linkage and amount of toxophores. Furthermore, conjugates revealed higher potency than MTX on MTX-resistant cells. These results emphasize that peptide−drug conjugates can overcome drug resistance and that the attachment of multiple cleavable toxophores enhances the efficiency of this smart delivery system.

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INTRODUCTION Classical chemotherapy uses potent toxic agents to treat cancer. However, the therapeutic window of these toxophores is small, leading to severe side effects.1 Additionally, tumor cells are often resistant to these compounds. The mechanisms of drug resistance include decreased drug transport, increased efflux, availability of alternative targets, and avoiding apoptosis.2 This desensitization of tumors toward chemotherapeutic drugs lowers their efficiency. Peptide−drug conjugates are promising structures to overcome these restrictions of classical chemotherapy. These compounds consist of peptides that are linked to toxic agents by intracellularly cleavable chemical bonds. The peptides are endogenous ligands for receptors that are overexpressed at the surface of cancer cells.3 Such a receptor is the human Y1-receptor (hY1R), that is overexpressed at the surface of breast tumors, in contrast to the human Y2-receptor (hY2R), that is expressed in healthy breast tissue.4 Both receptors belong to the neuropeptide Y (NPY) receptor family, which are G-protein coupled receptors.5 Binding of their endogenous ligand NPY induces internalization by receptormediated endocytosis.6 This situation enables an efficient targeting of hY1R-expressing breast tumors with hY1R-specific analogues of NPY. This was impressively shown by using a 99m Tc-labeled derivative of the hY1R-preferring [F7,P34]-NPY.7 Another important aspect of such a delivery system is the linkage between peptide and drug, which should be stable at extracellular sites but should also release the toxic agent after internalization in a fast manner.8,9 Furthermore, peptide−drug conjugates have impressively proven to be able to overcome © XXXX American Chemical Society

drug resistance by avoiding the altered efflux and entry mechanism involved in multidrug resistance.10 Previously, analogues of the tumor-targeting [F7,P34]-NPY modified at Lys4 with the toxophore methotrexate (MTX) were shown to efficiently induce hY1R-mediated toxicity in breast cancer cells depending on the linkage between MTX and the peptide.11 MTX is a chemotherapeutic agent that inhibits the metabolism of folic acid, which leads to the breakdown of the synthesis of key metabolites such as dTMP, purines, and some amino acids.12 An approach to increase the potency of peptide−drug conjugates is the attachment of more than one toxophore per peptide molecule. This manuscript describes the design of [F7,P34]-NPY analogues carrying two MTX molecules. In addition to the already identified modification site at position four of [F7,P34]-NPY,11 the suitability of position 22 as potential MTX modification point was examined. First, the fluorophore carboxytetramethylrhodamine (TAMRA) was connected to an introduced lysine at position 22 of [F7,P34]NPY by an amide or enzymatically cleavable bond. To evaluate the applicability of these different linkages for their subsequent use in MTX-[F7,P34]-NPY conjugates, their intracellular feasibility and extracellular stability were monitored. The enzymatically cleavable linker displayed optimal stability properties showing a fast intracellular release and high resistance against extracellular enzymatic degradation. In a second step, TAMRA was replaced by MTX, which was linked by an amide linkage or enzymatic cleavage site. Furthermore, Received: January 16, 2016

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DOI: 10.1021/acs.jmedchem.6b00043 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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two MTX molecules were connected to Lys4 and Lys22 of [F7,P34]-NPY by two amide bonds or enzymatic cleavage sites by using exclusively solid phase peptide synthesis. Signal transduction and receptor internalization experiments with hYR subtype expressing cell lines showed high and hY1R-preferring activity and uptake. Cell viability studies with hY1R-expressing breast cancer cells revealed a hY1R-mediated toxicity of the conjugates that was dependent on the linkage type and the amount of attached toxophores, whereby conjugates were able to overcome drug resistance in MTX-resistant cells.

sopropylethylamine (DIPEA). Peptide [F7,K22(GFLG-TAMRA),P34]-NPY (4b), containing an enzymatic cleavage site, was built up by coupling of the amino acids GFLG with N,Ń diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) and subsequent conjugation of 6 with HBTU/DIPEA. Toxic analogues [F 7 ,K 22 (MTX),P 34 ]-NPY (5a) and [F7,K22(GFLG-MTX),P34]-NPY (5b), containing one molecule of 3 linked by an amide or enzymatic cleavage site were prepared in a similar manner. Additionally to the modification at position 22, Lys4 of peptide 2 can also be readily modified without changing the hY1R-preferring properties of 2.11,15 Therefore, Lys4 and Lys22 were used to attach two molecules of 3 to peptide 2 by amide linkage or the enzymatic cleavage site GFLG. Double amide-linked compound [K4(MTX),F7,K22(MTX),P34]-NPY (5c) was synthesized by hydrazine-induced Dde deprotection in Boc-[K4(Dde),F7,K22(Dde),P34]-NPY and subsequent simultaneous coupling of 3 to Lys4 and Lys22 with DIC/HOBt activation. The synthesis of double GFLG-linked conjugate [K4(GFLG-MTX),F7,K22(GFLG-MTX),P34]-NPY (5d) is illustrated in Scheme 1. After Dde deprotection with

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RESULTS Peptide Synthesis. Fluorescent and toxic [F7,P34]-NPY conjugates were synthesized by solid phase peptide synthesis by applying the Nα-9-fluorenylmethoxycarbonyl (Fmoc)/tBu strategy. Hereby, either the fluorophore TAMRA or the toxic agent MTX was linked to [F7,P34]-NPY by different chemical structures (Figure 1).

Scheme 1. Synthesis of Double GFLG-MTX-Modified [F7,P34]-NPY Conjugate 5da

Conditions: (a) Dde cleavage: 3% (v/v) hydrazine in DMF; 14 × 10 min. (b) Subsequent coupling of the amino acids Fmoc-Gly, FmocPhe, Fmoc-Leu, Fmoc-Gly: 8 eq Fmoc-amino acid, HOBt, DIC in DMF for 2 h. (c) Subsequent removal of Fmoc-protecting groups: DBU/piperidine/DMF (1:1:8, v/v); 2 × 20 min. (d) Coupling of MTX: 2 eq MTX, HOBt, DIC in DMF; 2 × 2 h. (e) Full cleavage: TFA/thioanisole/thiocresol (18:1:1, v/v) for 135 min. SPPS, solid phase peptide synthesis; Boc, tert-butyloxycarbonyl; Fmoc, Nα-9fluorenylmethoxycarbonyl; MTX, methotrexate; HOBt, 1-hydroxybenzotriazole; DIC, N,N’-diisopropylcarbodiimide. a

hydrazine, the side chains of Lys4 and Lys22 were simultaneously elongated with the amino acids GFLG by coupling with DIC/HOBt and subsequent Fmoc deprotection with DBU/ piperidine. Next, 3 was simultaneously coupled to both elongated side chains by DIC/HOBt activation. After full cleavage from Rink amide resin under acidic conditions, peptides were purified by reversed-phase (RP)HPLC. Purity ≥95% was confirmed by analytical RP-HPLC, and identification was performed by MALDI- and ESI-mass spectrometry (Table S1). Analytical data are exemplarily shown for compound 5d (Figure 2). Intracellular Linker Cleavage and Stability in Human Blood Plasma. The time-dependent linker cleavage of fluorescent analogues 4a,b was analyzed by fluorescence microscopy (Figure 3A), as described elsewhere.11 Briefly, after stimulation of HEK293 cells stably expressing hY1R Cterminally fused to enhanced yellow fluorescent protein (EYFP) with fluorescent peptides, cells were washed to remove noninternalized peptide. After further incubation in Opti-MEM, a loss of intracellular fluorescence can be detected by

Figure 1. (A) Peptide sequences of NPY (1), [F7,P34]-NPY (2), and novel [F7,K22(X-Y),P34]-NPY analogues and chemical structures of the fluorescent dye TAMRA and toxic agent MTX (3). (B) Linker structures used to connect TAMRA (6) for fluorescently labeled analogues (4a,b) and MTX for single (5a,b) and double modified toxic conjugates (5c,d).

It was shown that Ser22 in peptide 2 can be readily exchanged by other modifiable amino acids such as lysine.13 Modification of introduced Lys22 with carbaboranes led to no change of receptor activity and selectivity.14 Therefore, 6 was linked to Lys22 by different chemical linkages. Site-specific modification was achieved by selective removal of the orthogonal 4,4dimethyl-2,6-dioxocyclohex-1-ylidenethyl (Dde)-protecting group in Boc-[F7,K22(Dde),P34]-NPY with hydrazine. The amide-linked compound [F7,K22(TAMRA),P34]-NPY (4a) was obtained by conjugation of 6 with O-benzotriazole-N,N,N′,N′tetramethyluronium-hexafluoro-phosphate (HBTU)/N,N-diiB



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Figure 2. Analytical data of [K 4(GFLG-MTX),F7 ,K22(GFLGMTX),P34]-NPY (5d). (A) Analytical RP-HPLC by using a Jupiter 4 μ Proteo C12, 90 Å, and (B) a VariTide RPC column with a linear gradient of 20−60% (v/v) eluent B in eluent A in 40 min used for both columns. (C) ESI mass spectrum displaying the five-, six-, seven-, eight-, and nine-fold charged molecular ions. (D) MALDI-TOF mass spectrum displaying the [M + H]+ and [M + H]2+ signals (Mcalc. = 5914.9 Da). Signals at m/z 5741.8, 5567.4, and 2784.0 are corresponding to a laser-induced MTX fragmentation that was not detected by ESI mass spectrometry.

Figure 3. (A) Time-dependent linker cleavage recorded by using livecell microscopy after stimulation of stably transfected HEK293-hY1REYFP cells with 100 nM of TAMRA-labeled analogues 4a,b for 1 h. Decrease in TAMRA fluorescence intensity was measured at different time points after stimulation. Measurements were normalized by using fluorescence intensity after stimulation (set at 100%) and fluorescence intensity of untreated cells (set at 0%). Results are presented as the mean fluorescence intensity ± standard error of the mean (SEM), and assays were performed in at least three independent experiments in quadruplet. (B) In vitro stability tests of fluorescent analogues performed in human blood plasma. At the indicated time points, degradation was followed by using RP-HPLC with fluorescence detection and was referred to the control at 0 h (set at 100%). Values represent the mean ± SEM of at least three independent experiments.

microscopically measuring the fluorescence intensity at distinct time points after the washing step. This decrease of fluorescence correlates to the cleavage of the bond connecting 6 and peptide 2. The experiments revealed that the release of 6 was faster for the enzymatic cleavage site-linked peptide 4b, compared to the amide-linked peptide 4a. After 2 h, the amidelinked compound still showed a fluorescence of (76 ± 7)%, whereas only (46 ± 6)% for the enzymatically linked peptide were left. The extracellular stability of the fluorescent analogues was evaluated by stability tests in human blood plasma (Figure 3B). The amide-linked compound 4a displayed a moderate half-life of (6.2 ± 0.4) h. Hereby, degradation of the peptide from the N-terminus was observed, while no release of 6 could be detected. The analogue 4b containing the enzymatic cleavage site GFLG showed a higher stability with a half-life of (26.4 ± 1.8) h. Here, only N-terminal degradation was observed, but no release of 6. These data indicate ideal stability properties of the enzymatic cleavage site GFLG-linker with a fast intracellular release and high extracellular stability. Receptor Activation and Internalization Studies of Toxic Analogues. Inositol phosphate (IP) accumulation was used to study receptor activation of toxic conjugates 5a−d in COS-7 cells stably expressing hY1R or hY2R with a chimeric GαΔ6qi4myr-protein, leading to an activation of phospholipase C and the production of inositol trisphosphate. After labeling with myo-[2-3H]inositol, cells were stimulated with different peptide concentrations. The hY1R-preferring properties of peptide 2 and all derived analogues modified with compound 3 were potent agonists at the hY1R (Figure S1A), as shown by EC50 values that were similar to that of the endogenous ligand 1

(Table 1). In contrast, at the hY2R all toxic conjugates derived from peptide 2 showed much lower activity (Figure S1B) with EC50 values >200 nM, compared to 1 with an EC50 value of 0.4 nM, which demonstrates their strong hY1R activity and selectivity. The hY1R/hY2R subtype selectivity was further analyzed by live-cell imaging to study the ligand-induced receptor internalization by using stably transfected HEK293 cells expressing hY1R or hY2R C terminally labeled with EYFP (Figure 4). Before stimulation, the hYR subtypes were mainly present in the cellular membrane. After stimulation with the endogenous ligand 1, both hYR subtypes were internalized, which can be clearly seen by the disappearance of the green cell surface fluorescence and the increase of the fluorescence in intracellular vesicles. All analogues modified with compound 3 induced only internalization of the hY1R, whereas the hY2R remains on the cell surface, thereby demonstrating the efficient and selective delivery of 3 into hY1R-expressing cells. Cell Viability Studies. The toxic activity and selectivity of the analogues 5a−d modified with compound 3 was compared to unconjugated 3 by using the hY1R-expressing cell lines MDA-MB-468, MDA-MB-231, and T-47D, all representing C



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Table 1. Receptor Activation of Peptides Determined by Inositol Phosphate Accumulation Assay EC50 [nM]a (pEC50 ± SEM)b No.

Compound

hY1R

1 2 5a 5b 5c 5d

NPY [F7,P34]-NPY [F7,K22(MTX),P34]-NPY [F7,K22(GFLG-MTX),P34]-NPY [K4(MTX),F7,K22(MTX),P34]-NPY [K4(GFLG-MTX,F7,K22(GFLG-MTX),P34]-NPY

3.6 5.0 2.9 3.0 2.8 8.9

(8.4 (8.3 (8.5 (8.5 (8.5 (8.1

± ± ± ± ± ±

hY2R 0.1) 0.1) 0.1) 0.1) 0.1) 0.1)

0.4 (9.4 ± 0.1) 87 (7.1 ± 0.1) 209 (6.7 ± 0.1) 226 (6.6 ± 0.1) n.d. n.d.

a

Values represent the total mean of the EC50 values determined in at least two independent experiments, each performed in duplicate, and were calculated by using GraphPad Prism 5.0 software by nonlinear regression. bpEC50 ± SEM corresponds to the negative decadic logarithm of the EC50 value. n.d.: EC50 and pEC50 values could not be determined, since no full receptor activation was observed up to 10 μM. MTX, methotrexate.

Lys4-modified [F7,P34]-NPY analogues [K4(MTX)F7,P34]-NPY and [K4(GFLG-MTX)F7,P34]-NPY, which are described elsewhere (Figure S2),11 induced lower toxicity compared to their Lys22-modified counterparts 5a and 5b, carrying the same linkage types (Table 2 and Figure S2A). Furthermore, the amide-linked conjugate 5c, containing to molecules of 3, showed higher toxic activity, compared to the single amidelinked analogues, with an IC50 value of 4.0 μM. The GFLGmodified analogue 5d, containing two molecules of 3, displayed the highest toxic activity of all peptides with an IC50 value of 2.0 μM. Toxic activity of the conjugates in T-47D cells relates well to the results obtained for MDA-MB-468 cells. Hereby, compound 3 showed again the highest potency with an IC50 value of 0.5 μM, and conjugate 5d displayed the highest activity of all peptides, with an IC50 value of 5 μM (Table 2 and Figure S3). In comparison, the single enzymatically linked peptide 5b showed a slightly reduced activity (IC50 = 7.7 μM), as well as the double amide-linked compound 5c (IC50 > 10 μM) and [K4(GFLG-MTX)F7,P34]-NPY (IC50 = 12 μM, Figure S2b). The single modified analogues 5a and [K4(MTX),F7,P34]-NPY, containing an amide linker, demonstrated a further decreased activity (IC50 > 25 μM). In MDA-MB-231 cells, peptide−drug conjugates remarkably showed an increased toxic activity compared to compound 3 (IC50 = 15 μM, Table 2 and Figure 5B). In particular, double amide- and double enzymatic-linked conjugates 5c and 5d demonstrated approximately 4-fold decreased IC50 values with 3.8 μM and 4.2 μM, respectively. Conjugates 5b and [K4(GFLG-MTX),F7,P34]-NPY (Figure S2C), containing one GFLG-linked molecule 3, displayed 2fold decreased IC50 values compared to compound 3, with 8.1 μM and 8.3 μM, respectively. Only the single amide-linked conjugates 5a and [K4(MTX),F7,P34]-NPY showed a lower activity than 3 (IC50 > 25 μM) by using this cell line. Additionally, the toxicity of peptide 2 was tested on MDA-MB468 cells (Figure S4). The carrier peptide displayed no toxic effect, demonstrating that the toxicity of the conjugates results from MTX and not from the NPY activity. In order to prove that the toxicity of the MTX-[F7,P34]-NPY conjugates is mediated by the hY1R, MDA-MB-468 cells were incubated with 3 and the toxic conjugates in the presence of 10 equiv of the hY1R-preferring peptide 2 (Figure 5C). The toxicity of the conjugates was significantly reduced with [F7,P34]-NPY, whereas the activity of 3 remained high. The same results were obtained by incubation of MDA-MB-231 and T-47D cells with the MTX conjugates in the presence of peptide 2 (Figure S5). To support these findings, non-hYRexpressing HEK293 cells were incubated with the conjugates and 3 (Figure 5D). Compound 3 as unselective molecule showed the highest toxic activity with a significant reduction in

Figure 4. Receptor internalization studies of MTX-containing [F7,P34]-NPY conjugates 5a−d in comparison to NPY (1) on HEK293 cells stably expressing either hY1R or hY2R fused to EYFP (green). Cell nuclei were stained with Hoechst33342 (blue). Cells were stimulated with 100 nM of peptides for 1 h. Representative pictures of three independent experiments are shown. Scale bar = 10 μm.

standard breast cancer cell lines and non-hYR-expressing HEK293 cells.16,17 Cells were stimulated with different compound concentrations for 8 h and proliferated in cell culture medium for a further 72 h. Cell viability was evaluated by resazurin assay.18 In MDA-MB-468 cells (Table 2 and Figure 5A), compound 3 showed the highest toxicity with an IC50 value of 0.2 μM. The single modified, amide-linked conjugate 5a showed an IC50 value of 18 μM. In comparison, the single modified peptide 5b, containing the enzymatic cleavage site GFLG, displayed a higher toxicity with a 5-fold decreased IC50 value of 3.6 μM. The amide- and GFLG-linked D



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Table 2. Toxicity of MTX (3), MTX-[F7,P34]-NPY Conjugates 5a−d, and Lys4-Modified MTX-[F7,P34]-NPY Analogues on hY1R-Expressing Breast Cancer Cell Lines MDA-MB-468, T-47D, and MDA-MB-231 IC50 [μM]a (pIC50 ± SEM)b No.

Compound

MDA-MB-468

T-47D

MDA-MB-231

3 5a 5b 5c 5d

MTX [F7,K22(MTX),P34]-NPY [F7,K22(GFLG-MTX),P34]-NPY [K4(MTX),F7,K22(MTX),P34]-NPY [K4(GFLG-MTX,F7,K22(GFLG-MTX),P34]-NPY [K4(MTX),F7,P34]-NPYc [K4(GFLG-MTX),F7,P34]-NPYc

0.2 (6.7 ± 0.1) 18 (4.8 ± 0.1) 3.6 (5.4 ± 0.1) 4.0 (5.4 ± 0.1) 2.0 (5.7 ± 0.1) >25 μM (n.d.)d 8.1 (5.1 ± 0.1)

0.5 (6.3 ± 0.1) >25 μM (n.d.)d 7.7 (5.1 ± 0.1) >10 μM (n.d.)d 5.0 (5.3 ± 0.1) >25 μM (n.d.)d 12 (4.9 ± 0.1)

15 (4.8 ± 0.1) >25 μM (n.d.)d 8.1 (5.1 ± 0.1) 3.8 (5.4 ± 0.1) 4.2 (5.4 ± 0.1) >25 μM (n.d.)d 8.3 (5.1 ± 0.1)

a

Values represent the total mean of the half-maximal inhibitory concentration (IC50) obtained from concentration response curves. Mean curves were determined in at least two independent experiments, each performed in triplicate, and were calculated by using GraphPad Prism 5.0 software by nonlinear regression. Measurements were normalized by using only medium treated cells (set at 100%) and ethanol treated cells (set at 0%). bpIC50 ± SEM corresponds to the negative decadic logarithm of the IC50 value. See Figure 5a−c for toxicity profiles. cSynthesis and characterization of these peptides are described elsewhere.11 See Figure S2 for toxicity profiles. dn.d.: pIC50 could not be determined, since no full toxicity was observed up to the limit of solubility.

Figure 5. Toxicity of MTX (3) and conjugates 5a−d on hY1R-expressing breast cancer cell lines MDA-MB-468 (A), MDA-MB-231 (B), and nonhYR-expressing HEK293 cells (D). Statistical significances refer to only medium treated cells. (C) Toxicity on hY1R-expressing MDA-MB-468 cells in the presence or absence of 10 equiv hY1R agonist [F7,P34]-NPY. A concentration of 25 μM for conjugate 5a and a concentration of 10 μM for compounds 3 and 5b−d were used. Bars represent the mean ± SEM of at least two independent experiments performed in triplicate. Measurements were normalized by using medium treated cells with or without [F7,P34]-NPY (set at 100%) and ethanol treated cells (set at 0%).

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cell viability by using concentrations of 1 μM [viability: (65 ± 3)%] or higher. In untransfected HEK293 cells that do not express hYR, the overall toxicity of 3 was lower compared to the breast cancer cell lines MDA-MB-468 and T-47D, which might result from the different genetic makeup of the cell lines. It was shown that the folate binding protein, which is important for the transport of 3 into cells, is overexpressed in breast cancer cells.12,19,20 All MTX-[F7,P34]-NPY conjugates induced no significant toxic effect at any of the tested concentrations. These data emphasize the selective toxic effect induced by the conjugates, in contrast to the unselective activity of compound 3.

DISCUSSION Nowadays, the concept of antibody−drug conjugates is transferred to peptide−drug conjugates,21 which are becoming a real opportunity for the targeted therapy of tumors, with the first compounds being evaluated in clinical trials.22 Especially the opportunity to synthesize and modify peptides in a controlled yet complex fashion by solid phase peptide synthesis makes these compounds ideal delivery agents,23 which is also demonstrated by their use for tumor imaging.24 Since the number of receptors in the cell membrane mediating drug transport is limited, a strategy to enhance the potency of peptide−drug conjugates is the attachment of more than one toxophore to one carrier molecule.25 For example, it was shown E



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obtained for Lys4-modified MTX-[F7,P34]-NPY analogues and carbaborane-containing conjugates.11,14,33 In cell viability studies with the hY1R-expressing breast cancer cell lines MDA-MB-468 and T-47D, free compound 3 showed the highest toxicity, because of its unspecific uptake by diffusion and active transporters.12 In contrast, the MTX[F7,P34]-NPY conjugates have to be specifically taken up by internalization and release molecule 3 intracellularly, which results in a decreased toxicity of the conjugated toxophore compared to the free toxic agent, as shown recently for cytolysin-[F7,P34]-NPY analogues.17 The single GFLG-MTXmodified peptide 5b displayed a higher toxicity than the single amide-linked peptide 5a, which correlates remarkably well to the release profiles observed for the corresponding fluorescent compounds 4a and 4b. Additionally, these peptides modified at Lys22 induced a stronger toxic effect, compared to the analogues modified at Lys4 of peptide 2 carrying the same linker structures.11 This might result from a lower stability of the Lys22-modified peptides already mentioned above. The modified analogues containing two molecules 3 were more toxic than the corresponding single modified peptides, whereby the GFLG-linked conjugate 5d was the most potent compound. This nicely shows that the use of a cleavable linker and the incorporation of two toxophores into one peptide molecule enhance the potency of peptide−drug conjugates. However, the IC50 value of the double modified GFLG-linked compound 5d was only slightly lower than the IC50 value of the corresponding single modified analogue 5b. This might result from a decreased release rate for the peptide containing two molecules 3, which was recently shown for cell-penetrating peptides containing multiple doxorubicin molecules connected by the GFLG-linker.34 The breast cancer cell line MDA-MB-231 is described as resistant to compound 3, owing to a lack expression of the reduced folate carrier, which is responsible for the uptake of 3 into cells.35 Therefore, an alternative delivery pathway can overcome drug resistance. This was previously shown by a proline prodrug of compound 3 and the conjugation of 3 to a cell penetrating peptide showing higher toxic activity than molecule 3 itself on MDA-MB-231 cells.36,37 However, prodrug and cell penetrating peptide do not offer selective delivery of 3 to breast cancer cells. In contrast, peptide 2 enables this targeted delivery of 3, while maintaining a different transport route by endocytosis. The single enzymatically linked peptides 5b and [K4(GFLG-MTX)F7,P34]-NPY showed a 2-fold higher toxicity than free MTX. The double amide and double enzymatic conjugates 5c and 5d even displayed a 4-fold increased potency than compound 3, which emphasizes that these analogues can overcome resistance to 3 in breast cancer cells. Furthermore, in competition experiments the hY1R agonist 2 clearly inhibited the toxicity of all peptide conjugates, whereas the potency of 3 was maintained. This underlines the selective delivery of 3 by peptide 2 via the hY1R. These results were supported by a low toxicity detected for the conjugates on nonhYR-expressing HEK293 cells, in contrast to the high activity of unselective compound 3.

that analogues of the tumor targeting gonadotropin-releasing hormone (GnRH), which were linked to two daunorubicin toxophores by oxime linkages, induced an up to 5-fold higher toxicity than the corresponding single modified compounds.26 Herein, we aimed to develop a potent drug conjugate, based on peptide 2, containing two toxophores. Since analogues of 2 linked to the toxophore 3 at Lys4 showed a potent hY1Rmediated toxicity,11 only a second modification site needed to be identified. The effect of alanine substitution in the sequence of native peptide 1 on receptor binding was investigated previously, revealing that especially an exchange of the native amino acids at position four and 22 had no influence on hY1R binding.13 Furthermore, it was also shown that a double modification of Lys4 and an introduced lysine at position 22 with carbaboranes led to no change of receptor activity and selectivity of the hY1R-preferring peptide 2.14 Therefore, an inserted lysine residue at position 22 of 2 was used as second modification site. First, conjugates were synthesized containing the fluorophore 6 connected to Lys22 by amide or enzymatic linker to compare the intra- and extracellular stability of these chemical structures. Intracellular release profiles revealed that the enzymatically cleavable GFLG linker 4b was cleaved more rapidly than the amide-linked peptide 4a. These results were in perfect agreement with previously published data where the fluorophore was linked to Lys4 of 2 by the same linker bonds.11 Stability tests with these fluorescent analogues in human blood plasma revealed high stability for the enzymatically cleavage site-linked conjugate 4b and moderate stability for the amide-linked peptide 4a. The higher stability of the GFLG compared to the amide-linked compound might be attributed to a better partial protection of the N-terminus by the spatially more demanding GFLG-linker, since the Nterminus was the first degradation site observed for both compounds. In order to further increase the bioavailability of the conjugates for a prospective application of these compounds in clinics, stabilizing structures such as palmitic acid or polyethylene glycol could be attached to the peptides.23 Especially the attachment of palmitic acid can enhance stability and additionally increase the internalization of the peptide, which is a prerequisite for a peptide−drug conjugate.27 Next, the chemotherapeutic agent 3 was coupled to peptide 2. Compound 3 is an antimetabolite with the enzyme dihydrofolate reductase as its primary target.28 This inhibits the metabolism of folic acid, which is an indispensable cofactor for the biosynthesis of purine and pyrimidine bases, resulting in the induction of apoptosis.12,29 Owing to its adverse effects during therapy,30 molecule 3 was linked to 2 to obtain selective delivery. Similar to the fluorescent peptides, 3 was connected to Lys22 of 2 by GFLG 5b or amide linkage 5a. Furthermore, [F7,P34]-NPY analogues containing two molecules of 3 were synthesized, where 3 is linked to Lys4 and Lys22 by amide 5c or enzymatic linkages 5d by using exclusively solid phase peptide synthesis. Although the α-carboxy group of 3 is important for its cytotoxic activity,31 it was shown that conjugation to the αor γ-carboxy group of 3 leads to no difference in conjugate toxicity in case unmodified molecule 3 is released.11,32 All analogues displayed strong and selective hY1R activity and internalization, although 3 was introduced at different positions and linkages. These findings indicate that the introduction of 3 at positions four and 22 of peptide 2 does not influence the hY1R-preferring properties of 2. This is in line with data

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CONCLUSION In summary, we developed a potent conjugate of peptide 2 that contained two selectively introduced toxophore molecules. For this purpose, the tumor targeting peptide was connected to fluorophores and toxophores via different chemical linkages by using exclusively solid phase peptide synthesis. The fluorescent F



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for Fmoc deprotection. Then, reaction with 6 was performed by HBTU/DIPEA activation as described for compound 4a. Toxic conjugates were synthesized by connecting molecule 3 to the Nε-group of Lys22 in Boc-[F7,K22,P34]-NPY or to the Nε-groups of Lys4 and Lys22 in Boc-[K4,F7,K22,P34]-NPY by amide bond or enzymatic cleavage site. Compound 5a was prepared by conjugation with 5 eq 3, DIC and HOBt in DMF for 2 h. For conjugate 5b, the Nε-group of Lys22 was elongated with the amino acids GFLG by using standard DIC/HOBt activation for amino acid coupling and piperidine for Fmoc deprotection. Subsequently, MTX was coupled by using standard DIC/HOBt activation. For the synthesis of 5c, MTX was simultaneously linked to Lys4 and Lys22 with 2 eq MTX, DIC and HOBt in DMF, twice for 2 h. For conjugate 5d, the Nε-groups of Lys4 and Lys22 were simultaneously elongated with the amino acids GFLG by using standard DIC/HOBt activation for amino acid coupling and by using DBU/piperidine/DMF (1:1:8, v/v) twice for 20 min for Fmoc deprotection. Afterward, 3 was simultaneously coupled twice to the elongated side chains of Lys4 and Lys22 by using 2 eq 3, HOBt, DIC in DMF for 2 h. Peptides were cleaved from resin by using TFA/TA/TC (18:1:1, v/ v) for 135 min. Then, peptides were precipitated from ice-cold diethyl ether, washed and collected by centrifugation. Crude product purification was performed by preparative RP-HPLC on a Shimadzu HPLC system (Columbia, MD, USA) with a Jupiter 10 μ Proteo C18 column (90 Å, 7.78 μm, 250 × 21.2 mm; Phenomenex, Aschaffenburg, Germany) by using a flow rate of 13 mL/min and a linear gradient system containing 0.1% (v/v) TFA in water (eluent A) and 0.08% (v/ v) TFA in ACN (eluent B). Pure products were characterized by analytical RP-HPLC, MALDITOF (Ultraflex III MALDI-ToF/ToF, Bruker Daltonics, Billerica, MA, USA) and ESI-HCT (High-capacity ion trap ESI-MS, Bruker Daltonics) mass spectrometry. For analytical RP-HPLC, the HPLC system LaChrome Elite (VWR) equipped with a Jupiter 4 μ Proteo C12 (90 Å, 5 μm, 250 × 4.6 mm, 0.6 mL/min; Phenomenex) and a VariTide RPC (200 Å, 6 μm, 250 × 4.6 mm, 1.0 mL/min; Agilent, Santa Clara, CA, USA) column were used by applying a linear gradient of 20−60% (v/v) eluent B in eluent A in 40 min for both columns. Detection was performed at 220 nm. Peaks were integrated by using EZ Chrome Elite software (VWR). Analysis by analytical RP-HPLC confirmed a purity ≥95% for all synthesized compounds. Cell Culture. HEK293 (human embryonic kidney) cells were cultured in standard Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose and L-glutamine and HAM’s F-12 (1:1) supplied with 15% (v/v) heat-inactivated FCS. HEK293 cells stably expressing hY1R or hY2R C-terminally tagged with EYFP were maintained in DMEM/HAM’s F-12 (1:1) supplemented with 15% (v/v) heatinactivated FCS and 100 mg/mL hygromycin. COS-7 (African green monkey, kidney) cells stably expressing either hY1R or hY2R Cterminally fused to EYFP and the chimeric G-protein GαΔ6qi4myr were grown in DMEM with 10% (v/v) heat-inactivated FCS, 100 units/mL penicillin, 100 mg/mL streptomycin, 146 mg/mL hygromycin B, and 1.5 mg/mL G418-sulfate. MDA-MB-468, MDA-MB-231 and T-47D cells were maintained in RPMI 1640 containing L-glutamine supplied with 10% (v/v) heat-inactivated FCS and 1 mg/mL human insulin. All cell lines were cultured under a humidified atmosphere at 37 °C and 5% CO2. Live-Cell Microscopy. hYR and peptide internalization was investigated by using stably transfected HEK293 cells (300000 cells per well), which were grown on μ-slide 8 well (ibidi) to full confluency overnight. Live-cell imaging was performed by using an Axio Observer.Z1 microscope equipped with an ApoTome Imaging System and a Heating Insert P Lab-Tek S1 unit (Zeiss, Oberkochen, Germany), as previously described.38,39 Prior to ligand stimulation, cells were starved for 30 min at 37 °C in Opti-MEM containing 0.5 mg/mL Hoechst33342 for nuclei staining. Cells were then stimulated with 100 nM peptide solution in Opti-MEM for 1 h. Subsequently, cells were washed once with acidic wash solution (50 mM glycine and 100 mM NaCl at pH 3) and twice with HBSS to remove noninternalized peptide. Cells were maintained in Opti-MEM and images were taken directly after washing. For experiments measuring

analogues containing the enzymatically cleavable GFLG-linker showed optimal stability properties. The toxic conjugates showed high and hY1R-specific activation and internalization, which showed the suitability of positions four and 22 of 2 for the attachment of the toxic agent 3. In cell viability studies, a hY1R-mediated toxicity was detected which was stronger for analogues containing the cleavable GFLG-linker and two instead of one molecule of 3. Furthermore, conjugates displayed an up to 4-fold higher potency than compound 3 on the resistant breast cancer cell line MDA-MB-231, thereby restoring the toxic effect of 3 and the tumor cell sensitivity for chemotherapy. This highlights directed targeting of toxophores toward hY1R-expressing breast cancer cells with analogues of peptide 2 containing more than one toxic agent for a prospective in vivo application.

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EXPERIMENTAL SECTION

Materials. Fmoc- and tert-butyloxycarbonyl (Boc)-protected amino acids were purchased from Orpegen (Heidelberg, Germany) and Iris Biotech (Marktredwitz, Germany). Thiocresol (TC) and thioanisole (TA) were obtained from Fluka (Buchs, Switzerland); HOBt, HBTU, DIC, and 2-cyano-2-(hydroxylimino)acetic acid ethyl ester (Oxyma) were purchased from Iris Biotech. NovaSyn TG resin was obtained from Merck KGaA (Darmstadt, Germany) and TAMRA from emp Biotech GmbH (Berlin, Germany). Acetonitrile (ACN) was purchased from VWR (Darmstadt, Germany) and DMF and DCM from Biosolve (Valkenswaard, The Netherlands). Diethyl ether and ethanol were obtained from Scharlau (Barcelona, Spain) and MTX from Selleck Chemicals (Houston, TX, USA) and DMSO, DIPEA, hydrazine, piperidine, TFA, and DBU from Sigma-Aldrich (Taufkirchen, Germany). Cell culture media, as well as trypsin-EDTA, Dulbecco’s Phosphate-Buffered Saline (DPBS), and Hank’s Balanced Salt Solution (HBSS) were purchased from Lonza (Basel, Switzerland). Fetal calf serum (FCS) and G418-sulfate were obtained from Biochrom GmbH (Berlin, Germany) and human insulin from Roche (Basel, Switzerland). Penicillin, streptomycin and hygromycin B were purchased from Invivogen (Toulouse, France) and Opti-MEM was obtained from Life Technologies (Basel, Switzerland). Glycine was purchased from Serva (Heidelberg, Germany); Hoechst33342, NaCl, LiCl and resazurin (In Vitro Toxicology Assay Kit, Resazurin based) were obtained from Sigma-Aldrich and myo-[2-3H]-inositol and scintillation cocktail Optiphase HiSafe from PerkinElmer (Boston, MA, USA). Peptide Synthesis. C-terminally amidated peptides were synthesized by a combination of automated solid phase peptide synthesis (SPPS) on a Syro II peptide synthesizer (MultiSynTech, Bochum, Germany) and manual coupling by applying orthogonal Fmoc/tBu strategy on NovaSyn TG resin (15 μmol scale, loading 0.18 mmol/g). With robot-assisted synthesis unmodified peptides 1, 2 and core peptides Boc-[F7,K22(Dde),P34]-NPY and Boc[K4(Dde),F7,K22(Dde),P34]-NPY were obtained. Reactive side chains of amino acids were protected by tert-butyl (tBu for Tyr, Ser, Asp, Glu, Thr), trityl (Trt for Asn, Gln, His), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf for Arg) and 4,4-dimethyl-2,6-dioxocyclohex-1-ylidenethyl (Dde for Lys). For automated synthesis, coupling reactions were performed twice with 8 eq Nα-protected amino acid, in situ activated with equimolar amounts of oxyma (2 min preincubation on resin) and DIC in DMF for 30 min. Automated Fmoc deprotection was carried out with 40% (v/v) piperidine in DMF for 3 min and 20% (v/v) piperidine in DMF for 10 min. Subsequent Dde deprotection for Boc-[F7,K22(Dde),P34]-NPY and Boc-[K4(Dde),F7,K22(Dde),P34]NPY was performed 14 times by using 3% (v/v) hydrazine in DMF for 10 min. For fluorescent peptides, 6 was connected to the Nε-group of Lys22 in Boc-[F7,K22,P34]-NPY by amide or enzymatic linkage. For compound 4a, coupling was performed by using 3 eq 6, HBTU and DIPEA with 0.1 eq HOBt in DMF for 2 h. For conjugate 4b, the Nεgroup of Lys22 was elongated with the amino acids GFLG by using standard DIC/HOBt activation for amino acid coupling and piperidine G



Article

the relative linker cleavage, additional images were taken 1, 2, 4, and 6 h after the washing step. Fluorescence was recorded by using the same settings and a light exposure time of 500 ms. ImageJ was used to measure intracellular raw intensity densities of four independent full images (image section: 1300 × 1030 pixel) per peptide. Measurements were normalized by using fluorescence intensity after stimulation (set at 100%) and fluorescence intensity of only Opti-MEM treated cells (set at 0%). Assays were performed in three independent experiments. Stability Tests in Human Blood Plasma. For in vitro stability tests, 10 μM peptide solutions of fluorescently labeled analogues 4a and 4b were incubated in human blood plasma at 37 °C and continuous shaking at 500 rpm (rpm). Samples were taken after 0, 0.5, 1, 2, 4, 8, and 24 h, and serum proteins were precipitated with 150 μL acetonitrile/ethanol (1:1, v/v) at −20 °C for 2 h. After centrifugation at 14000 rpm for 30 s, supernatants were processed by using Costar Spin-X tubes (0.22 μm membrane pore size). Degradation was analyzed by RP-HPLC with a Varian-VariTide RPC column and fluorescence detection (λex = 525 nm, λem = 572 nm) by using a linear gradient of 20 to 50% (v/v) of eluent B in eluent A over 50 min. Quantity of intact peptide was referred to the control at 0 h (set at 100%). Values represent the mean with SEM of at least three independent experiments. An exponential decay was revealed for the first data points that allowed determination of half-lives according to an enzymatic degradation of first order. Y-Receptor Activation Studies. Signal transduction inositol phosphate (IP) accumulation assay was performed like described before.40 Briefly, stably transfected COS-7 cells were seeded into 48well plates (50000 cells per well) and cultured overnight. After labeling cells with 2 μCi/mL myo-[2-3H]-inositol in culture medium without penicillin and streptomycin, cells were washed and incubated with peptide concentrations ranging from 10−5 to 10−12 M in DMEM supplied with 10 mM LiCl for 1 h at 37 °C. Radioactive IP species were isolated by anion exchange chromatography and measured in a scintillation counter. EC50 and pEC50 values were calculated from concentration−response curves by using nonlinear regression (GraphPad Prism 5.0). Assays were performed in duplicate in at least two independent experiments. Cell Viability Studies. Cytotoxic activities of free compound 3 and conjugates 5a-d were analyzed by resazurin assay.18 MDA-MB-231 (6000 cells per well), T-47D (10000 cells per well), MDA-MB-468 and HEK293 cells (8000 cells per well) were seeded in 96-well plates and grown in culture medium. After 24 h, medium was replaced by culture medium without FCS containing compound 3 or peptides in concentrations ranging from 50 to 0.01 μM. Cells were incubated at 37 °C for 8 h. For toxicity experiments with the hY1R agonist 2, MDAMB-468 cells were incubated with toxic agents in the presence of 10 eq 2. Cells treated only with medium in absence or presence of 2 were used as negative control. Subsequently, cells were washed twice with medium and maintained in culture medium. After 72 h, cells were washed twice with culture medium without FCS and incubated with 10% (v/v) resazurin solution in culture medium without FCS at 37 °C for 2 h. As a positive control cells were treated with 70% (v/v) ethanol in water for 10 min. Fluorescence of formed resorufin was detected at 550 nm excitation and 595 nm emission with the plate reader Infinite M200 (Tecan, Männedorf, Switzerland). Measurements were normalized by using cells treated only with medium in presence or absence of 2 (set at 100%) and ethanol treated cells (set at 0%). IC50 and pIC50 values were calculated from concentration−response curves by using nonlinear regression (GraphPad Prism 5.0). Assays were performed in triplicate in at least two independent experiments.

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Lys4-modified MTX-[F7,P34]-NPY conjugates on hY1Rexpressing breast cancer cell lines. Toxicity of all peptides on non-hYR-expressing HEK293 cells and toxicity of [F7,P34]-NPY on MDA-MB-468 cells. (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: 0049-341-9736900. Fax: 0049-341-9736909. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Kristin Löbner, Regina Reppich-Sacher, and Ronny Müller are acknowledged for technical assistance during cell culture and synthesis. We express our thanks to the Staatsministerium für Kunst und Wissenschaft (SMWK), the European Union, the Free State of Saxony (ESF) and the Graduate School Leipzig School of Natural Sciencesec-Building with Molecules and Nano-objects (BuildMoNa) for generous funding.

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ABBREVIATIONS USED ACN, acetonitrile; Boc, tert-butyloxycarbonyl; Dde, 4,4dimethyl-2,6-dioxocyclohex-1-ylidenethyl; DIC, N,Ń -diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; EYFP, enhanced yellow fluorescent protein; FCS, fetal calf serum; Fmoc, Nα-9-fluorenyl-methoxycarbonyl; HBTU, O-benzotriazole-N,N,N′,N′-tetramethyluronium-hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; hYxR, human Yx-receptor; IP, inositol phosphate; MTX, methotrexate; NPY, neuropeptide Y; RP, reversed-phase; rpm, revolutions per minute; TA, thioanisole; TAMRA, carboxytetramethylrhodamine; TC, thiocresol; SEM, standard error of the mean; SPPS, solid phase peptide synthesis

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00043. Analytical characterization of synthesized peptides. hY1R and hY2R activation curves of all peptides. Toxicity of H



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Double Methotrexate-Modified Neuropeptide Y Analogues Express

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