Chemical Modification for Proteolytic Stabilization of the Selective

Article Cite This: J. Med. Chem. 2017, 60, 9874−9884

pubs.acs.org/jmc

Chemical Modification for Proteolytic Stabilization of the Selective αvβ3 Integrin RGDechi Peptide: in Vitro and in Vivo Activities on Malignant Melanoma Cells Daniela Comegna,†,▽ Antonella Zannetti,‡,▽ Annarita Del Gatto,†,§ Ivan de Paola,† Luigi Russo,∥ Sonia Di Gaetano,†,§ Annamaria Liguoro,† Domenica Capasso,*,⊥ Michele Saviano,*,§,@ and Laura Zaccaro*,†,§ †

Institute of Biostructures and Bioimaging-CNR, Via Mezzocannone 16, 80134 Naples, Italy Institute of Biostructures and Bioimaging-CNR, Via De Amicis 95, 80145 Naples, Italy § Interdepartmental Center of Bioactive Peptide, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy ∥ Department of Environmental, Biological and Pharmaceutical Science and Technology, University of Campania Luigi Vanvitelli, via Vivaldi 43, 81100 Caserta, Italy ⊥ Department of Pharmacy, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy @ Institute of Crystallography-CNR, Via Amendola 122/O, 70126 Bari, Italy ‡

S Supporting Information *

ABSTRACT: Herein, we report the synthesis and biological characterization of the new peptide ψRGDechi as the first step toward noveltargeted theranostics in melanoma. This pseudopeptide is designed from our previously reported RGDechi peptide, known to bind selectively αvβ3 integrin, and differs for a modified amide bond at the main protease cleavage site. This chemical modification drastically reduces the enzymatic degradation in serum, compared to its parental peptide, resulting in an overall magnification of the biological activity on a highly expressing αvβ3 human metastatic melanoma cell line. Selective inhibition of cell adhesion, wound healing, and invasion are demonstrated; nearinfrared fluorescent ψRGDechi derivative is able to detect αvβ3 integrin in human melanoma xenografts in a selective fashion. More, molecular docking studies confirm that ψRGDechi recognizes the receptor similarly to RGDechi. All these findings pave the way for the future employment of this novel peptide as promising targeting probe and therapeutic agent in melanoma disease.

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INTRODUCTION

integrin as marker of cancer stem cells and its involvement in drug resistance and tumor recurrence.10,11 All these findings validate αvβ3 as diagnostic and prognostic biomarker and strongly promote its targeting in drug and molecular imaging probe design for highly aggressive tumors,12 such as melanoma. The relevance of αvβ3 has also been witnessed by several αvβ3 antibodies currently in clinical trials for their antitumor, antiangiogenic, and antimetastatic properties.13,14 The majority of αvβ3 integrin-targeted ligands are based on the tripeptide Arg-Gly-Asp (RGD) sequence because of its high affinity and specificity for this receptor. These molecules have been developed as tumor endothelium-targeted diagnostic agents for different imaging strategies15−22 investigated for clinical translation23,24 and also used in selective delivery of chemotherapeutics or radionuclides in tumors,25,26 thus

Malignant melanoma is the form of skin cancer responsible for the majority of deaths caused by tumors affecting this tissue.1 Melanoma is a highly metastatic carcinoma often resistant not only to conventional 2 but also to targeted therapies, consequently early visualization of the tumor and the inhibition of a secondary tumor site onset are the first goals to improve melanoma therapy. Among the cell adhesion proteins for the extracellular matrix, αvβ3 integrin is well-known to be crucially important in tumor progression due to its role in cell migration, stemness, and angiogenesis.3 Actually, high expression of αvβ3 integrin is associated with tumor invasion and poor prognosis in melanomas4,5 but also in glioma,6 prostate,7 and breast cancer;8 more, the receptor is highly expressed on activated endothelial cells and new-born vessels in tumors, but it is completely absent in the rest of endothelial cells and in most normal organs.9 In addition, very recently Seguin et al. reported the role of αvβ3 © 2017 American Chemical Society

Received: October 27, 2017 Published: November 16, 2017 9874

DOI: 10.1021/acs.jmedchem.7b01590 J. Med. Chem. 2017, 60, 9874−9884

Journal of Medicinal Chemistry

Article

Scheme 1. ψRGDechi Synthesisb

b

Conversion of 1 into the corresponding aldehyde 3 followed by on resin reductive amination and SPPS cycles to afford the pseudo-peptide 5. Isolated overall yields.

a

reducing the systemic side effects. The first developed integrin peptide antagonist, Cilengitide, is currently in the phase III study in patients with newly diagnosed glioblastoma,27 and recently, its capability to suppress the invasion in αvβ3 overexpressing malignant pleural mesothelioma cells has been demonstrated.28 Furthermore, some RGD-peptidomimetic agents are able to interfere with both early and late steps of metastasis in animal models.29 Russo et al. demonstrated that the treatment of human glioblastoma cell lines with a new small-molecule RGD antagonist strongly inhibited cell attachment and migration.30 Unfortunately, most of the available αvβ3 ligands do not bind specifically to the receptor,31,32 getting difficult to target αvβ3mediated processes for diagnostic and/or therapeutic applications without toxicity to normal cells; in addition, although several of them have been found effective in controlling integrin-mediated cell proliferation,13 just a few have been demonstrated to be able to control the different steps involved in the metastatic cascade. Over the past decade, we focused our attention on RGDechi peptide, which selectively targets the αvβ3 receptor without cross-reacting with αvβ5 and αIIbβ3 integrins.33 The peptide is a chimeric molecule with a cyclic portion containing the RGD motif recognized by all RGD integrins and a linear sequence derived from the C-terminal portion of the echistatin protein needed to confer specificity for the β3 subunit.33,34 We demonstrated antiangiogenic activity of the peptide in animal models of wound healing,35 antiadhesive, and proapoptotic effects on melanoma cells.36 To provide structural insights into the molecular basis of RGDechi selectivity, very recently we performed a structural analysis in isolated cell membranes of highly expressing αvβ3 melanoma by a combination of NMR and computational studies,37 showing the involvement of some residues located at the C-terminus, deriving from the echistatin portion, in the receptor recognition and mainly the role of hCit15 in conferring selectivity to the molecule by specific interactions with β3 subunit. This last observation was supported by the lacking ability to bind αvβ3 integrin of the RGDechi derivative containing the Asp residue in place of hCit at position 15.37

Furthermore, SPECT and PET imaging studies on nude mice bearing αvβ3- or αvβ5-expressing tumors confirmed the ability of RGDechi peptide to selectively visualize αvβ3 and proved that a derivative of peptide, lacking the last five residues located at the C-terminus, loses in vivo the selectivity in agreement with our structural analysis of the RGDechi-αvβ3 complex.34 In PET analysis, biodistribution studies indicated that unfortunately the absolute tumor uptake of RGDechi was suboptimal,34 probably due to the low stability in serum of the peptide.35 Luckily, nowadays several site-specific chemical modifications are suitable to be introduced in a peptide sequence to increase plasma residence time.38 Among them, the most straightforward and commonly used is the isosteric replacement of the peptide bond with a reduced amide.39 The resulting aminoethylene group confers the pseudopeptide a higher enzymatic resistance but also an increased hydrophilicity. With the aim to improve the drawbacks of resistance to current therapeutics and to significantly prolong survival in melanoma patients, novel targeted theranostics could help to lay out future directions in treatment of this skin cancer. Therefore, encouraged by the RGDechi peptide receptor selectivity, as well as by its therapeutic and diagnostic features, and taking into account its low serum stability, we designed a pseudopeptide analogue, namely ψRGDechi, and performed molecular docking studies which suggested that the insertion of the reduced amide bond did not interfere with the receptor recognition mechanism. The peptide was synthesized and biologically characterized, paving the way for the development of new theranostics.

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RESULTS Design and Synthesis. In vivo long-term function of RGDechi peptide is limited by the low resistance to the proteases. Our previous studies allowed the identification of the main enzymatic cleavage site located between Pro17 and Ala18 of the peptide sequence.35 Thus, in order to improve the proteolytic resistance, we designed and synthesized the pseudopeptide ψRGDechi (5, Scheme 1) containing the reduced Pro-ψ[CH2−NH]-Ala synthon at the RGDechi proteolytic site. This modification, however, is reasonably 9875



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the absolute absence of degradation peaks. The RP-HPLC profile clearly shows that ψRGDechi is not hydrolyzed even after 48 h incubation, indicating that the modification introduced in parental sequence by reducing the natural amide bond worked well in conferring high serum stability. Recognition Mechanism of the αvβ3 Integrin by ψRGDechi. In order to describe the structural details of the recognition mechanism of the αvβ3 receptor by ψRGDechi, we performed, as reported in the materials and methods section, a series of Molecular Docking studies. Following the optimized docking procedure,37 100 solutions were generated that were successively sorted into clusters based on the binding energy (Figure 2 and Figure S7). The cluster analysis selected four most populated clusters (named Cluster 1, 2, 3, 4), representing more than 70% of the entire conformational ensemble obtained by the Molecular Docking calculation (Figure S7). Notably, in all representative structures, the recognition process of the αvβ3 receptor by the ψRGDechi peptide is principally modulated by the RGD motif that is embedded into a cleft between the αv-subunit and the βA domain on the integrin head. Only in the third cluster, containing ten structures (10%), the RGD cycle appears to be less buried inside the RGD binding cavity with respect to the other three representative structures. Moreover, the docking models demonstrate that the αvβ3/ψRGDechi is further stabilized by additional residues of the C-terminal echistatin moiety. In particular, in cluster 1 (33%), cluster 2 (21%), and cluster 4 (9%), the residues Lys1, Arg2, Gly3, Asp4, and DGlu5 of the RGD cycle contacts both αv and the β3 subunits, especially interacting with αv‑Asp148, αv‑Asp148, αv‑Asp150 and with the residues located inside the specificity determining SDL loop of the β3 subunit (β3‑Tyr166, β3‑Asp179, β3‑Arg214 and β3‑Arg216), whereas in the cluster 3 (10%), the RGD motif makes contact principally with β3 subunit. Interestingly, in all four clusters the C-terminal part of the peptide contributes to stabilize the αvβ3/ψRGDechi complex. In particular, the cluster 1, containing more than 30 structures, indicates that the hCit15 mainly establishes interaction with the β3 subunit, suggesting that the modified ψRGDechi presents a αvβ3 recognition mechanism similar to that observed for the RGDechi. Effect of ψRGDechi on Cell Adhesion. To investigate the effect of ψRGDechi on tumor cell adhesion, human metastatic melanoma cell line WM266 overexpressing αvβ3 (4.4 ± 0.4 × 104 of αvβ3 vs 3.8 ± 1.4 × 103 of αvβ5 receptors per cell) and human cervix cell line HeLa not expressing αvβ3, whereas they express high levels of αvβ5 integrin (1.8 ± 0.1 × 104 per cell),37 were seeded on different extracellular matrices. To prevent receptor internalization, the cell treatments with ψRGDechi, RGDechi, cRGDfK (an αvβ3/αvβ5 antagonist used as positive control),42 scrambled peptide (negative control) and αvβ3 antibody (LM609) were carried out at 4 °C for 30 min before seeding cells onto fibronectin, vitronectin, and collagen I, respectively.37,43−45 As shown in Figure 3A, ψRGDechi strongly reduces the adhesion of WM266 cells plated onto fibronectin and vitronectin of 33% and 55%, respectively, while no effect is observed on cells plated onto collagen I which does not interact with αv integrins. As expected, the effect of ψRGDechi on cell adhesion to different extracellular components is similar to that previously observed for RGDechi.46 Furthermore, we observed that ψRGDechi is also able to inhibit cell adhesion in a concentration-dependent manner, showing an IC50 of 5.2 and 20.7 μM for vitronectin and fibronectin, respectively (Figure

expected to have no effect on the binding mechanism as supported by the docking studies shown below. The reduced peptide bond was successfully obtained by on-resin reductive amination as shown in Scheme 1. First of all, the Fmocprotected proline aldehyde has been prepared as follows. The commercially available N-Fmoc proline 1 was converted into the corresponding ethanethiol ester 2 by the Steglich’s method40 in 70% yield followed by Fukuyama’s reduction with Pd/C of the thiol ester affording the corresponding aldehyde 341 in 80% yield. N-Fmoc proline aldehyde 3 was allowed to react with the free amino group of Ala-Thr peptidyl-resin 4, and the in situ formed iminium species was then reduced by NaBH3CN in dimethylformamide containing 1% acetic acid to form the corresponding pseudotripeptide. Therefore, the final pseudopeptide 5 was synthesized by solid-phase protocols and obtained in 25% yield. Serum Stability Studies. RGDechi and ψRGDechi stabilities were evaluated in human serum at 37 °C. The degradation was followed by LC−MS through the disappearance of the peaks corresponding to the intact peptides from 0 to 48 h after the incubation. These studies confirmed that RGDechi peptide is already degraded after 3 h of incubation, as shown by the presence of two peaks corresponding to the complete sequence at tr = 12.5 min (theoretical MW = 2100.1 g mol−1, [M + 2H]+2 m/z at 1052.4, and [M + 3H]+3 m/z at 702.1) and to the fragment lacking the dipeptide Ala18-Thr19 at tr = 12.7 min (theoretical MW = 1929.1 g mol−1, [M + 2H]+2 m/z at 966.4 and [M + 3H]+3 m/z at 644.9), respectively (Figure 1A). After 24 h, a further peak appears in the RP-HPLC

Figure 1. In serum stability studies. (A) RP-HPLC chromatograms and mass spectra for RGDechi. The chromatographic peaks at tr = 12.3 min (☆), 12.5 min (○), and 12.7 min (□) are marked; (B) RP-HPLC chromatograms and mass spectra for ψRGDechi; the chromatographic peak at tr = 11.7 min (⬠) is marked.

profile ascribable to peptide sequence lacking the tetrapeptide Gly16-Thr19 at tr = 12.3 min (theoretical MW = 1774.1 g mol−1, [M + 2H]+2 m/z at 889.1, and [M + 3H]+3 m/z at 593.3). Conversely, the modification introduced in ψRGDechi confers high protease stabilization to the molecule (tr = 11.7 min, theoretical MW = 2088.7 g mol−1, [M + 2H]2+ m/z at 1045.4, and [M + 3H]+3 m/z at 697.6, Figure 1B) confirmed by 9876



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Figure 2. The Molecular Docking studies of the ψRGDechi-αvβ3 complex. The representative structural models of the ψRGDechi-αvβ3 complex from the (A) cluster 1 (light blue), (B) cluster 2 (magenta), (C) cluster 3 (green), and (D) cluster 4 (yellow) as reported by the cluster analysis. The αv and β3 subunits are depicted in light pink and light violet, respectively.

and scrambled treated cells (*p < 0.05) and comparable to cRGDfK. Effect of ψRGDechi on Cell Invasion. Cell invasion is another important step in the process of metastasis. We examined the effect of ψRGDechi on the invasiveness of WM266 cells using trans-well chambers coated with ECL Cell Attachment. As shown in Figure 6A, significant decrease in tumor cell invasiveness is observed when tumor cells are treated with 50 μM cRGDfK, RGDechi, and ψRGDechi with respect to untreated and scrambled treated cells. In particular, ψRGDechi peptide is able to inhibit tumor cell invasion better than RGDechi (over 2,5-fold), demonstrating that the stability enclosed from the new peptide significantly affects the tumor cell invasiveness, whereas linear and scrambled peptides have no effect on the invasion of WM266 cells (Figure 6, right panel). In Vivo Tracking of NIR-Labeled ψRGDechi and RGDechi. To demonstrate the improvement in vivo of tumor uptake of ψRGDechi with respect to RGDechi, we labeled peptides with VivoTag-S 750, an amine-reactive NIR fluorochrome which allows for deeper tissue penetration, reduced autofluorescence, and minimal scattering. Preliminary, we observed the ability of ψRGDechi-NIR750 to colocalize with αvβ3 integrin in vitro on WM266 cells (Manders’coefficients: M1= 0.799; M2 = 0.996) and we verified that the ψRGDechi-NIR750 specifically binds to receptor by competition studies using an excess of unlabeled parent compound (Figure S5). Following these findings, we used the peptide to target αvβ3 integrin in WM266 tumor-bearing mice. When tumors reached an appropriate and similar size (0.5 cm3), mice (3 for each group) were administrated i.v. with 1 nmol of ψRGDechi-NIR750, RGDechi-NIR750, or VivoTag-S 750,

3B). These values are slightly improved with respect to the values previously reported for RGDechi (IC50 vitronectin: 25.5 μM; IC50 fibronectin: 53 μM).46 Incubation of cells with scrambled peptide does not decrease cell adhesion on all tested matrices, whereas cRGDfK inhibits adhesion similarly on fibronectin and vitronectin in accordance with its ability to bind both αvβ3 and αvβ5. To confirm the specificity of ψRGDechi toward αvβ3, competition studies were carried out. After treatment with the peptides, WM266 cells were seeded on anti αvβ3 and Hela cells on anti-αvβ5 antibodies coated plates. As shown in Figure 4, we observed that both ψRGDechi and RGDechi inhibit WM266 binding (about 40%) to anti-αvβ3 antibody while very slightly reduce HeLa binding to anti-αvβ5 antibody thus confirming their specificity versus the αvβ3 receptor. As expected, cRGDfK is able to equally inhibit both WM266 and HeLa cell adhesion to antibodies since it binds both αvβ3 and αvβ5 integrins. Effect of ψRGDechi on Wound Healing. Given the importance of cell migration activity in cancer progression,47 in vitro scratch assay was performed on WM266 to assess the effects of ψRGDechi. Monolayers of WM266 cells, grown in the presence of serum, were scratched and images were taken at 0, 24, and 48 h after wounding (Figure 5A). When cells were grown in the presence of ψRGDechi or RGDechi, the wound healing was significantly delayed compared to untreated cells (*p < 0.001). Notably, ψRGDechi strongly inhibits closure of the gap at 24 and 48 h after the scratch (Figure 5B), and its effect is higher than that observed with RGDechi (54% vs 31% at 24 h; 33% vs 20% at 48 h). Both peptides show a significant inhibition of wound healing activity with respect to untreated 9877



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Figure 3. Inhibition of WM266 adhesion on extracellular matrix coated plates. (A) Cells were preincubated with peptides (50 μM) or anti-αvβ3 antibody (10 μg/mL) and then seeded on extracellular matrix precoated plates. Cell adhesion is evaluated considering the slope of each curve; results are presented as the percentage of slope of adherent cells vs control (untreated cells). (B) ψRGDechi dose−effect on WM266 cell adhesion. Cells were preincubated with increasing concentrations of ψRGDechi then seeded on vitronectin or fibronectin (10 μg/mL) precoated plates. The cell adhesion was evaluated after 1 h of incubation using crystal violet reagent. The results are presented as the percentage of adherent cells with respect to the control and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using student’s t test (*p < 0.05).

Figure 5. Effect of peptides on wound healing activity. (A) The effect was determined by the scratch assay. Monolayers of WM266 were scratched linearly. The cells were treated with different peptides (50 μM) and then photographed using phase-contrast microscopy. (B) Quantification of wound closure. Graphic represents the wound width for each treatment expressed as the mean ± SE of the percentage of the wound width at 24 or 48 h with respect to t = 0. Similar results were obtained in three experiments performed in triplicate. *p < 0.05.

3 to 24 h (Figure 7A), whereas VivoTag-S 750 alone fails in allowing tumor detection (Figure S6). Interestingly, we observed that ψRGDechi-NIR750 tumor uptake was significantly higher than RGDechi-NIR750 at 5 and 24 h. (Figure 7B). Furthermore, ex vivo imaging analysis of tumors, excised from euthanized animals 24 h post injection with peptides, confirmed these results showing a stronger persistent fluorescence of ψRGDechi-NIR750 in the tumor with respect to RGDechi-NIR750 (Figure 7C). Taken together these data indicate that the modified peptide shows a higher efficiency in targeting melanoma in respect to RGDechi.

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Figure 4. Inhibition of cell adhesion on antibody-coated plates. The cells were preincubated with peptides (50 μM) then seeded on anti αvβ3 or anti-αvβ5 antibody precoated plates. WM266 adhesion is evaluated using crystal violet reagent; results are presented as the percentage of adherent cells with respect to the control. HeLa adhesion was evaluated considering the slope of each curve; results are presented as the percentage of slope of adherent cells vs control. Data are expressed as mean ± SE from at least three independent experiments performed in duplicate (*p < 0.05, **p < 0.01).

DISCUSSION AND CONCLUSION To best exploit the potential of RGDechi as bioprobe platform in melanoma cancer, we reported the novel peptide ψRGDechi, a derivative where a reduced amide bond (ψ[CH2−NH]) has been introduced at the proteolytic degradation site to improve the protease stability, preserving at the same time its proved selectivity for αvβ3 integrin. Several procedures have been established so far for both in solution and on solid-phase synthesis of pseudopeptides; however, most of them are based on a reductive amination reaction.39 In a typical procedure, an α-amino aldehyde reacts with the free amino group of the growing peptide to form an iminium species that is in turn reduced to the methylene amino linkage of the pseudopeptide.

respectively. Then, these were subjected to NIR fluorescence tomography at different time points 3, 5, and 24 h. The results of our preliminary in vivo imaging studies showed that both labeled peptides allow the visualization of WM266 tumor from 9878



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Figure 6. Effect of peptides on cell invasion. (Left) WM266 cell invasion was assessed in transwell chambers coated with ECL Cell Attachment. Cells were treated with 50 μM of different peptides. Cells that invaded the ECL-coated insert were fixed, stained, and captured at 20× magnification; (right) the invaded WM266 cells was counted and their number (±SE) reported in the scheme (*p < 0.05).

through on-solid phase reductive amination using NaBH3CN as a reductive agent. LC−MS studies clearly indicate that ψRGDechi gains high stability in serum beyond 24 h, assessing that the chemical modification introduced in the new sequence confers notable enzymatic resistance. The molecular docking studies indicate that the ψRGDechi recognizes the αvβ3 integrin with a binding mechanism similar to that observed for the RGDechi. In particular, the recognition process is principally mediated by the RGD cycle that is deeply embedded inside the RGD binding cavity delimited by the αv and β3 subunits. Additionally, the molecular docking models suggest that the complex αvβ3/ψRGDechi is further stabilized by the residues of the C-terminal echistatin moiety in which the hCit15 plays a crucial role in the selectivity of the recognition process. In fact, as observed for the RGDechi, the residue hCit15 interacts with the β3‑Asp179 of the SDL loop that is very important for the selectivity for the αvβ3. The essential role of SDL in the selectivity of ψRGDechi for the αvβ3 integrin is also proved by the fact that this region is poorly conserved among the various RGD-integrins (αvβ5, αvβ6, α5β1, and αIIbβ3) (Figure S8). For the evaluation of ψRGDechi theranostic features, the WM266 melanoma cell line, characterized by predominant mutation in the BRAF gene occurring in 48% of metastatic melanomas,49 was selected as a good model expressing high level of αvβ3 with respect to αvβ5 integrin. In our study, we demonstrated that ψRGDechi, as well as RGDechi, evidently inhibits cell adhesion to vitronectin, to a lesser extent to fibronectin and has no effect on cells seeded on collagen I.37 As it is reported, vitronectin interacts only with a few types of integrins such as αvβ3 and αvβ5; on the contrary fibronectin interacts with a wide integrin repertoire including αvβ3 and αvβ5,50 and thus the different antiadhesion effects of ψRGDechi can be explained considering the capability of fibronectin to bind WM266 cells more efficiently thanks to the diverse integrins most likely present on their surface. The inhibition of adhesion by ψRGDechi occurs in a concentration-dependent way. In addition, as expected, the clear different ability of ψRGDechi to compete with anti-αvβ3 and anti-αvβ5 antibodies for the binding to the corresponding receptors is in perfect accordance with its selectivity. The effect of ψRGDechi on wound healing activity was evaluated by the scratch assay. Interestingly, the peptide induces an evident inhibition of cell motility with respect to RGDechi due to its definitely increased stability which is most detectable in the experiment performed

Figure 7. In vivo imaging of αvβ3 expressing tumors by ψRGDechiNIR750 and RGDechi-NIR750. (A) WM266 melanoma cells were implanted s.c. into the right flank of CD-1 nu/nu athymic mice. One nmole ψRGDechi-NIR750 or RGDechi-NIR750 was injected i.v., and total body NIR fluorescence was monitored after 3, 5, and 24 h using fluorescence molecular tomography FMT4000. (B) Uptake and persistence of ψRGDechi-NIR750 and RGDechi-NIR750 in melanoma xenografts at different time points is reported as counts/energy. Bars depict means ± SD of three independent experiments. Mice injected with ψRGDechi-NIR750 have a significantly higher signal as compared to those injected with RGDechi-NIR750 at 5 and 24 h (**p < 0.01; *p < 0.05). (C) After imaging, study mice were euthanized and tumors explanted were observed ex vivo by FMT4000.

Amino acid aldehydes can be prepared by reduction of Weinreb amides, acyl imidazolides,48 α-amino acid halides, and esters and thiol esters or by oxidation of β-amino alcohols. Taking into account that these compounds tend to racemize both under acidic and basic conditions, for our purpose, we decided to take advantage of the Fukuyama’s reduction of thiol esters under mild conditions (Et3SiH, Pd/C),41 converting an NFmoc proline for the first time into the corresponding thiol ester by Steglich thioesterification (DCC, DMAP, and EtSH).40 The reaction, as expected, gave the desired product in high yield and without racemization as confirmed by analytical chiral analysis (Figure S4). The so-prepared N-Fmoc proline aldehyde was effectively assembled into the peptide backbone 9879



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gradient from 1% to 70% B in 20 min. Serum stability was performed by a LCQ Deca XP Max liquid chromatography−mass spectrometry (LC−MS) system equipped with a diode-array detector combined with an electrospray ion source and ion trap mass analyzer (ThermoFinnigan, San Jose, CA), using a Phenomenex C18 column (5 μm; 300 Å; 250 × 2 mm) and a linear gradient of H2O (0.01% TFA)/CH3CN (0.01% TFA) at a flow rate of 200 μL/min. Confocal microscopy were performed on Zeiss LSM 700 META equipped with an oil immersion Plan-Neofluar 63 × 1.40 objective. FMT4000 Quantitative Tomography Imaging In Vivo Imaging System and 3D were performed using the TrueQuant software package (PerkinElmer, Inc.). Synthesis. 1-Pyrrolidinecarboxylic Acid, 2-[(Ethylthio)carbonyl]-, 9H-Fluoren-9-ylmethyl ester (2). Fmoc-L-Pro-OH (1) (675 mg, 2 mmol) was dissolved in DCM dry (10 mL) under argon and cooled to 0 °C and then treated with DCC (454 mg, 2.2 mmol) and DMAP (24.4 mg, 0.2 mmol) and, subsequently, dropwise with ethanethiol (216 μL, 3.0 mmol). The solution was allowed to warm to room temperature and stirred for 3 h until TLC indicated full conversion in thioethylester derivative 2.40 The reaction mixture was then diluted with ethyl acetate (50 mL) and extracted with HCl 0.1 M (50 mL). The organic layer was washed twice with brine, dried over anhydrous Na2SO4, filtered, concentrated and purified via flash chromatography (SiO2, dichloromethane) providing 533 mg (1.40 mmol, 70% yield) of the desired product. The mass [M + H]+1 observed by HRMS-TOF (calcd. for C22H24NO3S 382.1477) was 382.1475. 1H NMR (400 MHz, CDCl3, mixture of two conformers): δ = 7.81−7.30 (m, 8H, Ar−H), 4.59−4.44 (m, 2H), 4.38−4.19 (m, 2H), 3.73−3.51 (m, 2H), 2.94−2.83 (m, 2H, CH3CH2), 2.28−1.86 (m, 4H), 1.27 and 1.23 (t, J = 7.4 Hz, 3H, CH3CH2); 13C NMR (100 MHz, CDCl3, mixture of two conformers): δ = 202.1 and 201.5 (s, 1C, CO), 154.9 and 154.5 (s, 1C, CO), 144.0 (s, 1C, Cq), 143.7 and 143.6 (s, 1C, Cq), 141.2 and 141.1 (s, 2C, Cq), 127.6 (s, 2C), 126.9 (s, 2C), 125.8 and 125.0 (s, 2C), 119.8 (s, 2C), 67.6 and 67.4 (s, 1C), 66.3 and 65.0 (s, 1C), 47.2 (s, 1C), 46.7 (s, 1C), 31.6 and 31.5 (s, 1C), 23.9 (s, 1C), 23.1 and 23.0 (s, 1C), 14.4 and 14.3 (s, 1C). 1-Pyrrolidinecarboxylic Acid, 2-Formyl-, 9H-Fluoren-9-ylmethyl Ester (3). To a solution of 2 (500 mg, 1.3 mmol) in THF dry 10% Pd/ C (0.026 mmol) was added under argon, and the resulting suspension was treated dropwise with triethylsilane (600 μL, 3.9 mmol) at room temperature. The mixture was stirred overnight then filtered over a Celite pad and purified via flash chromatography (SiO2, Hexane/ EtOAc 95:5 to 90:10) provided 334 mg (1.04 mmol, 80% yield) of the 341 as a colorless oil. The mass [M + H]+1 observed by HRMS-TOF (calcd for C20H20NO3 = 322.1443) was 322.1561. Peptide Synthesis. RGDechi, linear peptide (corresponding to the linear precursor of RGDechi), scrambled peptide, and cRGDfK were synthesized as previously reported (purity obtained: 95.7%, 95.6%, 95.8%, and 95.8%, respectively).46,52 ψRGDechi peptide 5 (0.05 mmol) was manually synthesized by Fmoc solid phase strategy on NovaSyn TGA resin (loading 0.26 mmol/g) in a polypropylene reaction vessel fitted with a sintered polyethylene frit using all standard amino acids except for Fmoc-D-Glu-OAll and Fmoc-L-Pro aldehyde. The first amino acid was bound to the support by treating with FmocThr(tBu)−OH (5 equiv)/MSNT (5 equiv)/MeIm (3.75 equiv) in DCM for 3 h. The amino acids in 5-fold excess were preactivated with HBTU (4.9 equiv)/Oxyma (4.9 equiv)/DIPEA (10 equiv) in DMF and then added to the resin suspended in DMF. The reaction was performed for 1 h, and the coupling efficiency was assessed by the Kaiser test. The Fmoc protecting group was removed with 30% piperidine in DMF (2 × 10 min). At the dipeptide stage, Fmoc-L-Pro aldehyde (50 mg, 1.5 mmol) was dissolved in 2 mL of 1% acetic acid in DMF and added to the peptidyl resin. This was followed by dropwise addition of NaBH3CN 1 M in THF (176 μL, 0.175 mmol).53 The reaction mixture was stirred at room temperature for 24 h, washed with DMF and subjected to another reductive amination cycle. Then, the coupling reaction with the following amino acid (glycine) were performed. An aliquot of this sample was used for the analytical assessment (see Supporting Information). The consecutive coupling steps, cyclization reactions, and cleavage from the resin were carried

in the presence of serum, as the scratch assay is. What is more, invasion studies clearly indicate the ψRGDechi is able to inhibit tumor cell invasion better than RGDechi. In our opinion, all these experiments highlight promising features of the novel pseudopeptide since it is well-known that cell adhesion, migration, and invasion are closely correlated with the metastatic cascade. The potential use of ψRGDechi for targeted molecular imaging was also investigated in vivo on WM266 melanoma xenografts by NIRF, a current emerging technology for noninvasive mapping of molecular events, assessment of therapeutic efficacy, and monitoring of disease progression in cancers.51 Over the past decade, NIRF dyes have been studied extensively for biomedical application since the NIR region is a suitable optical window for deep tissue imaging. In comparison with radioisotope-based PET or SPECT imaging, NIRF technology does not require use of ionizing radiation or radioactive materials, is relatively low cost, safe, and easy to use, and is thus a promising technique for clinical application. Our studies show that both ψRGDechi and RGDechi peptides labeled with NIR dye are able to visualize the tumors, but NIRψRGDechi tumor uptake is definitely higher for its improved stability in serum. In conclusion, we found that an easy chemical modification introduced in our selective peptide considerably improves its biological activities simply by preventing the protease degradation. In particular, we demonstrated an enhanced effect on the two critical steps of the metastatic process, migration and invasion, in highly metastatic melanoma cells. Furthermore, the ability of the peptide to detect αvβ3 in overexpressing human melanoma xenografts was analyzed by near-infrared fluorescence. All these features strongly support the evidence that this new molecule is a promising targeting peptide probe with application as new and selective theranostic agent for melanoma disease.

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EXPERIMENTAL SECTION

Chemistry: General Methods. All solvents (Sigma-Aldrich), the amino acids (Iris Biotech), and the resin (Novabiochem) were purchased from the indicated suppliers. Ultrapure Milli-Q water (18.2 MΩ cm at 25 °C, Millipore) was used for the experiments. Analytical thin layer chromatography (TLC) was performed on aluminum plates precoated with Merck Silica Gel 60 F254 as the adsorbent. The plates were developed with 5% H2SO4 ethanolic solution and then heated to 130 °C. Column chromatography was performed on Merck Kieselgel 60 (63−200 mesh). 1H and 13C NMR spectra were recorded on Varian Unity Inova 400 NMR spectrometer equipped with dual probe (400.13 MHz for 1H, 100.13 MHz for 13C) and Varian Unity Inova 600 MHz NMR spectrometer equipped with cryo probe. Chemical shifts (δ) are reported in ppm relatively to the residual solvent peak (CHCl3, δ = 7.26, 13CDCl3, δ = 77.0). Peptide purification was performed by RP-HPLC on a Agilent 1200 system (equipped with UV−vis diode array detector set at 210 nm) using a Phenomenex Jupiter Proteo C12 column (10 μm; 90 Å; 250 × 10 mm) with a flow rate of 10 mL/min and a linear gradient starting from 5% to 70% B in 40 min. High-resolution ESI-MS spectra and analytical RP-HPLC analyses were performed on a AGILENT Q-TOF LC−MS instrument equipped with DUAL ESI source and diode array detector using Phenomenex Aeris Peptides C18 column (3.6 μm, 2.1 × 50 mm). For all RP-HPLC procedures, the system solvent was H2O 0.1% TFA (A) and CH3CN 0.1% TFA (B). The percentage of purity for all compounds was assessed by analytical analysis performed with a linear gradient from 5% to 70% B in 12 min and determined to be ≥95% pure through integration at 210 nm. Enantiomeric purity of compound 3 was assessed on a Phenomenex Lux Cellulose-2 chiral column (3 μm, 50 × 4.60 mm) with a flow rate of 0.8 mL/min and a linear 9880



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out as reported before.46 The resin was filtered, the crude peptide was precipitated with diethyl ether, dissolved in H2O/ACN solution, lyophilized, and purified by RP-HPLC obtaining a white solid in 25% yield (purity obtained 96.0%). The mass observed by HRMS-TOF (calcd. for [M + H]+1, 2088.9997) was [M + 2H]2+, 1045.5202. Serum Stability. RGDechi and ψRGDechi peptides degradation in serum were performed as previously reported,35 introducing slight modifications in order to favor serum protein precipitation. Briefly, 400 μL of human serum were added to 160 μL of a 1 mg/mL solution of RGDechi and ψRGDechi, respectively, at 37 °C. After 1, 2, 4, 8, 24, and 48 h, samples of 15 μL of each incubation mixture were added to a 15 μL of 15% TFA aqueous solution and centrifuged for 5 min at 13000g. Supernatants were then analyzed by LCQ Deca XP Max LC− MS system. Cell Lines and Culture Conditions. HeLa (ATCC U.S.) were grown in DMEM supplemented with 10% fetal bovin serum, 1% glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Euroclone, Milano, Italy). WM266, provided by Dr. Palmieri (ICBCNR-Sassari Italy), were grown in RPMI, supplemented with heatinactivated 10% FBS, 1% glutamine, 100 U/mL penicillin, and 100 μg/ mL streptomycin. The cells were maintained in humidified air containing 5% CO2 at 37 °C. Characterization of αvβ3 and αvβ5 cell surface density was performed by quantitative flow cytometry as previously reported.52 Cell Adhesion Assay. The real-time cell analyzer (RTCA) and Eplate 16 system (xCELLigence, Roche, San Diego, CA) allow monitoring the viability of cultured cells using impedance by a labelfree, real time, and noninvasive analysis.54 This platform uses gold electrodes at the bottom of microplate wells as sensors to which an alternating current is applied. Cells are adherently grown in monolayers on top of the electrodes, affecting the alternating current at the electrodes by changing the electrical impedance, denoted as cell index (CI). Cell index values are proportional to the area of cells attached and to the total number of cells.55,56 The effect of peptides, on WM266 adhesion on extracellular matrices (vitronectin, fibronectin, or collagen type I) and on HeLa cells adhesion on anti-αvβ5 antibody, was monitored using the RTCA methodology. A total of 350 μL of cell culture media was added to Eplates coated with 10 μg/mL of matrices or antibody (Millipore). Coating was carried out overnight at 4 °C, and then a blocking step for nonspecific binding was performed with 1.5% BSA in PBS for 1 h at room temperature. After measurement of the background intensity, adhesion capacity of cells, visualized in increments of the CI, was determined using the manufacturer’s instructions. In brief, cells (4 × 104 WM266 or 6 × 104 HeLa) were suspended in 250 μL of adhesion buffer prepared as previously reported46 in the presence of peptides (50 μM) for 30 min at 4 °C and then seeded on precoated plates. Immediately, the E-plate was inserted into the RTCA system, and the CI was measured every minute in a range of 0−24 h. The rate of cell adherence was determined by calculating the slope of the line between two selected time points. The end-point of the experiments corresponds to the maximum CI values observed for each adhesion curves. Differently, the effect of peptides on WM266 adhesion on anti-αvβ3 antibody (LM609) was tested on NUNC MaxiSorp 96 well plates (Dasit Sciences, Milano, Italy) coated with LM609 (10 μg/mL) (Millipore). 1.5 × 104 cells/well were seeded and incubated in the presence of peptides (50 μM) for 1 h. Nonadherent cells were gently removed by repeated washings and adherent cell number was evaluated by crystal violet assay, which correlates optical density with cell number.57 The same method was used to evaluate the IC50 of the peptide. For both protocols the mean value ± SE of adherent cells for each treatment was expressed as relative percentage of cell number versus cells not treated (control). Statistical differences were determined by student’s t test, paired, two-sided. All experiments were performed at least in duplicate and repeated at least 3 times; a p < 0.05 was considered to be significant. In Vitro Scratch Assay. In vitro wound model was performed using a scratch assay.58 Confluent monolayers of WM266, cultured in

6-well plates, were linearly scratched with a plastic pipet tip to create a cell-free area. Cultures were gently washed 3 times with warmed PBS to remove loose cells. The medium containing 50 μM of peptides was then added, and cells were incubated in a CO2 incubator. Each scratch area was photographed at 0, 24, and 48 h. The wound size for the different peptides were calculated as percent width at each end-points with respect to their value at 0 h. Transwell Invasion Assay. Cell invasion was assayed using transwell chambers (8 μm) coated with ECL Cell Attachment (Millipore Corporation) used according to the manufacturer’s instructions. Two duplicates were set for the assay. Cells at the logarithmic phase were detached using 1 mM EDTA and washed twice with serum-free RPMI. A total of 1 × 105 cells/150 μL were seeded in the upper chamber. In the lower chamber, 600 μL of RPMI medium containing 10% fetal bovine serum was added for incubation at 37 °C in a 5% CO2 incubator for 18 h. After removal of the upper chamber, the lower chamber was washed twice with PBS, and migrated cells were fixed with 10% formalin for 10 min. The not migrated cells were removed with cotton swabs. Next, the cells on the bottom surface of the membrane were stained with crystal violet for 30 min. Cell images were obtained under a phase contrast microscope. To plot the number of invasive cells, four different fields were counted for each chamber and mediated. Peptides Labeling. ψRGDechi (1 nmol) or RGDechi (1 nmol) were incubated in 50 μL 0.1 M Na2CO3 buffer, pH 8.6, with 5 nmol VivoTag-S 750-N-hydroxysuccinimide (NHS) ester (PerkinElmer, Inc., Boston, MA), dissolved in 5 μL dimethyl sulfoxide, for 2 h at room temperature in the dark. Excess reactive groups were saturated by addition of 2 μL 1 M Tris−HCl buffer, pH 8.6, and incubated for additional 15 min. Then, conjugates were separated on Zeba Spin Desalting Columns (Thermo Scientific, Rockford, IL) and eluted with 250 μL 0.14 M NaCl in 20 mM Hepes buffer, pH 7.4, under centrifugation (2 min, 1000g) and obtained with a purity of 95.4% and 95.7%, respectively. Cell Binding Assay. WM266 cells (85000) were seeded in 24 well plate on glass coverslips and were allowed to attach overnight. After washing the cells with DPBS, these were incubated with 1 nmole of ψRGDechi-NIR750 in DPBS containing 5% FBS at 4 °C for 1 h. Cells were washed in DPBS and fixed in 4% PFA/DPBS for 20 min at room temperature, incubated with 1.5 μM DAPI, permeabilized with 0.1% Triton X-100/DPBS for 5 min and then subjected to blocking in 5% FBS/DPBS for 30 min at RT. The cells were then incubated overnight at 4 °C with anti-αvβ3 primary antibody and subsequently with Alexa Fluor 488 antimouse secondary antibody for 1 h at RT. For the competition study, the cells were preincubated with an excess of parent compound (1000 fold) for 1 h at 4 °C. Samples were visualized on Zeiss LSM 700 META confocal microscopy equipped with an oil immersion Plan-Neofluar 63 × 1.40 objective. To quantify the yellowmerged spots, ImageJ plugin JACoP was used and Manders’ coefficients M1 and M2 were calculated.59 In Vivo Imaging of αvβ3 Expressing Xenografts with Molecular Fluorescence Tomography (FMT). All experimental procedures complied with the European Communities Council directives (2010/63/EU) and national regulations (D.L. 116/92), were performed in accordance with National Institutes of Health (NIH) recommendations. The present study was approved by the Italian Ministry of Health (authorization number 2013/0100808). All efforts were made to minimize animal suffering and the number of animals necessary to produce reliable results. All experimental procedures described were performed under general anesthesia with 2% isoflurane in 100% oxygen at 0.8 L/min. Briefly, 10 × 106 WM266 cells expressing high level of αvβ3 were injected in right flank of 9 CD1 nu/nu athymic mice. To perform imaging studies by molecular fluorescence tomography, tumor-bearing mice were maintained on a diet with a purified, alfalfa-free rodent chow for 15 days before fluorescent imaging to minimize fluorescence in the gut. When the tumor reached a volume, about 0.5 cm3 mice (3 for each group) were i.v. injected with ψRGDechi-NIR750 (1 nmol), RGDechi-NIR750 (1 nmol), or VivoTag-S 750 (1 nmol) alone and total body imaged after 3, 5, and 24 h with the FMT4000 Quantitative Tomography Imaging 9881



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ORCID

In Vivo Imaging System (PerkinElmer, Inc.). Mice were placed in a biplanar imaging cassette supplied with the instrument and transilluminated with laser light. Resulting transmission and fluorescence patterns were captured with a thermoelectrically cooled CCD camera, and the position and intensity of fluorescence sources were reconstructed in 3D using the TrueQuant software package (PerkinElmer, Inc.), supplied with the FMT4000. 3D Regions of interest were drawn around tumor regions, and a threshold was applied equal to 30% of the maximum value of fluorescence in the adjoining nontumor area. Statistical Analysis. Results were obtained from at least three independent experiments and are expressed as the mean ± SD or mean ± SE, as indicated in the legend of figures. Statistical values were determined using the two-tailed unpaired student’s t-test. Differences were considered significant at the p value < 0.05. Molecular Docking Protocol. The Molecular Docking investigation was carried out using the software AutoDock 4.0.60 The Molecular Docking involved the following step in accordance with the optimized protocol reported in a previous manuscript.37 (1) Preparing αvβ3 receptor and ψRGDechi peptide coordinates: we created the ligand and receptor coordinate files in order to include all the information needed as spatial changes, atom types, polar hydrogen atoms, and torsional degrees of freedom. In particular, for the αvβ3 receptor we used as reference structural model the coordinate reported in the X-ray crystal structure of the αvβ3 in complex with cyclo(RGDf[NMe]), “cilengitide” (PDB ID code: 1L5G), whereas for ψRGDechi, we properly modified the representative structure of the RGDechi peptide used in the molecular docking studies reported by Farina et al.37 In addition, the polar hydrogen atoms were added to both structures, and the rotatable bonds for the ligand were automatically selected by using the tools of the AutoDock 4.0 program. (2) Defining of the grid maps by using the tool AutoGrid that is included in the AutoDock software package. In particular, the grid map, centered on the region of interest of the receptor, was set to be 64 × 64 × 64 and the grid space was 0.658 Å. (3) Running the docking calculation using the AutoDockTools that specifies the input parameters for the docking. In the docking protocol, we used the Lamarckian genetic algorithm (LGA) search methods. Minimized ligands were randomly placed inside the grid box, and the docking process initiated with a quaternion and torsion steps of 58, torsional degrees of freedom of 3, number of energy evaluations of 2500000, and run number 100. The structures obtained by the docking simulation were clustered by evaluation of the binding free energy of the complex. The representative structural model of the αvβ3/ ψRGDechi was visualized and analyzed by using the software PyMoL61 and MolMol.62

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Michele Saviano: 0000-0001-5086-2459 Laura Zaccaro: 0000-0001-6843-0152 Author Contributions ▽

This work was funded by grants from Ministero dell’Istruzione, dell’Università e della Ricerca, PRIN 2010−2011 (no. 2010C4R8M8), FIRB 2012−2017 (no. RBFR124FEN), Programma Operativo Nazionale Ricerca e Competitività 2007−2013 PON 01_ 02388, and Programma Operativo Nazionale Ricerca e Competitività 2007−2013 PON 01_01078. Notes

The authors declare no competing financial interest. Authors will release the atomic coordinates and experimental data upon article publication.

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ACKNOWLEDGMENTS We are grateful to Matteo Gramanzini and Billy Samuel Hill for in vivo imaging and confocal analysis, respectively. The authors thank Leopoldo Zona, Luca De Luca, Maurizio Amendola, and Florinda Pignatiello for the excellent technical/administrative support and Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (C.I.R.C.M.S.B.).

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ABBREVIATIONS HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; oxyma, (ethyl cyano(hydroxyimino)acetate); PyBop, benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate; MSNT, 1-(mesitylene-2-sulfonyl)3-nitro-1H-1,2,4-triazole; DIPEA, N,N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DCC, N,N′-dicyclohexylcarbodiimide; DCM, dichloromethane; DMF, N,N-dimethylformamide; BSA, bovin serum albumin; FBS, fetal bovin serum; EDTA, ethylenediaminetetraacetic acid; RTCA, real time cell analyzer; CI, cell index; LC−MS, liquid chromatography−mass spectrometry; SPECT, single-photon emission computed tomography; NIR, near-infrared; NIRF, near-infrared fluorescence; FMT, fluorescent molecular tomography; DMEN, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; DPBS, Dulbecco’s phosphate-buffered saline; DAPI, 4′,6diamidino-2-phenylindole; RT, room temperature; NIH, National Institutes of Health

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01590.

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D.C. and A.Z. contributed equally to this work.

Funding

REFERENCES

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Structure 1 (PDB) Structure 2 (PDB) Structure 3 (PDB) Structure 4 (PDB) NMR, LC−MS spectra, PDB files as well as supplementay cell binding and in vivo imaging (PDF) SMILE results (CSV)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. 9882



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