Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells

University of Groningen Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells Hahn, Eva M.; Estrada Ortiz, Natalia; Han, Jiaying; Ferreira, Vera F. C.; Kapp, Tobias G.; Correia, Joao D. G.; Casini, Angela; Kuehn, Fritz E. Published in: European Journal of Inorganic Chemistry DOI: 10.1002/ejic.201601094 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Hahn, E. M., Estrada Ortiz, N., Han, J., Ferreira, V. F. C., Kapp, T. G., Correia, J. D. G., Casini, A., & Kuehn, F. E. (2017). Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells. European Journal of Inorganic Chemistry, 2017(12), 16671672. https://doi.org/10.1002/ejic.201601094 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverneamendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 02-05-2026 DOI: 10.1002/ejic.201601094 Communication CLUSTER ISSUE Targeting Integrin Receptors Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides To Target Integrin Receptors in Cancer Cells Eva M. Hahn,[a,b] Natalia Estrada-Ortiz,[c] Jiaying Han,[c] Vera F. C. Ferreira,[d] Tobias G. Kapp,[e] João D. G. Correia,[d] Angela Casini,*[b,c,e] and Fritz E. Kühn*[a] Abstract: The lack of selectivity for cancer cells and the resulting negative impact on healthy tissue is a severe drawback of actual cancer chemotherapy. Tethering of cytotoxic drugs to targeting vectors such as peptides, which recognize receptors overexpressed on the surface of tumor cells, is one possible strategy to overcome such a problem. The pentapeptide cyc(RGDfK) targets the integrin receptor αvβ3, important for tumor growth and metastasis formation. In this work, two terpyridine-based RuII complexes were prepared and for the first time conjugated to cyc(RGDfK) through amide bond formation, which resulted in a monomeric and a dimeric bioconjugate. Both RuII complexes were found to bind strongly and selectively to integrin αvβ3, and the dimeric molecule displayed a 20-fold higher affinity to the receptor than the monomeric one. However, the cytotoxicity of the complexes and related bioconjugates against human A549 and SKOV-3 cell lines is still not sufficient for application as anticancer agents. Nevertheless, considering the high selectivity for integrin receptor αvβ3, the synthesis of Ru-based bioconjugates with cyc(RGDfK) paves a promising way towards the design of effective targeted anticancer agents. Introduction appeared in recent years.[6] Specifically, the functionalization of metallodrugs is aimed at improving tumor selectivity and/or minimizing systemic toxicity to enhance their cellular accumulation and to overcome tumor resistance. Moreover, a synergistic anticancer effect of different therapeutic modalities would also be welcome. In some cases, the use of imaging tags conjugated to the metal compounds allows visualization of the drug molecules in vitro or in vivo, which thus leads to the design of theranostic agents.[7] Platinum anticancer drugs are widely used for chemotherapy of various cancers. However, indiscriminate distribution or poor selectivity often results in severe side effects and drug resistance.[1] Therefore, enhancing the tumor selectivity has become a major goal for the development of platinum-based cytotoxic agents. Similar issues are encountered with the new generation of experimental anticancer metal complexes, including, among others, compounds based on ruthenium,[2] gold,[3] iron,[4] and copper.[5] Thus, the development of so-called targeting and drug-delivery strategies of metallodrugs has become a priority in the field, together with the design of new chemical scaffolds. Within this framework, an increasing number of reports on tethering metal complexes to a wide range of functional molecules or nanoparticles with or without targeting groups has [a] Molecular Catalysis, Catalysis Research Center and Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching bei München, Germany E-mail: fritz.kuehn@ch.tum.de [b] School of Chemistry, Cardiff University, Park Place, Cardiff CF103AT, United Kingdom E-mail: casinia@cardiff.ac.uk [c] Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands [d] Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, CTN, Estrada Nacional 10 (km 139.7), 2695-066 Bobadela LRS, Portugal [e] Institute for Advanced Study, Technische Universität München, Lichtenbergstr. 2a, 85748 Garching, Germany Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejic.201601094. Eur. J. Inorg. Chem. 2017, 1667–1672 Among the various strategies explored so far to actively target cytotoxic metallodrugs to cancer cells, tumor-targeting peptides (TTPs), which are specific for tumor-related surface markers, such as membrane receptors, can be used.[8] Integrin receptors have been largely explored as drug targets, as they are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules, and signal transduction platforms in a multitude of biological processes.[9] Integrins interact with the extracellular matrix (ECM) and thereby regulate many cellular functions, such as proliferation, migration, and survival. Integrins are also involved in cell-to-cell interactions. Through cell–cell and cell–ECM contacts, integrins transduce the information from the external environment into the cell and vice versa to promote cell adhesion, spreading, and motility.[10] One common feature of the integrin family is a heterodimeric structure that consists of α and β subunits.[11] These structures form 24 different subtypes in mammals, which can be classified according to their binding partners (e.g. laminin, collagen). Different integrins are also associated with tumor angiogenesis and metastasis,[12] which are upregulated in tumor cells relative to the low levels in normal endothelial 1667 © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Communication cells. The integrin receptor αvβ3 plays a crucial role in these processes[13,14] and has become an attractive target for pharmaceutical research.[15] In 1984, Pierschbacher and Ruoslahti discovered that the amino acid sequence Arg-Gly-Asp-Ser (RGDS) is essential for binding integrin receptors.[16] In fact, eight of the abovementioned integrin subtypes form the RGD-binding class.[17] Since then, a wide screening of peptide libraries has been performed to discover ligands including the RGD sequence and targeting integrin receptors with even higher selectivity. Interestingly, the cyclic pentapeptide cyc[RGDfK] (Figure 1) was found to have increased selectivity for integrin αvβ3.[18] Among the metal-based radiopharmaceuticals tethered to cyclic RGD peptides, the majority of the reported examples were evaluated as single-photon emission computed tomography (SPECT) and positron emission tomography (PET) radiotracers for tumor imaging.[7a,21] Recently, preclinical evaluation of the potential theranostic radiopharmaceutical 66Ga-DOTAE(cyc[RGDfK])2 compound was reported.[22] As an example of targeted anticancer metal complexes, recent reports describe the synthesis and biological evaluation of PtIV prodrugs, the axial positions of which could be functionalized with cyclic RGD tripeptides that bind selectively to the integrin receptor αvβ3.[19,23] In a more elaborated approach, Lippard et al. synthesized a cisplatin prodrug encapsulated into poly(D,L-lactic-co-glycolic acid)-block-polyethylene glycol (PLGAPEG) nanoparticles tethered to cyc[RGDfK]. The prodrug shows a significant increase in cytotoxicity towards αvβ3 integrin-expressing cancer cell lines, comparable to cisplatin. In vivo studies also revealed equivalent tumor growth inhibition (ca. 60 %) by both the prodrug and cisplatin in mice bearing ovarian cancer xenografts.[20] Concerning anticancer ruthenium complexes coupled to peptides, some examples have already been reported,[8] including luminescent RuII complexes linked through the mitochondrial penetrating peptide (MPP)[24] as well as to the nuclear localization sequence (NLS),[25] the latter of which enables the active transport of drugs into the cell nucleus as confirmed by fluorescence microscopy studies. Interestingly, Keyes et al. developed ruthenium(II) polypyridyl luminophores anchored to peptide sequences as a new class of stimulated emission depletion (STED) microscopy probes for the imaging of key cell organelles.[26] Ueyama et al. also described a peptide-labeling approach by using RuII terpyridine complexes to implement the mass spectrometry detection of proteolytic peptides.[27] As far as it concerns RGD-type peptides, only a few examples are described. Thus, Sadler et al. reported the synthesis of a RuII arene complex attached to the linear RGD tripeptide[28] that dissociated from the peptide by irradiation with visible light to form an aqua complex that generated monofunctional adducts with the guanine bases of DNA. Furthermore, Adamson et al. designed luminescent RuII polypyridyl complexes attached to the linear RGD tripeptide, which acted as molecular probes for reporting the presence and conformation of integrins.[29] Livecell studies with confocal microscopy confirmed the selective binding to an integrin receptor, but no cytotoxicity studies were described. Finally, fluorescent ruthenium polypyridyl complexes were attached to RGD-functionalized mesoporous silica nanoparticles,[30] the uptake and subcellular distribution of which could be followed by fluorescence microscopy. Interestingly, the RDG peptide on the nanoparticle surface induced an increased selectivity for cancer cells. After internalization of the nanoparticle, the ruthenium species were released and induced changes Figure 1. Cyclic pentapeptide cyc[RGDfK] (A) and two representative targeted Pt constructs: the PtIV conjugate[19] (B) and nanoparticles encapsulating a cisplatin prodrug[20] (C). Eur. J. Inorg. Chem. 2017, 1667–1672 www.eurjic.org 1668 © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Communication in the phosphorylation pathway of certain protein kinases, which led to apoptosis. To the best of our knowledge, besides the abovementioned publications, there are no other studies about the conjugation of ruthenium complexes to RGD-type peptides. Therefore, in this work the successful bioconjugation of two RuII terpyridine complexes to the cyclic RGD peptide cyc[RGDfK] for targeting integrin αvβ3 is reported. The two RuII compounds were designed to feature carboxylic acid groups for conjugation to the lysine residue of the RGD peptide through amide bond formation. Thus, the compounds were tethered to one or two peptides. In the latter case, anchoring to two cyc[RGDfK] was intended to enhance the binding affinity to the αvβ3 integrin receptor.[13] The binding affinities of the ruthenium–RGD conjugates for both the αvβ3 and α5β1 integrin receptors were evaluated by integrin binding assays. The anticancer effects of the “free” ruthenium complexes and their respective conjugates were evaluated in vitro against human cancer cell lines with different expression levels of integrin αvβ3, namely, human lung cancer A549 cells (scarce αvβ3 integrin expression) and human mammary carcinoma SKOV3 cells (moderate αvβ3 integrin expression).[31] Results and Discussion The experimental procedures can be found in the Supporting Information. The two ligands used in this work are 2,2′:6′,2′′terpyridine (terpy, 1a) and [2,2′:6′,2′′-terpyridine]-4′-carboxylic acid (terpy*, 1b). For the synthesis of 1b, a reported two-step procedure was followed.[32] In the first step, 2-acetylpyridine and furfural were combined in ethanol under basic conditions to yield 4′-(furan-2-yl)terpyridine, which was oxidized in the following step with KMnO4 to obtain [2,2′:6′,2′′-terpyridine]-4′carboxylic acid (terpy*, 1b) (see Scheme 1). Complexes 3a and 3b were prepared by a novel synthetic route based on literature procedures[33] (Scheme 2). Heating RuCl3·3H2O with 1a or 1b in dry ethanol yielded brown complex 2a or 2b, respectively, after 1 h in the dark. Afterwards, the complexes were separately treated with 1b, triethylamine, and LiCl for chloride abstraction and reduction of RuIII to RuII. Upon the addition of 1 M KPF6, the [Ru(terpy)(terpy*)](PF6)2 (3a) and [Ru(terpy*)2](PF6)2 (3b) complexes bearing one and two carboxylic acid groups, respectively, precipitated. Conjugation of 3a and 3b to the cyclic peptide cyc[R(Pbf )GD(tBu)fK] was accomplished by reaction of the free carboxylic acid groups of the complexes with the primary amine group of the lysine side chain in the presence of a mixture of the activating agents 1-[bis(dimethylamino)methylene]1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) (Scheme 3). The success of the bioconjugation reaction was confirmed by electrospray ionization mass spectrometry (ESI-MS), which allowed identification of the intermediate products at m/z = 752.78 for [Ru(terpy){terpy-cyc{R(Pbf )GD(tBu)fK}}]2+ and 1221.58 for [Ru{terpy-cyc{R(Pbf )GD(tBu)fK}}2]2+, respectively. Afterwards, the remaining protecting groups of Arg and Asp were cleaved by using a cleavage cocktail as detailed in the Experimental Section. For purification of the crude product, size-exclusion chromatography with Sephadex G-15 was used, as the compounds decomposed during reverse-phase (RP)-HPLC. Finally, the products were precipitated by the addition of solid KPF6 to give [Ru(terpy){terpy-cyc(RGDfK)}](PF6)2 (4a) and [Ru{terpy-cyc(RGDfK)}2](PF6)2 (4b) as red solids. Scheme 1. Synthesis of the [2,2′:6′,2′′-terpyridine]-4′-carboxylic acid ligand (1b). Scheme 2. Two-step procedure for the synthesis of [Ru(terpy)(terpy*)](PF6)2 (3a) and [Ru(terpy*)2](PF6)2 (3b). Eur. J. Inorg. Chem. 2017, 1667–1672 www.eurjic.org 1669 © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Communication Scheme 3. Synthesis of bioconjugate products 4a and 4b (PG = protecting group; DIPEA = N,N-diisopropylethylamine). Characterization of Ligand 1b Complexes 3a/3b and 4a/4b The ligands and the corresponding complexes were characterized by 1H NMR, 13C NMR, and 31P NMR spectroscopy and ESIMS. Comparing the 1H NMR spectra of ligand 1b with 3a, several signal shifts are observed owing to complex formation (Figure 2). The signals of H3′,5′ and H3,3′′ are shifted downfield by around Δδ = +0.61 and +0.49 ppm. In contrast, the signal of H4,4′′ remains, and the signals of H6,6′′ and H5,5′′ show a strong upfield shift of Δδ = –1.25 and –0.28 ppm. Nearly the same values are observed for complex 3b containing two ligands 1b. The downfield shifts of H3′,5′ and H3,3′′ are about Δδ = +0.62 and +0.47 ppm, whereas the signal of H4,4′′ remains, and the signals of H6,6′′ and H5,5′′ are shifted upfield by about Δδ = –1.20 and –0.26 ppm. For these observations, two effects have to be taken into account: first, the deshielding effect of the carboxylic acid group; second, the increase in electron density in the aromatic system through coordination of ruthenium. The remaining signals in the spectrum of 3a can be assigned to coordinated ligand 1a. In the 31P NMR spectra, the presence of the PF6– counterions in complexes 3a and 3b is confirmed by the characteristic septet. Complexes 3a and 3b and their conjugation derivatives 4a and 4b were characterized by ESI-MS, and the characteristic isotopic patterns are consistent with the assigned structures (Figures S3–S14 in the Supporting Information). The ESI mass spectra of the complexes show signals at m/z = 757.05 and 306.04 for 3a and at m/z = 801.04 and 328.04 for 3b, which indicate the loss of one or two PF6– anions, and this leads to a singly or doubly positive charged cationic species. Similarly, for coupling products 4a and 4b, the loss of the PF6– anions is observed. The characteristic isotopic patterns of the signals match perfectly with the calculated ones, which can be seen in the Supporting Information. Figure 2. 1H NMR spectra of 1b and complexes 3a and 3b (in [D6]DMSO). Eur. J. Inorg. Chem. 2017, 1667–1672 www.eurjic.org 1670 © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Communication Integrin Binding Assay The impact of conjugation of the RuII complexes to cyc[RGDfK] on the binding affinity to the integrin receptors αvβ3 and α5β1 was evaluated. The binding affinities for 4a, 4b, and benchmark Cilengitide[34] are shown in Table 1. Table 1. Results of integrin binding assays for bioconjugates 4a and 4b in comparison to the benchmark Cilengitide.[a] Compound IC50 [nM] ± SD ανβ3 α5β1 Cilengitide[34] 4a 4b 0.54 ± 0.06 49 ± 4.3 2.5 ± 0.3 15.4 ± 0.2 >1000 595 ± 67 [a] The reported IC50 values were determined by using a solid-phase binding assay (see the Supporting Information for details). Bioconjugate 4a exhibits a median inhibitory concentration (IC50) value of (49 ± 4.3) nM, which is 90-fold higher than that of Cilengitide [(0.54 ± 0.06) nM]. However, the selectivity for αvβ3 is reasonably high, which reflects the fact that the bioconjugate does not bind the α5β1 receptor at all (IC50 > 1000 nM), whereas Cilengitide still has an affinity of (15.4 ± 0.2) nM. Considering bioconjugate 4b, enhanced binding affinities are predicted owing to its dimeric character. Indeed, the binding affinity for integrin αvβ3 is (2.5 ± 0.3) nM, and it presents an affinity that is 20 times higher than that of the monomeric product and nearly approaches the value of Cilengitide. Given that the affinity for the α5β1 receptor shows merely a value of about (595 ± 67) nM, the high selectivity of 4b for αvβ3 is demonstrated. Antiproliferative Activity Ruthenium compounds 3a and 3b and respective cyc[RGDfK] bioconjugates 4a and 4b were evaluated for their antiproliferative properties on two human cancer cell lines with scarce (A549) or moderate (SKOV3) expression of integrin αvβ3.[31] Unfortunately, both ruthenium(II) complexes and their targeted derivatives showed similarly very low cytotoxic effects against both cell lines, independent of the presence of the RGD domains (Table 2). This could be attributed to the intrinsic limited anticancer effects of the selected RuII derivatives. Therefore, although their cell uptake should be favored by the presence of cyc[RGDfK] domains, in the end, no toxic effects were observed. Table 2. IC50 values of Ru complexes and their RGD bioconjugates against human A549 and SKOV-3 cell lines. Compound IC50[a] [μM] A549 SKOV-3 3a 4a 3b 4b 70.3 ± 9.8 87.7 ± 5.4 >100 >100 74.5 ± 13.7 85.2 ± 18.7 >100 >100 [a] The reported values are the mean ± SD of at least three determinations. Conclusions In summary, two novel ruthenium(II) polypyridyl complexes coupled to the cyclic pentapeptide cyc[RGDfK] with monomeric Eur. J. Inorg. Chem. 2017, 1667–1672 www.eurjic.org or dimeric character were prepared to deliver anticancer metallodrugs directly to tumors cells overexpressing the αvβ3 integrin receptor. The preparation of terpy-based ruthenium complexes 3a and 3b bearing one and two carboxylic acid groups, respectively, was performed by using a novel synthetic strategy. The compounds were coupled to a protected derivative of the cyclic pentapeptide through amide bond formation between the carboxylic acid of the complex and the amine group of the lysine side chain. Purification of resulting monomeric bioconjugate 4a and dimeric bioconjugate 4b was achieved by sizeexclusion chromatography followed by precipitation as their PF6 salts. Considering the binding affinities of the bioconjugates towards the integrin receptors, a high selectivity for the αvβ3 integrin receptor and a negligible impact on the α5β1 receptor was observed. Still, the cytotoxicity of all the reported bioconjugates was low, most likely as a result of still-scarce uptake in cancer cells. Hence, whereas the reported strategy holds promise to achieve targeted metallodrugs, future studies have to focus on tethering to the RGD peptide of ruthenium complexes with an intrinsically higher cytotoxic potency, such as similar types of ruthenium complexes with terpyridine-type ligands.[35] Acknowledgments A.C. acknowledges support from Cardiff University, the AugustWilhelm Scheer Visiting Professorship at the Technical University of Munich (TUM), and a Hans Fischer Senior Fellowship of TUM IAS (Institute for Advanced Study) funded by the German Excellence Initiative and the European Union Seventh Framework Program under grant agreement n° 291763. E. M. H. acknowledges Prof. H. Kessler for helpful discussions and is grateful for financial support of the TUM Graduate School of Chemistry and the Short Term Scientific Mission supported by COST action CM1105. V. F. C. F. and J. D. G. C. gratefully acknowledge the FCT (Portugal) support through the UID/Multi/04349/2013 project. 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