Anti-adhesive action of novel ruthenium(II) chlorophenyl terpyridine complexes with a high affinity for double-stranded DNA: in vitro and in silico
Graphical abstract
Best docked poses of complex-[Ru(Cl-Ph-tpy)(dach)Cl]+ (Cl-Ph-tpy, 4′-(4-chlorophenyl)-2,2′:6′,2′′-terpyridine; dach, 1,2-diaminocyclohexane) on human serum albumin and dodecamer double-stranded oligonucleotide as assessed by molecular docking. Linear dichroism study of intercalation of the complex into a full-length double-helical DNA. The complex inhibits the adhesion of normal cells -HTR-8/SVneo and tumour cells - JAr choriocarcinoma.
Introduction
Transition metal coordination compounds have been used in the clinic for decades for the treatment of various diseases [1,2]. Silver compounds showed efficacy as antimicrobial agents, gold compounds are commonly used as anti-arthritic drugs, bismuth has been applied to treat the ulcers, few insulin-mimetics are vanadium complexes, one iron complex was approved as an antihypertensive drug, the majority of contrasting agents in magnetic resonance imaging are based on Gd(III) etc. [1,2]. However, the most widely used metallotherapeutics are those based on Pt (cisplatin, carboplatin and oxaliplatin), which are administered to patients with solid cancers such as breast, lung, ovarian, liver, bladder and testicular cancer [1,3]. Not only that Pt-based drugs often cause adverse effects, but also tumours find their way to develop resistance to these drugs [4]. That is why the ongoing search for anticancer activity often turns to other metals, such as ruthenium, which became “a new chemical hope” [5,6]. The two iconic Ru-based complexes have been tested in clinical trials: NAMI-A ((ImH)[trans-RuCl4(dmso-S)(Im)], Im = imidazole, dmso = dimethylsulfoxide) and KP1019/KP1339 (KP1019 = (IndH)[trans-RuCl4(Ind)2], Ind = indazole; KP1339 = Na[trans-RuCl4(Ind)2]), one possibly about to obtain approval for the clinical use [7].
The most effective oncological drugs in clinical use are those that target DNA [8]. On the other hand, the main targets of investigational ruthenium drugs seem to be both nuclear DNA and proteins, such as protein kinases, histones, integrins, and ion channels [9]. Hence, to reveal the mechanism of action of Ru(II) based drugs, one must seek to understand both drug/DNA and drug/protein interactions. As any metallotherapeutic would be preferably administered intravenously, its interaction with serum proteins is of crucial importance [10]. The favourable binding of complexes/drugs to plasma proteins is essential for adjusting the effective drug concentration at pharmacological target sites [11]. Human serum albumin (HSA) is a non-glycosylated globular protein of 585 amino acids [12], and about 60% of the total protein in blood serum comes from it [13]. HSA transports fatty acids, hormones, various metabolites, drugs, as well as transition metals [13]. The adducts formed between HSA and a metallodrug affect the distribution, rate of metabolism and excretion of the latter [14].
Apart from organometallic coordination compounds of Ru(II) [15], ruthenium compounds carrying polypyridyl ligands are lead compounds for potential application in anticancer therapy [16,17]. Ru(II)-polypirydyl compounds most often contain 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), 2,2′:6′,2″-terpyridine (tpy) ligands or their derivatives [17]. Anticancer activities of numerous Ru(II) polypyridyl compounds have been examined [18], but their interactions with proteins have seldom been studied. Terpyridine ligand is of special interest as it forms highly stable complexes with transition metals [19] and may stabilize G-quadruplex structures in DNA [20].
It is generally thought that Ru(II) coordination compounds exert their antitumour activity by causing apoptosis, through one or more of the following pathways: mitochondria-mediated pathway, autophagy pathway or triggering ROS (reactive oxygen species)-mediated apoptosis [17]. Mechanisms of biological actions of Ru polypyridyl complexes in tumour cells were rarely examined, apart from in vitro testing of their cytotoxicity.
We have earlier synthesized a series of water-soluble ruthenium(II) terpyridine compounds, with the general formula mer-[Ru(L3)(N-N)X][Y]n, in which L3 is either tpy or Cl-tpy; X is Cl or dmso-S; N-N is en (ethylenediamine), dach (1,2-diaminocyclohexane) or bpy; Y is Cl, PF6 or CF3SO3, and n = 1 or 2, depending on the nature of X [21]. They bind strongly to calf thymus (CT) DNA, both covalently and non-covalently [22]. We described a moderate-to-strong binding of the complexes containing en to HSA and much lower affinity for human serum transferrin, whereas bpy-containing compound bound weakly to these two serum metal transporters [23]. The target amino acid sequences on both bovine serum albumin (BSA) and HSA were identified [24,25].
We have recently synthesized a series of ruthenium(II) tpy compounds with the additional functional group on the 4′-position of tpy, whereas other ligands around the Ru(II) centre remained unaltered: 4′-chlorophenyl-tpy (4′-Cl-Ph-tpy) instead of 4′-Cl-tpy. Their general formula is mer-[Ru(Cl-Ph-tpy)(N-N)Cl]Cl, where N-N = en (complex 1), dach (complex 2) or bpy (complex 3) [26]. These complexes appeared to be promising DNA intercalators with a significant cytotoxic activity [26]. We, thus, focused on their interactions with DNA, which we studied utilising linear dichroism (LD) and molecular docking. Secondly, we aimed to evaluate the binding parameters to HSA and to predict their binding sites on HSA. Finally, we tested their effect on the cellular adhesion, using two cell lines: extravillous trophoblast HTR-8/SVneo (normal) and JAr choriocarcinoma (tumour).
Section snippets
Chemicals and solutions
The compounds [Ru(Cl-Ph-tpy)(en)Cl]Cl (1), [Ru(Cl-Ph-tpy)(dach)Cl]Cl (2) and [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) were synthesized as described [26]. The chemical structures of their complex ions are displayed in Fig. 1. Microanalysis, ultraviolet-visible (UV–Vis) and 1H nuclear magnetic resonance (NMR) spectroscopy were used to check their purity and the data and spectra agreed well with those already reported [26]. Their molar masses are: 575.88 (1), 629.97 (2) and 671.97 g mol−1 (3). Stock solutions
MALDI TOF MS of the complexes
Apart from the elemental analysis and UV–Vis, NMR and infrared (IR) spectroscopies of 1, 2 and 3 [26], we checked their purity and stability by MALDI TOF MS, which is a quick tool for the analysis of transition metal complexes [24]. The mass spectra of the complex ions 1, 2 and 3 are shown in Fig. S2 and the assigned peaks are summarised in Table S1. The isotopic masses of the molecular ions are: [Ru(Cl-Ph-tpy)(en)Cl]+ 540.029; [Ru(Cl-Ph-tpy)(dach)Cl]+ 594.076 and [Ru(Cl-Ph-tpy)(bpy)Cl]+
Conclusions
The complexes 1 and 2 bound to HSA via medium-strength interactions (Kb fell within the range 104–105 M−1), whereas 3 bound weakly. It is highly probable that, after their intravenous application, concentrations of free 1 and 2 in human plasma would be relatively low. It was suggested that interactions of complexes with human and bovine albumin were different. We have also shown that Ru(II) Cl-phenyl-terpyridine complexes 1–3 used mostly π-π stacking and vdW interactions to bind to HSA, with
Abbreviations
- cisplatin
cis-diamminedichloroplatinum(II)
- carboplatin
cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)
- oxaliplatin
(1,2-diaminocyclohexane)oxalatoplatinum(II)
- NAMI-A
(ImH)[trans-RuCl4(dmso-S)(Im)]
- Im
imidazole
- dmso
dimethylsulfoxide
- KP1019
(IndH)[trans-RuCl4(Ind)2]
- KP1339
Na[trans-RuCl4(Ind)2]
- Ind
indazole
- HSA
human serum albumin
- bpy
2,2′-bipyridine
- phen
1,10-phenanthroline
- tpy
2,2′:6′,2′′-terpyridine
- ROS
reactive oxygen species
- en
1,2-diaminoethane
- dach
1,2-diaminocyclohexane
- CT DNA
calf thymus DNA
- BSA
bovine serum
Acknowledgements
The authors wish to thank dr Marija Matković (Department for Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia) for her help with the LD measurements.
Author contributions
Conceived and designed the experiments: RM, AR, IC. All authors performed the experiments and analyzed obtained data. Contributed reagents/materials/analysis tools: all authors. Wrote the paper: RM. All authors reviewed the manuscript critically.
Funding
This work was supported by Ministry of Education, Science and Technological Development of Republic of Serbia; Ministry of Science, Education and Sport of Croatia [grant No. 098-0982914-2918]; and FP7-REGPOT-2012-2013-1 (grant No. 316289-InnoMol). None of the listed funding sources had any role in the study design, collection, analysis and interpretation of data; in the writing of the report or in the decision to submit the article for publication.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (65)
- et al.
Cisplatin in cancer therapy: molecular mechanisms of action
Eur. J. Pharmacol.
(2014) - et al.
Organometallic ruthenium-based antitumor compounds with novel modes of action
J. Organomet. Chem.
(2011) - et al.
Chemistry and reactivity of ruthenium(II) complexes: DNA/protein binding mode and anticancer activity are related to the complex structure
Coord. Chem. Rev.
(2019) - et al.
Terpyridine-metal complexes: applications in catalysis and supramolecular chemistry
Coord. Coord. Chem. Rev.
(2019) - et al.
Highly water-soluble ruthenium(II) terpyridine coordination compounds form stable adducts with blood-borne metal-transporting proteins
Arab. J. Chem.
(2018) - et al.
Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin
J. Inorg. Biochem.
(2016) - et al.
New 4′-(4-chlorophenyl)-2, 2′:6′,2′′-terpyridine ruthenium(II) complexes: synthesis, characterization, interaction with DNA/BSA and cytotoxicity studies
J. Inorg. Biochem.
(2017) - et al.
Establishment and characterization of first trimester human trophoblast cells with extended lifespan
Exp. Cell Res.
(1993) - et al.
Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies
Biophys. J.
(1980) - et al.
Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry
J. Am. Soc. Mass Spectrom.
(2004)
Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: a combined spectroscopic and docking simulation approach
Bioorg. Chem.
Quantitation of species differences in albumin–ligand interactions for bovine, human and rat serum albumins using fluorescence spectroscopy: a test case with some Sudlow’s site I ligands
J. Luminisc.
Multiple spectroscopic and theoretical investigation of meso-tetra-(4-pyridyl)porphyrin-ruthenium(II) complexes in HSA-binding studies. Effect of Zn (II) in protein binding
J. Mol. Liquids
Structural basis and anticancer properties of ruthenium-based drug complexed with human serum albumin
Eur. J. Med. Chem.
Noncovalent interactions with DNA: an overview
Mutation Res
Non-intercalative binding mode of bridged binuclear chiral Ru(II) complexes to native duplex DNA
J. Inorg. Biochem.
Synthesis, solvatochromism, photochemistry, DNA binding, photocleavage, cytotoxicity and molecular docking studies of a ruthenium(II) complex bearing photoactive subunit
J. Photochem. Photobiol: Chem.
Synthesis, cytotoxic activity and DNA-binding properties of copper (II) complexes with terpyridine
Polyhedron
Spectroscopic and viscometric determination of DNA-binding modes of some bioactive dibenzodioxins and phenazines
Biochem. Biophys. Rep.
The structural basis of dynamic cell adhesion: heads, tails, and allostery
Exp. Cell Res.
Integrin signaling in control of tumor growth and progression
Int. J. Biochem. Cell Biol.
Actin-dependent tumour cell adhesion after short-term exposure to the antimetastasis ruthenium complex NAMI-A
Eur. J. Cancer
Inhibition of adhesion, migration and of α5β1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A
J. Inorg. Biochem.
Metal-based drugs
Sci. Prog.
Bioinorganic Medicinal Chemistry
Platinum anticancer drugs and photochemotherapeutic agents: recent advances and future developments
Sci. Prog.
Ru(II) compounds: next generation anticancer metallotherapeutics?
J. Med. Chem.
The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials
Chem. Soc. Rev.
NAMI-A and KP1019/1339, two iconic ruthenium anticancer drug candidates face-to-face: a case story in medicinal inorganic chemistry
Molecules
The path for metal complexes to a DNA target
Chem. Comm.
Designing ruthenium anticancer drugs: what have we learnt from the key drug candidates?
Inorganics
Antitumour metal compounds: more than theme and variations
Dalton Trans.
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