Anti-adhesive action of novel ruthenium(II) chlorophenyl terpyridine complexes with a high affinity for double-stranded DNA: in vitro and in silico

https://doi.org/10.1016/j.jinorgbio.2020.111090Get rights and content

Highlights

  • Ru(II) terpyridine coordination complexes are potential anti-tumour compounds.

  • Confident assessment of metallodrugs-to-DNA binding mode through linear dichroism.

  • Different binding of Ru(II) terpyridine complexes to human and bovine serum albumin.

  • Novel druggable site on human serum albumin for Ru(II) terpyridine complexes.

  • DNA accommodates Ru(II) terpyridines using an intercalation through minor groove.

Abstract

Interactions of three Ru(II) chlorophenyl terpyridine complexes: [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) (Cl-Ph-tpy = 4′-(4-chlorophenyl)-2,2′:6′,2′′-terpyridine, en = 1,2-diaminoethane, dach = 1,2-diaminocyclohexane, bpy = 2,2′-bipyridine) with human serum albumin (HSA), calf thymus DNA and a double-helical oligonucleotide d(CGCGAATTCGCG)2 (1BNA) were examined. Fluorescence emission studies were used to assess the interactions of complexes with HSA, which were of moderate strength for 1 and 2. Molecular docking allowed us to predict mostly π-π stacking and van der Waals interactions between the complexes and the protein. We suggest that the complexes bind to a novel site on HSA, which is different from its druggable sites I, II or III. We suggest a partial intercalation of complexes through the minor groove as a possible mode of interaction with double-helical DNA. Finally, when applied to normal extravillous cell line HTR8/SVneo and JAr choriocarcinoma cell line, complexes 1 and 2 exerted anti-adhesive properties at very low doses, whereas complex 3 had a negligible effect. The obtained results are completion of our studies of Ru(II) terpyridyl complexes that carry N-N ancillary ligands. We suggest a new research direction towards studying the cellular effects of Ru(II) polypyridyl compounds.

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.

Unlabelled Image
  1. Download : Download high-res image (356KB)
  2. Download : Download full-size image

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 13 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)

  • R. Joshi et al.

    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.

    (2017)
  • M. Poór et al.

    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.

    (2014)
  • O.A. Chaves et al.

    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

    (2019)
  • Y. Zhang et al.

    Structural basis and anticancer properties of ruthenium-based drug complexed with human serum albumin

    Eur. J. Med. Chem.

    (2014)
  • L. Strekowski et al.

    Noncovalent interactions with DNA: an overview

    Mutation Res

    (2007)
  • N. Chitrapriya et al.

    Non-intercalative binding mode of bridged binuclear chiral Ru(II) complexes to native duplex DNA

    J. Inorg. Biochem.

    (2011)
  • M. Mariappan et al.

    Synthesis, solvatochromism, photochemistry, DNA binding, photocleavage, cytotoxicity and molecular docking studies of a ruthenium(II) complex bearing photoactive subunit

    J. Photochem. Photobiol: Chem.

    (2018)
  • B.Đ. Glišić et al.

    Synthesis, cytotoxic activity and DNA-binding properties of copper (II) complexes with terpyridine

    Polyhedron

    (2018)
  • A.A. Phadte et al.

    Spectroscopic and viscometric determination of DNA-binding modes of some bioactive dibenzodioxins and phenazines

    Biochem. Biophys. Rep.

    (2019)
  • R.C. Liddington et al.

    The structural basis of dynamic cell adhesion: heads, tails, and allostery

    Exp. Cell Res.

    (2000)
  • J. Xiong et al.

    Integrin signaling in control of tumor growth and progression

    Int. J. Biochem. Cell Biol.

    (2013)
  • G. Sava et al.

    Actin-dependent tumour cell adhesion after short-term exposure to the antimetastasis ruthenium complex NAMI-A

    Eur. J. Cancer

    (2004)
  • C. Pelillo et al.

    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.

    (2016)
  • R.J. McQuitty

    Metal-based drugs

    Sci. Prog.

    (2014)
  • E. Alessio

    Bioinorganic Medicinal Chemistry

    (2011)
  • E. Shaili

    Platinum anticancer drugs and photochemotherapeutic agents: recent advances and future developments

    Sci. Prog.

    (2014)
  • S. Thota et al.

    Ru(II) compounds: next generation anticancer metallotherapeutics?

    J. Med. Chem.

    (2018)
  • L. Zheng et al.

    The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials

    Chem. Soc. Rev.

    (2017)
  • E. Alessio et al.

    NAMI-A and KP1019/1339, two iconic ruthenium anticancer drug candidates face-to-face: a case story in medicinal inorganic chemistry

    Molecules

    (2019)
  • A.C. Komor et al.

    The path for metal complexes to a DNA target

    Chem. Comm.

    (2013)
  • J.P.C. Coverdale et al.

    Designing ruthenium anticancer drugs: what have we learnt from the key drug candidates?

    Inorganics

    (2019)
  • M.A. Jakupec et al.

    Antitumour metal compounds: more than theme and variations

    Dalton Trans.

    (2008)
  • Cited by (6)

    • Orthopalladated tetralone oxime compounds bearing tertiary phosphines: Synthesis, structure, biological and in silico studies

      2022, Journal of Organometallic Chemistry
      Citation Excerpt :

      Compound 3 has been selected as representative in the HSA/DNA binding interactions assays. DNA represents one of the most studied therapeutic target in the search of new potential antitumor agents [74]. Several spectroscopic techniques are employed to evaluate the DNA-complex interaction such as circular dichroism (CD) and UV–vis titration [45,46,75].

    View full text