Lighting up metallohelices: from DNA binders to chemotherapy and photodynamic therapy

Xuezhao Li ab, Zhuolin Shi a, Jinguo Wu a, Jinlong Wu a, Cheng He *a, Xiaorou Hao a and Chunying Duan *ab
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, 116024, China. E-mail: hecheng@dlut.edu.cn; cyduan@dlut.edu.cn
bZhang Dayu College of Chemistry, Dalian University of Technology, Dalian 116024, China

Received 25th March 2020 , Accepted 9th June 2020

First published on 11th June 2020


The design of novel agents that specifically target DNA and interrupt its normal biological processes is an attractive goal in drug design. Among the promising metallodrugs, metal-directed self-assembled metallohelices with defined three-dimensional stereochemical structures display unique structure-inherent and unprecedented noncovalent targeting abilities towards DNA, resulting in excellent anticancer or antibiotic activities. A newly burgeoning hotspot is focusing on lighting them up by embedding luminescent metal ions as the vertices. The photoactive metallohelices that combine strong interactions toward DNA targets and efficient 1O2 quantum yield may provide new motivation in diagnostic and photodynamic therapy (PDT) areas. This perspective focuses on research progress on metallohelices as DNA binders and chemotherapeutic agents, and highlights recent advances in fabricating luminescent examples for PDT. The relative assembly strategies are also discussed and compared. Finally, perspectives on the future development of the lit-up metallohelices are presented.


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Xuezhao Li

Xuezhao Li received his PhD degree in 2016 from Dalian University of Technology (DUT). Then he worked with Prof. Chunying Duan as a postdoctoral researcher at State Key Laboratory of Fine Chemicals, DUT. He joined DLUT as an Associate Professor from 2020. His research interests focus on iridium complex-based supramolecular chemistry, photocatalysis and cancer therapy.

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Zhuolin Shi

Zhuolin Shi received her MS degrees from Henan Key Laboratory of Polyoxometalate Chemistry, Henan University. In 2019, she began to pursue her PhD degree under the supervision of Prof. Cheng He at State Key Laboratory of Fine Chemicals, Dalian University of Technology. Her research interests now focus on polyoxometalate-based metal organic cages/frameworks and relevant catalytic properties.

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Jinguo Wu

Jinguo Wu obtained his BS degree from Dalian University of Technology in 2014. Now, he is a PhD candidate under the supervision of Prof. Cheng He in State Key Laboratory of Fine Chemicals, Dalian University of Technology. His research interests focus on the construction of functional Metal–Organic Cages for photocatalysis and recognition.

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Cheng He

Cheng He received his PhD degree in 2000 from Nanjing University. After postdoctoral studies at Peking University and the Pohang University of Technology, he was awarded the Alexander von Humboldt fellowship and then worked with Prof. Herbert W. Roesky as a postdoctoral researcher at Goettingen University. He has worked at the Dalian University of Technology as a Professor since 2006. His research interest is in supramolecular coordination chemistry.

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Chunying Duan

Chunying Duan completed his PhD in 1992 at Nanjing University. Since 2006, he has worked at the DaLian University of Technology as a professor. His research interests cover aspects of coordination chemistry, supramolecular chemistry, molecular sensors, chiral materials, and enzyme mimics catalysis.


Introduction

DNA, with a double helix structure, encodes and regulates most aspects of life, and thus undoubtedly represents one of the main targets of many drugs.1 The interactions between small molecules and DNA can cause DNA damage, blocking the division of cells, and resulting in cell death.2 As one of the world's deadliest diseases, cancer continues to increase largely every year.3 The design of new effective drugs that target DNA combined with seeking successful approaches for cancer treatment has drawn substantial attention. Different from purely organic drugs which predominantly adopted linear (1D) or planar (2D) shape, the use of metallodrugs with diverse 3D topologies demonstrated a vital role in targeting DNA, which enhances the chemotherapeutic effect.4 Inspired by the clinical success of well-known platinum-based anticancer drugs including cisplatin,5 carboplatin6 and oxaliplatin,7 the exploration of new metallodrugs with distinct structural and mechanistic profiles able to reduce systemic effects, and even effective for drug-resistant cancer cell lines are highly desired.8

From further explorations beyond the versatile mononuclear metallodrugs,9 the results from binuclear metallodrugs where the metal centres are linked by an alkyl chain are also impressive. Their unique interactions with DNA could induce long-range inter- and intra-strand DNA cross-linking, displaying high activity and overcoming the problems of intrinsic cisplatin resistance.10 In particular, as promising mimics of protein α-helices,11 binuclear metallohelices with defined three-dimensional stereochemical structures display more sophisticated structural superiority.12 The metal–ligand coordination self-assembled double- or triple-stranded helical structural motifs with novel biochemical and photophysical properties can be rationally tailored in different shapes, sizes and enantiomers by introducing different metal ions and ligands.13 Their more sophisticated 3D structural skeletons impart structure-inherent and unprecedented noncovalent DNA-binding properties.14 These novel molecular-level interactions are distinct from the most effective clinical platinum metallodrugs that are believed to bind covalently to DNA through irreversibly inserting into DNA double-strands.15 Some metallohelices have displayed profound effects on cytotoxicity towards cancer cells or bacterial strains. In addition, with selection of luminescent d6-transition-metal ions (RuII/IrIII) as the metal vertices, the obtained metallohelices with photoactivities are able to generate singlet oxygen (1O2) under light irradiation. Therefore, these metallohelices can be used as photosensitizers in PDT. In fact, the clinically approved PDT procedure with minimally invasive characteristics presents a burgeoning approach for cancer treatment.16 Considering that metallohelices also bind with DNA, these PDT agents would result in an enhanced PDT effect via damaging DNA directly.

This perspective focuses on 3D metallohelice-based drugs, specifically bimetallic double- and triple-stranded metallohelices with kinetically labile metal ions (CuI/FeII/NiII/ZnII) and luminescent inert metal ions (RuII/IrIII) as the vertices. Discussion and comparison of the fundamentals in terms of the synthesis methodologies are presented first. Then an update on the state-of-the-art literature in this fascinating research area covering from DNA binders to anticancer chemotherapy and phototherapy is provided. Emphasis is given in particular to photoactive metallohelices as DNA recognition domains and applications in PDT. Some remaining challenges, especially of luminescent ones for further applications in phototherapy areas are discussed.

Metallohelices: general principles

In 1987, Lehn firstly introduced the term metallohelices to describe a bimetallic helical double-stranded complex, with two oligobipyridine ligands wrapped around two tetrahedral CuI ions.17 As shown in Fig. 1A, in principle, the mechanically coupled two tetrahedral metal centres with right-handed (+, P) or left-handed (−, M) configuration could result in either chiral double-stranded helicates (MM or PP) or an achiral meso-helicate (MP).18 Similarly, triple-stranded helicates with M2L3 stoichiometry can be formed by bridging three bis-bidentate ligands bound to two octahedral metal centres with Λ or Δ chirality, thereby generating the homochiral (ΛΛ or ΔΔ) helicate or the heterochiral (ΛΔ) complex with an achiral meso structure (Fig. 1B).19
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Fig. 1 Helical and chiral properties of bimetallic (A) double-stranded helicate and (B) triple-stranded helicate with C2 symmetrical ligand strands.

The predictive synthesis of chemical robust metallohelices with desired configurations and properties is prerequisite for further biological applications. The well-known classical metal-directed self-assembly approach commonly relies on the coordination process between pre-designed ligands with suitable labile metal ions (Fig. 2A). Many examples of short or rigid linkers decorated with 2,2′-bipyridines,20 catechols,21 or pyridylmethanimines22 chelate sites are well-known. However, the often complicated and inefficient organic synthesis procedure of ligands is an obstacle for further assembly. Alternatively, a simpler approach named the “subcomponent self-assembly” strategy was developed by Nitschke et al. (Fig. 2B).23 This unique technique allows the fabrication of specific architectures from simple building blocks (amines and aldehydes) through the simultaneous formation of dynamic covalent bonds (C[double bond, length as m-dash]N) around metal templates (M ← N). This optimized strategy has resulted in impressive supramolecular assemblies; however, the reversibly formed intra-ligand and metal–ligand covalent and coordinative bonds render them sensitive in the complicated physiological environments.24 To overcome the problems mentioned above, an updated programmed stepwise hierarchical assembly approach25 was carried out in our recently reported work (Fig. 2C).26 By first pre-synthesizing kinetically locked inert metal vertices with the required connective geometry and/or function, and then connecting them with suitable linkers by using dynamic imine coupling chemistry, chemically robust metallohelices were controllably obtained by the subsequent reduction of the imine linkages to amine linkages. The pre-fixed stereochemical structure of the inert metal vertices not only allows for an easier synthesis but also results in a stabilizer input channel to lock the desired structure configurations. Importantly, the incorporation of luminescent IrIII cores into the metallohelices by using amine-linkages ensures efficient phosphorescence induction.


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Fig. 2 Self-assembly of triple-stranded metallohelices (only exemplified with the ΛΛ-enantiomer). (A) Classical coordination-driven self-assembly approach between pre-designed ligands and suitable labile metal ions. (B) Subcomponent self-assembly approach combining dynamic covalent and coordinative bonds. (C) Stepwise hierarchical assembly approach employing kinetically locked inert organometallic metal vertices.

Metallohelices as DNA binders

The first exploration of interaction between metallohelices and DNA was described by Lehn's group.27 Their double-stranded polynuclear CuI helicates contained three oligo-bipyridine ligand strands with flexible alkyl-ether spacers, which were confirmed to bind strongly to DNA by spectroscopic, DNA-melting, and electrophoretic-mobility measurements. The binding affinity was proved to depend on the size of the helicates used. When increasing the size of the helicates, the number of metal centres and consequently the electrostatic contribution to binding increased. The author stated that groove binding might be present, but there was a lack of further experimental evidence to determine which groove.

Following this inspirational work, fantastic results in this field sprung up through Hannon and co-workers’ contribution.28 To obtain a large synthetic structure that closely mimics the dimensions of protein DNA recognition motifs, they constructed a novel tetracationic triple-strand helicate (also named as cylinder) [Fe2L3]4+via the classical self-assembly approach between bis-(pyridylimine) ligands and FeII ions. X-ray crystallographic analysis revealed that [Fe2L3]4+ possesses ∼2 nm length and ∼1 nm diameter (Fig. 3A).28b Such dimensions are close to those of the α-helical DNA recognition unit of zinc fingers, but too large to fit into the minor groove of DNA. Circular dichroism (CD), linear dichroism (LD) and atomic force microscope (AFM) studies confirmed that the [Fe2L3]4+ cylinder bound strongly to DNA with a binding constant in excess of 107 M−1; in addition, it also induced dramatic intramolecular DNA coiling, which is unexpected with conventional small synthetic DNA binders reported before (Fig. 3B). Further exploration of intermolecular NOEs between the [Fe2L3]4+ and DNA confirmed that the cylinder noncovalently binds to DNA in the major groove.


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Fig. 3 (A) Self-assembly of helicate [Fe2L3]4+. (B) AFM images showing the dramatic intra-molecular coiling induced by [Fe2L3]4+. Reproduced with permission from ref. 28b. Copyright 2006, Wiley-VCH. (C) Three-dimensional structure of the [Fe2L3]4+-DNA in different directions, showing [Fe2L3]4+ binding to the central cavity of a DNA three-way junction. Reproduced with permission from ref. 31a. Copyright 2006, Wiley-VCH.

Chiral recognition of DNA has been considered of great importance for drug design.29 It is noted that the synthetic [Fe2L3]4+ cylinder contains a pair of enantiopure M-Fe and P-Fe isomers; interestingly, the previous NMR data collected for the racemate confirmed the major groove binding mode, but only the M enantiomer was present in the refined structure.28b Subsequently, the relationship between the helicity of [Fe2L3]4+ and binding mode with DNA were investigated by Hannon and co-workers.30 The enantiopure M-Fe and P-Fe were obtained separately after chiral resolution by a cellulose column. A series of experiments including CD, LD and AFM demonstrated that the M-Fe preferentially bound the major groove and strongly induced coiling of DNA; while P-Fe adopted a more ambiguous binding mode and the interaction of P-Fe with DNA resulted in less pronounced coiling effects. The investigation of the helicity effect of metallohelices to bind DNA is particularly useful, which may guide further chiral drug design.

Moreover, the specific recognition abilities of the [Fe2L3]4+ cylinder towards various unusual DNA structures were also observed.31 When [Fe2L3]4+ was crystallised with a DNA palindromic hexanucleotide (5′-d-(CGTACG)-3′), a novel molecular recognition not of duplex DNA but rather of a three-way junction was observed by Hannon and Coll.31a As shown in Fig. 3C, single-crystal X-ray analysis unambiguously confirmed that the DNA arranged into a three-way Y-shaped junction, and [Fe2L3]4+ fitted perfectly into the central hydrophobic hollow cavity. The highly positively charged [Fe2L3]4+ was considered to facilitate the interactions with the negatively charged DNA. A more-detailed analysis of the structure demonstrated that the face-to-face π-stacking intercalation-type interactions, C–H⋯X type H-bond interactions and minor-groove sandwiching interactions between [Fe2L3]4+ and strands of DNA also played important roles. It is noteworthy that this work is the only structurally characterized interaction of helicates with any biomolecule, thus allowing the details of this new mode of DNA recognition to be explored. Such an unprecedented binding mode is quite different from previously identified intercalation, groove-binding, and DNA alkylation or metalation modes since the 1960s.32 This finding opens a route for the development of anti-DNA therapeutic agents with completely novel characteristics.

They have also controlled the helicity of [Fe2L3]4+-based cylinders with external chiral groups and shown the effect of that on 3-way junction binding.33 The enantiopure helicates P-[Fe2(L-Arg)3]10+ and M-[Fe2(D-Arg)3]10+ were controllably constructed via the subcomponent self-assembly approach in a one pot reaction of the respective arginine-functionalised pyridine-carboxaldehyde with 1,4-diamino-phenylmethane and FeII ions (Fig. 4). The grafted chiral arginine units at the extremities of the bis-pyridylimine ligands control the helicity of the cylinders absolutely. These two chiral cylinders all promoted the formation of the DNA 3-way junctions over the parent [Fe2L3]4+, and the M-[Fe2(D-Arg)3]10+ was slightly more effective than the P-[Fe2(L-Arg)3]10+. Similar trends of their cytotoxicity against A2780 ovarian cancer cells were also observed. It is noted that the IC50 of the M-[Fe2(D-Arg)3]10+ cylinder was very similar to that of cisplatin, despite the cylinders’ very different and non-covalent mode of DNA recognition.


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Fig. 4 Self-assembly of helicates P-[Fe2(L-Arg)3]10+ and M-[Fe2(D-Arg)3]10+. Reproduced with permission from ref. 28. Copyright 2011, the Royal Society of Chemistry.

Bulged DNA structures are suspected to be relevant to numerous diseases.34 Thus, the development of novel agents capable of targeting such noncanonical DNA structures is of general biological significance. They not only can be used as probes for studying their role in nucleic acid function, but could also even possess significant therapeutic potential.35 Hannon and co-workers demonstrated, for the first time, that the supramolecular helicates [Fe2L3]4+ are capable of recognizing and stabilizing bulged DNA.36 The enantiomers M-Fe and P-Fe both bound to DNA bulges containing at least two unpaired nucleotides. In addition, these helicates also showed considerably enhanced affinity for the duplexes containing unpaired pyrimidines in the bulge and/or pyrimidines flanking the bulge on both its sides.

Enlightening works of metallohelices about [Fe2L3]4+ and [Ni2L3]4+ that selectively bind to human telomeric G-quadruplex (G4) DNA based on their chirality were also studied by X. Qu and co-workers.37 The enantiopure isomers were obtained separately after chiral resolution of the racemic mixture. Structures and CD spectra of the two pairs of enantiomers, M and P, are shown in Fig. 5A–D. Both of them exhibited chiral selective binding with human telomeric G4, with the right-handed helicates selectively stabilizing antiparallel G4, while the left-handed helicates did not (Fig. 5E). These results provide new insights into the development of chiral anticancer metallohelices for targeting G4 DNA.


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Fig. 5 Structures of the M-enantiomer (left) and P-enantiomer (right) of (A) [Ni2L3]4+ and (B) [Fe2L3]4+, respectively. (C and D) CD spectra of the corresponding enantiomers. (E) Representative illustration of chiral supramolecular helicate selective recognition of human telomeric G-quadruplex DNA. Reproduced with permission from ref. 37. Copyright 2008, Oxford University Press.

Interestingly, subsequent work from Qu's group by using the same enantiopure NiII helicates to bind higher-order G4 structures resulted in inverse selectivity over the single G4.38 It is evidenced that the P-Ni enantiomer preferentially binds to the end of the monomeric G4 by external stacking, but M-Ni showed no specific binding. However, new findings demonstrated that M-Ni can bind to higher-order G4s (dimeric G4s, G2T1) 200-fold better than to monomeric G4. The specific binding to high-order G2T1 over G1 was confirmed by DNA UV melting studies. P-Ni showed negligible influence on the melting temperature (Tm) of G1 (Fig. 6A), whereas it significantly increased the Tm of G2T1 (Fig. 6B). Titrating fluorescent TMR-labeled G2T1 by M-Ni (Fig. 6C) and isothermal titration calorimetry (ITC, Fig. 6D) revealed a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 binding ratio for Ni–M and G2T1. The longer TTA linkers between two G4 subunits weakened the stabilization effect of M-Ni on dimeric G4s suggested that M-Ni simultaneously interacted with the two G-quadruplex subunits of G2T1, stacking on the end G-quartets of the two G-quadruplex motifs. In this sense, dimeric G-quadruplex units present as novel potential targets for the design of metallohelices targeting higher-order G-quadruplexes.


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Fig. 6 (A) Preferred binding of helicate M-Ni to higher-order G4s (dimeric G4s) over a monomeric G4 unit. UV melting profiles of (B) G1 and (C) G2T1 in the absence and presence of M-Ni. (D) Fluorescence emission spectra of TMR-G2T1 titrated by Ni–M. Inset: Job's plot for complexation of M-Ni with TMR-G2T1; (E) representative ITC profile for the titration of M-Ni into a solution of G2T1, and (inset) the corresponding normalized heat signals versus mole ratio. Reproduced with permission from ref. 38. Copyright 2017, American Chemical Society.

Metallohelices for chemotherapy

The unprecedented noncovalent interactions between metallohelices and DNA provide a promising approach to design effective agents against cancer cells or bacterial strains. Relative studies in these two areas were first explored by Hannon's group by using their [Fe2L3]4+ cylinder.39 Although cisplatin showed higher efficiency for killing nontumor MRC5 and several tumor cell lines (HBL100, SKOV3, T47D, and HL-60), it could lead to DNA damage and cannot be repaired. It is worth noting that the absence of genotoxicity and mutagenicity of the [Fe2L3]4+ cylinder may represent a significant step toward therapeutic advancement. The authors have shown for the first time, that such a noncovalent and nongenotoxic interaction mode of a supramolecular metallohelix with DNA is linked to an arrest of cells in the G0/G1 phase of the cell cycle and subsequent death through apoptosis. They also provided the first evidence of the synthetic [Fe2L3]4+ cylinder with antimicrobial activity against the Bacillus subtilis and Escherichia coli.40 The killing process was in an extremely fast manner by reaching and binding to its intended DNA target. The metallohelix [Fe2L3]4+ acted as a kind of novel antibiotic compared to other known antimicrobial molecules in structure and mode(s) of action, and is anticipated be effective against bacterial strains currently resistant to other antibiotics.

To investigate whether the methods routinely employed for toxicity testing can also be suitable to explore the activity of such a non-covalent DNA-binding metallo-drug [Fe2L3]4+ in a standard way, Hannon and co-workers examined both the effect of the incubation time and incubation volume on the IC50.41 They discovered that during 48 and 72 h both cisplatin and [Fe2L3]4+ had similar IC50 values, but over a longer time the cylinder showed twice the IC50 value of cisplatin in the end. Besides, the IC50 value decreased dramatically when the incubation volume of [Fe2L3]4+ was increased, but the IC50 value decreases relatively gradually with cisplatin under the same conditions. Based on the above results, the author inferred that the current methods employed to assess the toxicity of non-covalent binding drugs may not be appropriate, unless the incubation time and volume are carefully considered.

Recently, Scott et al. developed a straightforward approach to form enantiopure bimetallic helicate architectures known as “flexicates” by a subcomponent self-assembly process.42 The absolute configurations of the individual metal centres were fixed utilizing amino acid derivatives as the source of chirality. This represented a very different chirality induction process that introduces a greater flexibility in the design of the metallo-helical compounds, where various types of end groups and bridges can be used – not just rigid ones (Fig. 7).43 Importantly, they were readily water-soluble and remarkably stable in a variety of media. CD, and LD studies showed that the FeII triple-helicate binds DNA in the major groove. In particular, the iron-based flexicate complex with both iron centres in the Δ conformation showed a significant interaction with DNA and displayed good antibiotic activity.


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Fig. 7 A straightforward approach to form enantiopure flexicates by a sub-component self-assembly process and the schematic showing the flexicate sitting in the major groove of B-DNA. Reproduced with permission from ref. 42 and 43. Copyright 2012 Springer Nature; 2019 the Royal Society of Chemistry.

The following work from the same group demonstrated that these kinds of flexicates also have the ability to recognize and stabilize bulged DNA and RNA.44 Both enantiomers enhanced the thermal stability of DNA bulges containing at least one unpaired nucleotide and the stabilizing effects of the bound flexicates were raised with the increasing number of unpaired nucleotides. Specifically, the Λ-enantiomer was more efficient in stabilizing DNA as well as RNA bulges than the Δ-enantiomer. The author also indicated that only one dominant binding site existed for the flexicates on the DNA and RNA bulges and that the flexicates bound directly to the bulge or in its close proximity. The unique recognition phenomenon toward DNA and RNA bulges by enantiopure isomers may broaden their further application in the field of biology.

Another achievement for fabricating enantiomerically pure asymmetric helicates which formed from directional chiral pyridylimine/bipyridine ligands and FeII or ZnII ions against a range of even cisplatin-resistant cancer cell lines was also reported in the same group (Fig. 8).45 The anti-parallel head-to-head-to-tail ‘triplex’ strand arrangement created a similar amphipathic functional topology. These asymmetric helicates displayed high, structure-dependent toxicity to the human colon carcinoma cell-line HCT116 p53++, resulting in dramatic changes in the cell cycle without DNA damage. The author demonstrated that the asymmetric helicate displayed lower toxicity to human breast adenocarcinoma cells (MDA-MB-468) and, most remarkably, they showed no significant toxicity to the bacteria methicillin-resistant Staphylococcus aureus and Escherichia coli. Following these promising results, post-assembly modification of the enantiopure asymmetric helicates by click reactions was achieved recently.46 By these means, the in vitro anticancer activity and the selectivity of a triplex metallohelix FeII system was dramatically improved.


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Fig. 8 Sub-component self-assembly from versatile components of a wide range of functionalized helices in which the strands are arranged head-to-head-to-tail. Reproduced with permission from ref. 45. Copyright 2014, Springer Nature.

Cancer stem cells (CSCs) are responsible for drug resistance, metastasis and recurrence of cancers.47 However, the exploration of effective agents to eliminate CSCs still remains a great challenge.48 X. Qu and co-workers recently investigated the effects of chiral triple-strands helicates (P-Ni and M-Ni) on breast CSCs, and found that P-Ni, rather than M-Ni, preferentially inhibits cell growth in breast CSCs compared to the bulk cancer cells, and importantly displayed little effect on normal cells. Further studies indicated that P-Ni could repress CSC properties and induce apoptosis in breast CSCs.49 Studies in detail revealed that telomere uncapping with the delocalization of TRF2 and POT1 from telomeres, telomere DNA damage and the degradation of the 3′-overhang result in the apoptosis of breast CSCs (Fig. 9A). Moreover, P-Ni showed the ability to reduce tumorigenesis of breast CSCs in vivo. The number and size of the tumors in the P-Ni-treated group were less than those in PBS- and M-Ni-treated groups (Fig. 9B and C). Furthermore, there was no tumor formation in the mice seeded with 1 × 105 cells after treatment with P-Ni. In contrast, significant tumor formation was observed in control and M-Ni-treated groups injected with the same number of cells. The first example that chiral helicates showed contrasting enantioselectivity on eradicating CSCs would shed light on the application of chiral agents in anti-CSC therapy.


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Fig. 9 (A) Schematic illustration of telomerase activity inhibition, telomere DNA damage, breast CSC property reduction, apoptosis and tumorigenesis inhibition induced by P-Ni in breast CSCs; (B) photographs of the dissected tumors; (C) tumor growth curves of tumor-bearing mice after P-Ni or M-Ni treatment. Reproduced with permission from ref. 49. Copyright 2017, American Chemical Society.

To combine the striking DNA binding features of the metallohelices with the fact that ruthenium compounds represent a new and promising class of anticancer drugs,50,51 an unsaturated binuclear RuII double-helicate [Ru2L2]4+ in both the trans/transRu1 and the cis/transRu2 isomers were obtained by Hannon and co-workers (Fig. 10A).52 The bimetallic double-stranded ruthenium metallohelices were synthesized from [RuCl2(dmso)4] and a bis-bidentate ligand containing two azopyridine donor units. Column chromatography of the reaction mixture isolated Ru1 (in 3% yield) and Ru2 (in 1% yield with respect to starting materials), respectively. Interestingly, the author observed the conversion of a sample of Ru2 into a third cis/cis isomer Ru3 on standing in a solution of CHCl3. Initial cell-line experiments indicated that the Ru2 isomer displays similar activity to cisplatin in the HBL100 cell line and better activity in the T47D cell line, whereas the Ru1 isomer showed extremely high activity, with approximately 30 times more potency than cisplatin in the HBL100 cell line and 100 times more in the T47D cell line (Fig. 10B).


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Fig. 10 (a) Self-assembly of unsaturated dinuclear RuII double-helicate in the trans/trans, cis/trans and cis/cis isomers, respectively; (b) the corresponding IC50 values (μM) in breast-cancer cell lines. Reproduced with permission from ref. 52. Copyright 2006, Wiley-VCH.

Another example of dinuclear RuII double-stranded helicate and mesocate complexes controlled by the steric interactions between ligand strands for cancer chemotherapy were reported recently by the groups of R. M. Phillips and C. R. Rice.53 The assembly of a ligand that is decorated with two tridentate thiazole-bipyridine domains separated by a m-terphenyl spacer unit and Ru(dmso)4Cl2 in ethylene glycol at 200 °C produced double-stranded helicate Ru4 and mesocate Ru5 in 6.4% yield in total (Fig. 11A). Similarly, another ligand containing a methyl substituent on the central aryl ring formed separable dinuclear double-stranded helicate Ru6 and mesocate Ru7 in 7.7% yield in total (Fig. 11B). Interestingly, when reducing the ligand flexibility by a diphenylene spacer only double-stranded helicate isomer Ru8 was obtained in 16% yield (Fig. 11C). Cytotoxicity studies showed that the helicate isomer Ru4 favourably killed bulk colon cancer cells lacking p53 and the mesocate Ru5 was preferentially cytotoxic towards bulk colon cancer cells possessing p53. In contrast, structurally similar Ru6/Ru7 and Ru8 showed very little cytotoxic activity. This study demonstrated that alteration of the metallohelice structures could have profound effects on cytotoxicity towards cancer cells of different p53 status.


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Fig. 11 Self-assembly of dinuclear RuII double-helicates (Ru4, Ru6, Ru8) or double-mesocates (Ru5, Ru7) between Ru(dmso)4Cl2 and the corresponding ligands. Reproduced with permission from ref. 53. Copyright 2018, Wiley-VCH.

Metallohelices for photodynamic therapy

Different from the traditional treatment approaches of chemotherapy and radiotherapy, PDT generally relies on the predominantly in situ generated 1O2 to irreversibly damage tumors when the photosensitizers are activated under light irradiation.54 However, the short life-time of 1O2 limited its diffusion distance in cells, which largely restricted the efficiency of PDT.55 The key issue includes how rationally designed PDT agents could selectively target specific subcellular organelles,56 or even biomacromolecules, such as DNA,57 to enhance their PDT efficacy.

It is evident that the 3D helical structures of metallohelices with specific structure-inherent targeting ability towards DNA have shown exciting results as chemotherapy agents. When lighting them up by using d6-transition-metal ions (RuII/IrIII) as the vertices, the resulting photoactive metallohelices with 1O2 generation abilities under light irradiation may further stimulate novel candidates in PDT of cancer treatment. Meanwhile, these luminescent metallohelices can be used as bioimaging agents to improve the therapeutic accuracy as well. In this regard, although lanthanide bimetallic helicates have been widely synthesized and applied for vitro imaging and sensing,58 the lack of ability to sensitize O2 hindered their applications in the field of PDT. In this part, only luminescent metallohelices equipped with RuII and IrIII metal ions are presented.

The first example of a luminescent binuclear RuII triple-helicate [Ru2L3]4+ (Ru9) was reported by Hannon's group in a similar fashion as their [Fe2L3]4+ in a very low yield (about 1% with respect to the starting materials).59 CD, LD, and importantly, fluorescence studies showed that the RuII triple-helicate binds and coils DNA. In addition, the high stability of this compound, due to the inert RuII centres, makes this type of agent particularly suitable for use in biological studies. Ru9 exhibited cytotoxicity activity against human breast cancer cells HBL-100 (IC50 = 22 μM) and T47D (IC50 = 53 μM), and can be classified as a member of a new and promising group of noncovalent DNA-binding anticancer metallodrugs. Thanks to the photoactive properties, Ru9 showed highly effective DNA-photocleavage ability, a feature not accessible with [Fe2L3]4+.60 Mechanistic studies revealed that 1O2 may play an important role in DNA photocleavage, although the 1O2 quantum yields (Φs) were not determined. In addition, the triple-helicate Ru9 displayed sequence selectivity in DNA photocleavage with a preference for regularly alternating purine-pyrimidine nucleotides. Besides, compounds cleaving DNA on photoactivation usually show localized effects in therapeutic applications and are much less toxic in the absence of light; thus, they may potentially be useful in PDT for cancers.

Cyclometalated IrIII complexes are attractive anticancer PDT photosensitizers61,62 and diagnostic agents63 due to their good biocompatibility, excellent luminescent emissive property, and impressive 1O2 quantum yield. The functional versatility of the octahedral structures can be easily modulated by the rational modification of the ligands. In addition, the carbon–metal bonds between the inert metal centre and ligands render them with high chemical and thermal stability. Specifically, these octahedral complexes display intrinsic metal-centred geometrical chirality, existing as Δ and Λ-enantiomers.64 Thus, a well-designed IrIII-containing metallohelice combines strong interactions with DNA targets and efficient 1O2 quantum yield may provide new motivation to achieve enhanced PDT for cancer treatment. However, the coordination self-assembly process with kinetically inert IrIII ions, similar to RuII, may produce undesired kinetic products or polymeric structures, and the corresponding metallohelices remain largely unexamined.65

Very recently, our group firstly reported a series of binuclear IrIII-containing luminescent metallohelices in good yield (about 32% with respect to starting material IrCl3, Fig. 12) by a stepwise hierarchical assembly approach.26 These metallohelices were fabricated using the dynamic imine-coupling chemistry between aldehyde end-capped fac-Ir(ppy)3 handles and linear alkanediamine spacers, followed by reduction of the imine-linkages. Interestingly, X-ray crystallographic analysis confirmed that the odd–even character of the diamine alkyl linkers determined the final conformations of the structure (Fig. 13). While 1,2-ethylenediamine (or 1,4-butanediamine) with even number atoms induced the formation of helicate Ir1a (or Ir3a). In contrast, the diamine spacers with odd number atoms 1,3-propanediamine (or 1,5-pentanediamine) connected with two IrIII centres with the opposite chirality possessed the achiral mesocate geometry Ir2a (or Ir4a). Following reduction of the imine linkages, chemically robust and photostable Ir1b, Ir2b, Ir3b and Ir4b metallohelices were obtained equivalently. It is noted that these metallohelices with amine-linkages exhibited high photoluminescence quantum yields (93.5–99.3%) and similar 1O2 quantum yields (30–33%). Such novel features made them promising candidates in diagnostic and anticancer PDT fields.


image file: d0cc02194f-f12.tif
Fig. 12 Schematic illustration of the stepwise hierarchical assembly construction and post-synthetic reduction of IrIII-containing helicates and mesocates based on odd–even alternation of diamine spacers. Reproduced with permission from ref. 26. Copyright 2020, Wiley-VCH.

image file: d0cc02194f-f13.tif
Fig. 13 Representation of the X-ray crystal structures of the triple-stranded helicates (a) Ir1a and (b) Ir3a (only the ΛΛ-Ir1a and ΛΛ-Ir3a enantiomers are shown), and the triple-stranded mesocates (c) Ir2a and (d) Ir4a. Reproduced with permission from ref. 26. Copyright 2020, Wiley-VCH.

The amine-linked metallohelices all showed a high degree of colocalization in the mitochondria of MCF-7 cells. They all showed low dark toxicity (IC50 > 30 μM) against MCF-7 cells (Fig. 14A). However, the mesocates Ir2b and Ir4b showed stronger phototoxicity when compared with the helicates Ir1b and Ir3b. Surprisingly, Ir4b displayed the most efficient PDT efficacy (IC50 = 1.1 μM) with a photocytotoxicity index PI > 33.3 (Fig. 14B). The flow cytometry assay results were in accordance with the cell viability test, and clearly demonstrated that Ir4b is a promising PDT agent due to its negligible toxicity in the dark but strong toxic effect under light (Fig. 14C).


image file: d0cc02194f-f14.tif
Fig. 14 Relative viabilities of MCF-7 cells in the presence of different concentrations of IrIII complexes (A) in the dark and (B) under irradiation conditions; (C) Flow cytometry quantification of Annexin V-FITC- and PI-labeled MCF-7 cells. Reproduced with permission from ref. 26. Copyright 2020, Wiley-VCH.

A key point is that our metallohelices themselves are formally neutral, and this is in contrast with all the other cationic helicates described before in this review. Some of the IC50 and PI values could not be precisely measured due to solubility reasons. Although metallohelices with high positive charges were considered facilitating the interactions with the negatively charged DNA, an ethidium bromide (EB) displacement assay confirmed that the neutral IrIII-containing metallohelices have binding capabilities with natural calf-thymus DNA (ct-DNA). The calculated apparent binding constants (Kapp) to ct-DNA of H2b, M3b, H4b, and M5b were about 0.39 × 107, 0.67 × 107, 0.66 × 107, and 1.12 × 107 M−1, respectively. In consideration of their similar 1O2 quantum yield, the discrepant PDT efficacies could be safely attributed to their different DNA affinities. A simple molecular docking study using B-DNA dodecamer was conducted to present the possible interaction manner of the IrIII-containing metallohelices with DNA. The results indicated that Ir4b showed a stronger DNA-binding affinity compared to others, and a minor-groove binding mode was preferred rather than the major-groove mode. This study shows a controllable approach for developing rational design and tailored IrIII-containing luminescent metallohelices, and presents a platform to investigate the structure–function relationship among the metallohelices and PDT efficacy.

Summary and outlook

Metallohelices with structure-inherent targeting ability towards DNA as an emerging potential source of new therapeutic metallodrugs have been developed and the field is worthy of deep investigations in future. Their more sophisticated 3D structural skeletons impart unprecedented noncovalent DNA-binding properties. These novel molecular level interactions are distinct from the most effective clinical platinum metallodrug agents which bind with DNA covalently. A series of metallohelices with labile metal ions (CuI/FeII/NiII/ZnII) have shown profound cytotoxic effects against bacterial strains. In particular, some enantiopure metallohelices were also separately investigated since the biological activity of enantiomers can differ drastically in terms of toxicity and pharmacokinetics. In addition, the recent rebound of metallohelices concerns how to light them up. Considering that PDT presents the most promising approach for cancer treatment, without doubt, the incorporation of luminescent d6-transition-metal ions (RuII/IrIII) to expand the application of metallohelices as promising PDT agents will lead to a huge renaissance of this research area. The following summarizes several perspectives of the future development.

(a) The incorporation of photoactive innocent, kinetically liable metal ions into metallohelices has generated a lot of basic scaffolds as DNA-binders. During the past twenty years, however, these studies were limited to rather few compounds, and mostly concentrated on the [Fe2L3]4+ cylinder. To date, both the surface of the ligands and the handedness of the metallohelices have been shown to affect their DNA binding. There is still a need to further expand the structural and synthetic diversities to deeply investigate the structure–functional relationship. The ability to place functional groups at positions of choice in metallohelices by pre-designed ligands or by post-modification methods is still a few steps away.

(b) Efforts to combine the striking DNA-binding features of metallohelices with the intrinsic photoactive properties came to fruition recently. The use of RuII or IrIII inert metal ions allows structurally robust and photoactive metallohelices to be made as novel PDT agents. However, the Ru-containing metallohelices are commonly obtained in very low yields when using the classical self-assembly process, which inhibits their further applications. Exciting results came from the recent controllably constructed and high-yielded binuclear IrIII-containing metallohelices. The functional iridium modules with desired connective geometry for metallohelix formation can be easily accessible via rational ligand design and the full-fledged synthetic method. We look forward to this method being effective for guiding the synthesis of other inert-metal-based metallohelices as well.

(c) For application of photoactive metallohelices in PDT, a number of issues including optical purity, solubility in water, and irradiation with longer wavelength light need to be addressed. However, the reported RuII/IrIII metallohelices only concentrated the racemic mixtures and the effects of enantiopure isomers on PDT remained unexamined. Specifically, for the next generation of IrIII-containing metallohelices, enantiopure isomers can be controllably obtained by pre-resolution of the IrIII modules first. On the other hand, functionalization of the skeletons with PEG or sugar chains to increase the water solubility of these neutral skeletons is also expected. The ideal activation wavelength for PDT agents is at the NIR region where deep-tissue tumor therapy can also be effective. However, the absorption peaks of the reported metallohelice-based PDT agents are below the NIR region. How to red shift the activation wavelength should be under consideration.

(d) Reported metallohelices for chemotherapy or phototherapy of cancers commonly without specific tumor-targeting ability were examined only in vitro. Therefore, identification of new drug designs and therapeutic strategies that could target cancer cells leaving normal cells unaffected still continues to be a challenge. Further development in this area should focus on an attempt to achieve improved tumor targeting properties and practicable in vivo applications. The design strategy includes the conjugation of metallohelices with tumor-targeting agents, such as antibodies, peptides, or small molecules.66,67

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21701019, U1608224, and 21861132004) and China Postdoctoral Science Foundation (2018M630286).

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