Mechanistic investigation of developmental copper chemotherapeutics

The quest for new metal-based anticancer agents, alternative to clinically established chemotherapeutics, has been motivated by deficiencies observed in current treatment regimes. Coupled with the approach of sophisticated and targeted drug design, there is a clear need for comprehending the underlying biomolecular and cellular responses of new developmental therapeutics. Reported herein is a detailed analysis of redox active developmental metallodrugs containing 1,10-phenanthroline (Phen) ligands and their action as novel cytotoxins of human cancers. This body of research describes mechanistic investigations into the oxidative nuclease activity and redox-targeting properties of new Cu(II) and Mn(II) phenanthroline chemo-types. A number of the Cu(II) complexes have been developed and examined, in collaboration with the National Cancer Institute, USA, for their ability to induce cytotoxicity within a wide variety of cancer cells. To uncover these properties, a range of molecular biology and biophysical techniques were employed including, flow cytometry, confocal microscopy, electrophoresis, and immunohistochemistry. Replacing auxiliary 1,10-phenanthroline with phenazine-type (N,Nʹ) ligands in mononuclear systems, [Cu(N,Nʹ)(Phen)]2+ , was found to enhance intercalation and oxidative DNA scission in vitro. Alternatively, incorporation of dicarboxylates (O,Oʹ) has shown to increase redox potential and stability, thereby targeting both mitochondrial and genomic DNA in human ovarian cancer cells, SKOV3. Increased nuclearity and varying rigidity was explored in dinuclear chemo-types ([Cu2(O,Oʹ)(Phen) 4 ]2+) through the addition of aliphatic and aromatic bridging dicarboxylate ligands. In combination with NCI-60 analysis, the dinulcear complexes were shown to enhance both geno- and cyto-toxic effects when compared to the mononuclear analogue, leading to an apoptotic mode of cellular death; activated through intrinsic mitochondrial machinery. Finally, exchange of the metal centre in the form of di-manganese(II) complex significantly influenced the mode of programmed cell death, activating autophagic catabolism and self-digestion

Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents

Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and \u27clicked\u27 using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the \u27Tri-Click\u27 (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques-including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes-the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(ll)-TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles

A Click Chemistry-Based Artificial Metallo-Nuclease

Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction can be directed to build Cu-binding AMN scaffolds. We selected biologically inert reaction partners tris(azidomethyl)mesitylene and ethynyl-thiophene to develop TC-Thio, a bioactive C3-symmetric ligand in which three thiophene-triazole moieties are positioned around a central mesitylene core. The ligand was characterised by X-ray crystallography and forms multinuclear CuII and CuI complexes identified by mass spectrometry and rationalised by density functional theory (DFT). Upon Cu coordination, CuII-TC-Thio becomes a potent DNA binding and cleaving agent. Mechanistic studies reveal DNA recognition occurs exclusively at the minor groove with subsequent oxidative damage promoted through a superoxide- and peroxide-dependent pathway. Single molecule imaging of DNA isolated from peripheral blood mononuclear cells shows that the complex has comparable activity to the clinical drug temozolomide, causing DNA damage that is recognised by a combination of base excision repair (BER) enzymes

[Cu(<i>o</i>‑phthalate)(phenanthroline)] Exhibits Unique Superoxide-Mediated NCI-60 Chemotherapeutic Action through Genomic DNA Damage and Mitochondrial Dysfunction

The <i>in cellulo</i> catalytic production of reactive oxygen species (ROS) by copper­(II) and iron­(II) complexes is now recognized as a major mechanistic model in the design of effective cytotoxins of human cancer. The developmental complex, [Cu­(<i>o</i>-phthalate)­(1,10-phenanthroline)] (<b>Cu-Ph</b>), was recently reported as an intracellular ROS-active cytotoxic agent that induces double strand breaks in the genome of human cancer cells. In this work, we report the broad-spectrum action of <b>Cu-Ph</b> within the National Cancer Institute’s (NCI) Developmental Therapeutics Program (DTP), 60 human cancer cell line screen. The activity profile is compared to established clinical agentsvia the COMPARE algorithmand reveals a novel mode of action to existing metal-based therapeutics. In this study, we identify the mechanistic activity of <b>Cu-Ph</b> through a series of molecular biological studies that are compared directly to the clinical DNA intercalator and topoisomerase II poison doxorubicin. The presence of ROS-specific scavengers was employed for <i>in vitro</i> and intracellular evaluation of prevailing radical species responsible for DNA oxidation with superoxide identified as playing a critical role in this mechanism. The ROS targeting properties of <b>Cu-Ph</b> on mitochondrial membrane potential were investigated, which showed that it had comparable activity to the uncoupling ionophore, carbonyl cyanide <i>m</i>-chlorophenyl hydrazine. The induction and origins of apoptotic activation were probed through detection of Annexin V and the activation of initiator (8,9) and executioner caspases (3/7) and were structurally visualized using confocal microscopy. Results here confirm a unique radical-induced mechanistic profile with intracellular hallmarks of damage to both genomic DNA and mitochondria

A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

Free radical generation is an inevitable consequence of aerobic existence and is implicated in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing and neurodegenerative disorder. Free radicals can, however, be used to our advantage since their production is catalysed by synthetic inorganic molecules—termed artificial metallonucleases—that cut DNA strands by oxidative cleavage reactions. Here, we report the rational design and DNA binding interactions of a novel di-Cu2+ artificial metallonuclease [Cu2(tetra-(2-pyridyl)-NMe-naphthalene)Cl4] (Cu2TPNap). Cu2TPNap is a high-affinity binder of duplex DNA with an apparent binding constant (Kapp) of 107 M(bp)−1. The agent binds non-intercalatively in the major groove causing condensation and G-C specific destabilization. Artificial metallonuclease activity occurs in the absence of exogenous reductant, is dependent on superoxide and hydrogen peroxide, and gives rise to single strand DNA breaks. Pre-associative molecular docking studies with the 8-mer d(GGGGCCCC)2, a model for poly[d(G-C)2], identified selective major groove incorporation of the complex with ancillary Cu2+-phosphate backbone binding. Molecular mechanics methods then showed the d(GGGGCCCC)2 adduct to relax about the complex and this interaction is supported by UV melting experiments where poly[d(G-C)2] is selectively destabilized

A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

Free radical generation is an inevitable consequence of aerobic existence and is implicated in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing and neurodegenerative disorder. Free radicals can, however, be used to our advantage since their production is catalysed by synthetic inorganic molecules—termed artificial metallonucleases—that cut DNA strands by oxidative cleavage reactions. Here, we report the rational design and DNA binding interactions of a novel di-Cu2+ artificial metallonuclease [Cu2(tetra-(2-pyridyl)-NMe-naphthalene)Cl4] (Cu2TPNap). Cu2TPNap is a high-affinity binder of duplex DNA with an apparent binding constant (Kapp) of 107 M(bp)−1. The agent binds non-intercalatively in the major groove causing condensation and G-C specific destabilization. Artificial metallonuclease activity occurs in the absence of exogenous reductant, is dependent on superoxide and hydrogen peroxide, and gives rise to single strand DNA breaks. Pre-associative molecular docking studies with the 8-mer d(GGGGCCCC)2, a model for poly[d(G-C)2], identified selective major groove incorporation of the complex with ancillary Cu2+-phosphate backbone binding. Molecular mechanics methods then showed the d(GGGGCCCC)2 adduct to relax about the complex and this interaction is supported by UV melting experiments where poly[d(G-C)2] is selectively destabilized.A.K. and Z.M. acknowledge funding from Science Foundation Ireland Career Development Award (SFI-CDA) [15/CDA/3648]; Marie Skłodowska-Curie Innovative Training Network (ITN) ClickGene [H2020-MSCA-ITN-2014-642023]; Circular dichroism analysis was carried out at the Nano Research Facility in Dublin City University which was funded under the Programme for Research in Third Level Institutions (PRTLI) Cycle 5. The PRTLI is co-funded through the European Regional Development Fund (ERDF), part of the European Union Structural Funds Programme 2011–2015. D.M. acknowledges funding from the European Commission [Marie Curie FP7-IEF]. Funding for open access charge: H2020 Marie Skłodowska-Curie Actions. The authors wish to acknowledge the DJEI/DES/SFI/HEA Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities and support. This work was also supported by the Synthesis and Solid-State Pharmaceutical Centre (SSPC) and Science Foundation Ireland (SFI) under grant number 12/RC/2275

Polypyridyl-Based Copper Phenanthrene Complexes: A New Type of Stabilized Artificial Chemical Nuclease

The building of robust and versatile inorganic scaffolds with artificial metallo-nuclease (AMN) activity is an important goal for bioinorganic, biotechnology, and metallodrug research fields. Here, a new type of AMN combining a tris-(2-pyridylmethyl)amine (TPMA) scaffold with the copper(II) N,N\u2032-phenanthrene chemical nuclease core is reported. In designing these complexes, the stabilization and flexibility of TPMA together with the prominent chemical nuclease activity of copper 1,10-phenanthroline (Phen) were targeted. A second aspect was the opportunity to introduce designer phenazine DNA intercalators (e.g., dipyridophenazine; DPPZ) for improved DNA recognition. Five compounds of formula [Cu(TPMA)(N,N\u2032)]2+ (where N,N\u2032 is 2,2-bipyridine (Bipy), Phen, 1,10-phenanthroline-5,6-dione (PD), dipyridoquinoxaline (DPQ), or dipyridophenazine (DPPZ)) were developed and characterized by X-ray crystallography. Solution stabilities were studied by continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE), and Davies electron\u2013nuclear double resonance (ENDOR) spectroscopies, which demonstrated preferred geometries in which phenanthrene ligands were coordinated to the copper(II) TPMA core. Complexes with Phen, DPQ, and DPPZ ligands possessed enhanced DNA binding activity, with DPQ and DPPZ compounds showing excellent intercalative effects. These complexes are effective AMNs and analysis with spin-trapping scavengers of reactive oxygen species and DNA repair enzymes with glycosylase/endonuclease activity demonstrated a distinctive DNA oxidation activity compared to classical Sigman- and Fenton-type reagents