RESUMO
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.
Assuntos
Química Click , Cobre , Cobre/química , Leucócitos Mononucleares/metabolismo , Ligantes , DNA/química , Azidas/químicaRESUMO
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 'clicked' 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 'Tri-Click' (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(II)-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.
Assuntos
Química Click/métodos , Cobre/farmacologia , Dano ao DNA/efeitos dos fármacos , Nylons/farmacologia , Oxidantes/farmacologia , Humanos , Leucócitos Mononucleares , Estresse OxidativoRESUMO
Nucleic acid click chemistry was used to prepare a family of chemically modified triplex forming oligonucleotides (TFOs) for application as a new gene-targeted technology. Azide-bearing phenanthrene ligands-designed to promote triplex stability and copper binding-were 'clicked' to alkyne-modified parallel TFOs. Using this approach, a library of TFO hybrids was prepared and shown to effectively target purine-rich genetic elements in vitro. Several of the hybrids provide significant stabilisation toward melting in parallel triplexes (>20 °C) and DNA damage can be triggered upon copper binding in the presence of added reductant. Therefore, the TFO and 'clicked' ligands work synergistically to provide sequence-selectivity to the copper cutting unit which, in turn, confers high stabilisation to the DNA triplex. To extend the boundaries of this hybrid system further, a click chemistry-based di-copper binding ligand was developed to accommodate designer ancillary ligands such as DPQ and DPPZ. When this ligand was inserted into a TFO, a dramatic improvement in targeted oxidative cleavage is afforded.
Assuntos
Química Click , DNA/química , Marcação de Genes/métodos , Oligonucleotídeos/química , Cobre/química , Dano ao DNA , Ligantes , Conformação de Ácido Nucleico , OxirreduçãoRESUMO
The binding of small molecule metallodrugs to discrete regions of nucleic acids is an important branch of medicinal chemistry and the nature of these interactions, allied with sequence selectivity, forms part of the backbone of modern medicinal inorganic chemistry research. In this tutorial review we describe a range of molecular methods currently employed within our laboratories to explore novel metallodrug-DNA interactions. At the outset, an introduction to DNA from a structural perspective is provided along with descriptions of non-covalent DNA recognition focusing on intercalation, insertion, and phosphate binding. Molecular methods, described from a non-expert perspective, to identify non-covalent and pre-associative nucleic acid recognition are then demonstrated using a variety of techniques including direct (non-optical) and indirect (optical) methods. Direct methods include: X-ray crystallography; NMR spectroscopy; mass spectrometry; and viscosity while indirect approaches detail: competitive inhibition experiments; fluorescence and absorbance spectroscopy; circular dichroism; and electrophoresis-based techniques. For each method described we provide an overview of the technique, a detailed examination of results obtained and relevant follow-on of advanced biophysical/analytical techniques. To achieve this, a selection of relevant copper(ii) and platinum(ii) complexes developed within our laboratories are discussed and are compared, where possible, to classical DNA binding agents. Applying these molecular methods enables us to determine structure-activity factors important to rational metallodrug design. In many cases, combinations of molecular methods are required to comprehensively elucidate new metallodrug-DNA interactions and, from a drug discovery perspective, coupling this data with cellular responses helps to inform understanding of how metallodrug-DNA binding interactions manifest cytotoxic action.
Assuntos
Complexos de Coordenação/química , DNA/química , Substâncias Intercalantes/química , Fosfatos/química , Cobre/química , Desenho de Fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Platina/químicaRESUMO
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'-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')]2+ (where N,N' 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-nuclear 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.
Assuntos
Complexos de Coordenação/química , Cobre/química , Fenantrenos/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Endonucleases/química , Endonucleases/metabolismo , Espectroscopia de Ressonância Magnética , Conformação MolecularRESUMO
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.
Assuntos
Cobre/química , Clivagem do DNA/efeitos dos fármacos , DNA/química , DNA/metabolismo , Compostos Organometálicos/farmacocinética , Fosfatos/química , Cobre/farmacocinética , Cobre/farmacologia , DNA/efeitos dos fármacos , Radicais Livres/química , Radicais Livres/farmacocinética , Radicais Livres/farmacologia , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fosfatos/farmacocinética , Fosfatos/farmacologiaRESUMO
Cancer remains a global health challenge. There is an urgent need to develop innovative therapeutics that can overcome the shortcomings of existing cancer therapies. DNA enzymes involved in nucleic acid compaction and organization are an attractive cancer drug target for therapeutic exploitation. In this work, a family of Cu(II) prodrugs containing suberoylanilide hydroxamic acid (SAHA), a well-established histone deacetylase inhibitor (HDACi) and clinically approved cancer drug, and phenanthrene ligands as DNA intercalative components have been rationally developed. The complexes, of general formula [Cu(SAHA-1H)( N, N'-phenanthrene)]+, exhibit excellent DNA recognition with binding affinity of lead agents in the order of â¼107 M(bp)-1. Biophysical studies involving nucleic acid polymers indicate intercalative binding at both adenine-thymine (A-T) and guanine-cytosine (G-C) rich sequences but thermodynamically stable interactions are favored in G-C tracts. The complexes mediate DNA damage by producing reactive oxygen species (ROS) with spin trapping experiments showing that superoxide, the hydroxyl radical, and hydrogen peroxide play critical roles in strand scission. The agents were found to have promising antiproliferative effects against a panel of epithelial cancers, and in two representative cell lines possessing mutated p53 (SK-OV-3 and DU145), enhanced cytotoxicity was observed. Significantly, mechanistic experiments with the most promising candidates revealed HDAC inhibition activity was achieved over a shorter time frame as compared to clinical standards with DNA damage-response markers identifying upregulation of both DNA synthesis and nucleotide excision repair (NER) pathways. Finally, confocal imaging and gene expression analysis show this metallodrug class exerts cytotoxic activity predominantly through an apoptotic pathway.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Substâncias Intercalantes/farmacologia , Neoplasias/tratamento farmacológico , Compostos Organometálicos/farmacologia , Pró-Fármacos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Cobre/farmacologia , Cobre/uso terapêutico , DNA/química , Dano ao DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/uso terapêutico , Neoplasias/genética , Neoplasias/patologia , Compostos Organometálicos/química , Compostos Organometálicos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Fenantrenos/química , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Vorinostat/químicaRESUMO
In order to expand the current repertoire of cancer treatments and to help circumvent limitations associated with resistance, the identification of new metallodrugs with high potency and novel mechanisms of action is of significant importance. Here we present a class of di-copper(II) complex based on the synthetic chemical nuclease [Cu(Phen)2]+ (where Phen = 1,10-phenanthroline) that is selective against solid epithelial cancer cells from the National Cancer Institute's 60 human cell line panel (NCI-60). Two metallodrug leads are studied and in each case two [Cu(Phen)2]+ units are bridged by a dicarboxylate linker but the length and rigidity of the linkers differ distinctly. Both agents catalyze intracellular superoxide (O2â¢-) and singlet oxygen (1O2) formation with radical species mediating oxidative damage within nuclear DNA in the form of double strand breaks and to the mitochondria in terms of membrane depolarization. The complexes are effective DNA binders and can discriminate AT/AT from TA/TA steps of duplex DNA through induction of distinctive Z-like DNA or by intercalative interactions.
Assuntos
Complexos de Coordenação/farmacologia , Quebras de DNA de Cadeia Dupla , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oligonucleotídeos/metabolismo , Oxigênio Singlete/metabolismo , Superóxidos/metabolismo , Linhagem Celular Tumoral , Complexos de Coordenação/química , Cobre/química , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Oligonucleotídeos/genética , Fenantrolinas/químicaRESUMO
There is an unmet need for novel metal-based chemotherapeutics with alternative modes of action compared to clinical agents such as cisplatin and metallo-bleomycin. Recent attention in this field has focused on designing intracellular ROS-mediators as powerful cytotoxins of human cancers and identifying potentially unique toxic mechanisms underpinning their utility. Herein, we report the developmental di-manganese(II) therapeutic [Mn2(µ-oda)(phen)4(H2O)2][Mn2(µ-oda)(phen)4(oda)2]·4H2O (Mn-Oda) induces autophagy-promoted apoptosis in human ovarian cancer cells (SKOV3). The complex was initially identified to intercalate DNA by topoisomerase I unwinding and circular dichroism spectroscopy. Intracellular DNA damage, detected by γH2AX and the COMET assay, however, is not linked to direct Mn-Oda free radical generation, but is instead mediated through the promotion of intracellular reactive oxygen species (ROS) leading to autophagic vacuole formation and downstream nuclear degradation. To elucidate the cytotoxic profile of Mn-Oda, a wide range of biomarkers specific to apoptosis and autophagy including caspase release, mitochondrial membrane integrity, fluorogenic probe localisation, and cell cycle analysis were employed. Through these techniques, the activity of Mn-Oda was compared directly to i.) the pro-apoptotic clinical anticancer drug doxorubicin, ii.) the multimodal histone deacetylase inhibitor suberoyanilide hydroxamic acid, and iii.) the autophagy inducer rapamycin. In conjunction with ROS-specific trapping agents and established inhibitors of autophagy, we have identified autophagy-induction linked to mitochondrial superoxide production, with confocal image analysis of SKOV3 cells further supporting autophagosome formation.
Assuntos
Antineoplásicos/farmacologia , Autofagossomos/metabolismo , Manganês/farmacologia , Neoplasias/metabolismo , Compostos Organometálicos/farmacologia , Antineoplásicos/química , Autofagia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ácidos Hidroxâmicos/farmacologia , Manganês/química , Mitocôndrias/metabolismo , Estrutura Molecular , Neoplasias/tratamento farmacológico , Compostos Organometálicos/química , Espécies Reativas de Oxigênio/metabolismo , VorinostatRESUMO
The in cellulo 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(o-phthalate)(1,10-phenanthroline)] (Cu-Ph), 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 Cu-Ph 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 Cu-Ph 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 in vitro 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 Cu-Ph on mitochondrial membrane potential were investigated, which showed that it had comparable activity to the uncoupling ionophore, carbonyl cyanide m-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.
Assuntos
Dano ao DNA/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fenantrolinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Microscopia Confocal , Estrutura Molecular , Fenantrolinas/química , Espécies Reativas de OxigênioRESUMO
The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, aging, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids-as demonstrated by metal-activated bleomycin-has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)](2+) (Cu-DPQ-Phen), [Cu(DPPZ)(phen)](2+) (Cu-DPPZ-Phen), and [{Cu(phen)2}2(µ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman's reagent [Cu(phen)2](2+) throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine; Terph = terephthalate). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilizers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals ((â¢)OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited-particularly along A-T rich chains-through oxidative damage of template strands.
RESUMO
Here we report the synthesis and isolation of a series of bis-chelate Cu(2+) phenanthroline-phenazine cationic complexes of [Cu(DPQ)(Phen)](2+), [Cu(DPPZ)(Phen)](2+), and [Cu(DPPN)(Phen)](2+) (where Phen = 1,10-phenanthroline, DPQ = dipyridoquinoxaline, DPPZ = dipyridophenazine, and DPPN = benzo[i]dipyridophenazine). These compounds have enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2](2+) (bis-Phen), with calf thymus DNA binding constants of DPQ and DPPZ agents (â¼10(7) M(bp)(-1)) being the highest currently known for Cu(2+) phenanthrene compounds. Complex DNA binding follows DPQ ≈ DPPZ > DPPN > bis-Phen, with fluorescence quenching and thermal melting experiments on poly[d(A-T)2] and poly[d(G-C)2] supporting intercalation at both the minor and major groove. Phenazine complexes, however, show enhanced targeting and oxidative cleavage on cytosine-phosphate-guanine-rich DNA and have comparable in vitro cytotoxicity toward the cisplatin-resistant ovarian cancer line, SKOV3, as the clinical oxidative DNA-damaging drug doxorubicin (Adriamycin). In this study we also describe how a novel "on-chip" method devised for the Bioanalyser 2100 was employed to quantify double-stranded DNA damage, with high precision, by the complex series on pUC19 DNA (49% A-T, 51% G-C). Both DPQ and bis-Phen complexes are highly efficient oxidizers of pUC19, with DPQ being the most active of the overall series. It is apparent, therefore, that oxidative chemical nuclease activity on homogeneous canonical DNA is not entirely dependent on dynamic nucleotide binding affinity or intercalation, and this observation is corroborated through catalytic interactions with the superoxide anion radical and Fenton breakdown of hydrogen peroxide.