Mapping the DNA Damaging Effects of Polypyridyl Copper Complexes with DNA Electrochemical Biosensors.

Several classes of copper complexes are known to induce oxidative DNA damage that mediates cell death. These compounds are potentially useful anticancer agents and detailed investigation can reveal the mode of DNA interaction, binding strength, and type of oxidative lesion formed. We recently reported the development of a DNA electrochemical biosensor employed to quantify the DNA cleavage activity of the well-studied [Cu(phen)2]2+ chemical nuclease. However, to validate the broader compatibility of this sensor for use with more diverse-and biologically compatible-copper complexes, and to probe its use from a drug discovery perspective, analysis involving new compound libraries is required. Here, we report on the DNA binding and quantitative cleavage activity of the [Cu(TPMA)(N,N)]2+ class (where TPMA = tris-2-pyridylmethylamine) using a DNA electrochemical biosensor. TPMA is a tripodal copper caging ligand, while N,N represents a bidentate planar phenanthrene ligand capable of enhancing DNA interactions through intercalation. All complexes exhibited electroactivity and interact with DNA through partial (or semi-) intercalation but predominantly through electrostatic attraction. Although TPMA provides excellent solution stability, the bulky ligand enforces a non-planar geometry on the complex, which sterically impedes full interaction. [Cu(TPMA)(phen)]2+ and [Cu(TPMA)(DPQ)]2+ cleaved 39% and 48% of the DNA strands from the biosensor surface, respectively, while complexes [Cu(TPMA)(bipy)]2+ and [Cu(TPMA)(PD)]2+ exhibit comparatively moderate nuclease efficacy (ca. 26%). Comparing the nuclease activities of [Cu(TPMA)(phen)] 2+ and [Cu(phen)2]2+ (ca. 23%) confirms the presence of TPMA significantly enhances chemical nuclease activity. Therefore, the use of this DNA electrochemical biosensor is compatible with copper(II) polypyridyl complexes and reveals TPMA complexes as a promising class of DNA damaging agent with tuneable activity due to coordinated ancillary phenanthrene ligands.

Polypyridyl-Based Copper Phenanthrene Complexes: Combining Stability with Enhanced DNA Recognition.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp ) rising from 105 to 107 m-1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine-cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

Copper phenanthrene oxidative chemical nucleases.

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 (∼10(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.

Deposition of Au and Ag nanoparticles on PEDOT.

The deposition of Au and Ag, locally and from bulk solution, on poly(3,4-ethylenedioxythiophene) (PEDOT) was studied. Specifically, PEDOT was electrochemically polymerized onto a glassy carbon (GC) electrode and used for bulk deposition of Au and Ag from their respective ions dissolved in the solution as well as for the local deposition of these metals using scanning electrochemical microscopy (SECM). These two sets of experiments were utilized to investigate the difference between Au and Ag electrochemical deposition on PEDOT. In particular, SECM experiments, which were conducted by the controlled anodic dissolution of Au and Ag microelectrodes close to GC/PEDOT, probed the effect of different PEDOT oxidation states on local deposition. The current-time transients recorded during the deposition, combined with scanning electron microscopy and EDX analysis provided insight into the reduction processes. AuCl(4)(-) and Ag(+) ions were electrochemically reduced at a potential equal to and more negative than the ions redox potentials (0.4 and 0.2 V, respectively) and more positive than -0.7 V, where the PEDOT starts transforming into the reduced, i.e. insulating, state. We found that the electroreduction of Ag(+) ions was diffusion-controlled and the PEDOT film served as a simple conductor. On the other hand, the reduction of AuCl(4)(-) ions was enhanced on GC/PEDOT as compared with bare GC, indicating that PEDOT catalyzes the reduction of AuCl(4)(-) to Au.

Evaluating the anticancer properties and real-time electrochemical extracellular bio-speciation of bis (1,10-phenanthroline) silver (I) acetate monohydrate in the presence of A549 lung cancer cells.

According to the American Cancer Society report (2019-2021), the majority (63%) of stage III non-small cell lung cancer (NSCLC) patients are prescribed with chemo and/or radiation therapies, with 5-year relative survival rates of just 19%. Thus, directed drug development, toward personalised cancer treatment, is widely recognised as a necessary strategy in drug discovery research. However, broad generalisations on the modes of action of bioinorganic compounds are not conducive to tailored drug design, hence, fundamental mechanistic research is essential in realising personalised healthcare. In this work, anticancer properties of bis (1,10-phenanthroline) silver (I) acetate monohydrate (Ag-Phen), toward A549 lung cancer cells are presented. Biological assays were carried out to evaluate the effect of Ag-Phen on cell viability, reactive oxygen species generation and mitochondrial membrane potentials. In tandem with the biological assays, electrochemistry was employed to determine the real-time concentrations of intact Ag-Phen and dissociated Ag+ in the extracellular medium using platinum microelectrodes, as a function of cellular exposure time. Observations from the assays conducted include, Ag-Phen induced cytotoxicity (IC50 4.5 µM at 72 h) and 2-fold ROS generation, and a 50% decrease in mitochondrial membrane potentials with respect to equivalent concentrations of Ag+ and 1,10-phenanthroline. Bio-speciation studies, conducted electrochemically at platinum microelectrodes, revealed almost 50% of the Ag-Phen had dissociated after 2 h. Significant reductions in concentrations of dissociated Ag+ (from 67.7 µM to 6.7 µM), and the Ag-Phen complex (from 50.2 µM to 11.7 µM) between 4 and 24 h from the extracellular medium, indicate cellular uptake of both. This novel method facilitates the real-time identification and quantification of electroactive species, both the intact Ag-Phen and Ag+, in the presence of A549 cells.

Highly Selective Fluorimetric Turn-Off Detection of Copper(II) by Two Different Mechanisms in Calix[4]arene-Based Chemosensors and Chemodosimeters.

Isoxazolo-pyrene tethered calix[4]arenes selectively detect copper(II) ions without interference from related perchlorate ions. The fluorescence emission of the probes, synthesised by nitrile oxide alkyne cycloaddition, and characterised by spectroscopic and crystallographic data, is rapidly reduced by Cu(II) ions. Detection limits are in the micromolar or sub-micromolar range (0.3-3.6 µM) based on a 1 : 1 sensor:analyte interaction. Voltammetric behaviour and 1 H NMR data provide new insights into the sensing mechanism which is dependent on the calixarene substitution pattern. When the calixarene lower rim is fully substituted, Cu(II) detection occurs through a traditional chelation mechanism. In contrast, for calixarenes 1,3-disubstituted on the lower rim, detection takes place through a chemodosimetric redox reaction. The isolation of a calix[4]diquinone from the reaction with excess Cu(ClO4 )2 provides confirmation that the sensor-analyte interaction culminates in irreversible sensor oxidation.

A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor.

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([CuII(phen)2]2+), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [CuII(phen)2]2+, a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [CuII(phen)2]2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [CuI(phen)2]+, with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [CuII(phen)2]2+.

Water-soluble and photo-stable silver(I) dicarboxylate complexes containing 1,10-phenanthroline ligands: Antimicrobial and anticancer chemotherapeutic potential, DNA interactions and antioxidant activity.

The complexes [Ag2(OOC-(CH2)n-COO)] (n=1-10) (1-10) were synthesised and reacted with 1,10-phenanthroline (phen) to yield derivatives formulating as [Ag2(phen)x(OOC-(CH2)y-COO)]·zH2O (x=2 or 3; y=1-10; z=1-4) (11-20) which are highly water-soluble and photo-stable in aqueous solution. The phen derivatives 11-20 exhibit chemotherapeutic potential against Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and against cisplatin-sensitive breast (MCF-7) and resistant ovarian (SKOV-3) cancer cell lines. Cyclic voltammetric analysis and DNA binding and intercalation studies indicate that the mechanism of action of 11-20 is significantly different to that of their silver(I) dicarboxylate precursors and they do not induce DNA damage or ROS generation in mammalian cells. The representative complexes 9 and 19 (containing the undecanedioate ligand) were both found to significantly reduce superoxide and hydrogen peroxide induced oxidative stress in the yeast S. cerevisiae.

Regulating bioactivity of Cu2+ bis-1,10-phenanthroline artificial metallonucleases with sterically functionalized pendant carboxylates.

The synthetic chemical nuclease, [Cu(1,10-phenanthroline)2](2+), has stimulated research within metallonuclease development and in the area of cytotoxic metallodrug design. Our analysis reveals, however, that this agent is "promiscuous" as it binds both dsDNA and protein biomolecules, without specificity, and induces general toxicity to a diversity of cell lineages. Here, we describe the synthesis and characterization of small-molecule metallonucleases containing the redox-active cation, [Cu(RCOO)(1,10-phen)2](+), where 1,10-phen = 1,10-phenanthroline and R = -H, -CH3, -C2H5, -CH(CH3)2, and -C(CH3)3. The presence of coordinated carboxylate groups in the complex cation functions to enhance dsDNA recognition, reduce serum albumin binding, and offer control of toxicity toward human cancer cells, Gram positive and negative bacteria, and fungal pathogens. The induction of genomic dsDNA breaks (DSBs) were identified in ovarian adenocarcinoma cells using immunodetection of γ-H2AX. Formate, acetate, and pivalate functionalized complexes induced DSBs in a higher percentage of cells compared with [Cu(1,10-phen)2](2+), which supports the importance of inner-sphere modification toward enhancing targeted biological application.

A new phenanthroline-oxazine ligand: synthesis, coordination chemistry and atypical DNA binding interaction.

1,10-Phenanthroline-5,6-dione and l-tyrosine methyl ester react to form phenanthroline-oxazine (PDT) from which [Cu(PDT)(2)](ClO(4))(2) and [Ag(PDT)(2)]ClO(4)·2MeOH are obtained. Binding to calf-thymus DNA by Ag(I) and Cu(II) PDT complexes exceed bis-1,10-phenanthroline analogues and the minor groove binding drugs, pentamidine and netropsin. Furthermore, unlike the artificial metallonuclease, [Cu(phen)(2)](2+), the [Cu(PDT)(2)](2+) complex does not cleave DNA in the presence of added reductant indicating unique interaction with DNA.

Potent oxidative DNA cleavage by the di-copper cytotoxin: [Cu2(µ-terephthalate)(1,10-phen)4]2+.

The di-copper(II) cation, [Cu(2)(µ-terephthalate)(1,10-phen)(4)](2+), is a powerful, non-sequence-specific, minor-groove oxidizer of duplex DNA which, unlike copper(II) bis-1,10-phenanthroline chloride, operates independently of exogenous reagents. The agent displays excellent in vitro cytoxicity towards cisplatin-resistant ovarian cancer cells, producing intracellular reactive oxygen species upon nano-molar exposure.

Non-trivial solution chemistry between amido-pyridylcalix[4]arenes and some metal salts.

Mercury ion complexation reactions were carried out between 3 and various mercury(II) salts. (1)H NMR studies showed that the role of solvent, the anion chosen and the initial reaction conditions were critical and that the formation of a "simple" mercury(II) complex was non-trivial. The mercury(II) ion can cause either (i) the formation of an ion-pair system, which have a characteristic doubling of all signals in the (1)H NMR spectrum, (ii) a cleavage reaction to occur resulting in the reformation of the calix[4]arene diester compound 2, but only when the reaction is heated and (iii) "simple" mercury binding to the pyridine rings when the binding studies are carried out using NMR titration techniques. The electrochemistry results, on the same systems, show that the initial reaction involves the removal of the phenoxide protons followed by the resulting catalysis of the mercury species. This proton removal is not observed in the NMR spectra of any of the mercury reactions. It was also found that 3 could bind silver and zinc salts and was not selective for mercury(II) as was previously described.

Determinants of exercise blood pressure response in normotensive and hypertensive women: role of cardiorespiratory fitness.

PURPOSE: Exaggerated blood pressure (BP) response during physical exertion is associated with increased risk for cardiovascular events. Furthermore, it may be the predisposing factor for myocardial infarction triggered by physical exertion. The authors have shown that systolic BP achieved after 6 minutes of exercise is the strongest predictor of left ventricular hypertrophy. Furthermore, a 37 mm Hg increase in systolic BP above resting BP at 6 minutes of exercise was the threshold for left ventricular hypertrophy. The purpose of this study was to determine predictors of exercise BP response in normotensive and hypertensive women. METHODS: An exercise tolerance test (Bruce) was performed by 1411 normotensive (resting BP < 140/90 mm Hg) and hypertensive (resting BP > or = 140/90 mm Hg) women. These women were faculty, students, and staff at the University of Maryland, College Park, Maryland, and the George Washington University Medical Center, as well as patients undergoing a routine exercise tolerance test at West Coast Cardiology, Pinellas Park, Florida. Two fitness categories (low-fit and high-fit) were established on the basis of treadmill time to exhaustion adjusted for age. RESULTS: Significant associations were observed among the 6-minute exercise BP and age, body mass index, resting systolic and diastolic BP, heart rate, and exercise time to exhaustion. In a stepwise multiple-regression analysis, the determinants of BP after 6 minutes of exercise were resting systolic BP and treadmill time to exhaustion (R2 = 0.36) for normotensive women and treadmill time to exhaustion and resting systolic BP (R2 = 0.30) for hypertensive women. When fitness categories were contrasted, low-fit women in both the normotensive and hypertensive categories had higher BP and rate-pressure product after 6 minutes of exercise than the high-fit women (P <.05). CONCLUSIONS: Resting systolic BP and cardiorespiratory fitness are determinants of a submaximal exercise BP response for both hypertensive and normotensive women. Low cardiorespiratory fitness is associated with a higher BP response during submaximal exercise, suggesting that increased fitness may attenuate this abnormal rise in BP. Thus, low- to moderate-intensity physical activities for most days of the week should be encouraged for all women to increase cardiorespiratory fitness. This is likely to attenuate an abnormal rise in systolic BP that may occur during routine daily activities and protect against the associated health consequences.