Critical congenital heart disease: contemporary prenatal screening performance and outcomes in a multi-centre perinatology service.

BACKGROUND: Prenatal detection of critical congenital heart disease (CCHD) optimises perinatal decision-making and neonatal outcomes. The objective of this study was to determine the prenatal screening performance, care pathways and perinatal outcomes for prenatally and postnatally diagnosed cases of CCHD over a four-year period. STUDY DESIGN: This retrospective cohort study in a tertiary centre and its two affiliated secondary sites examined all cases of CCHD, including cases of pregnancy termination and in-utero fetal death, neonatal death and liveborn babies that underwent cardiac catheterization or surgery in the first six weeks of life. Prenatal and postnatal data were ascertained from the first trimester assessment for all patients diagnosed prenatally. Cases requiring intervention that were first identified in the postnatal period were included to determine prenatal detection rates. Follow-up for all cases of CCHD continued to one year of age. RESULTS: In a consecutive cohort of 49,950 pregnancies in a 4-year period 01/2019 to 12/2022, a prenatal diagnosis of CCHD was made in 96 cases, yielding a prevalence of 1.9 per 1000 births. The prenatal detection for right duct-dependant heart pathology and congenital heart block was 100%, 85% for left duct-dependant pathology and 93% for transposition of the great arteries (TGA). In the prenatally diagnosed group, 37% of cases were complicated by extracardiac structural abnormalities, a genetic diagnosis or both. All cases of prenatal detection were identified in the context of routine anatomy screening rather than specialist Fetal Cardiac screening services. Almost half of all pregnancies complicated by CCHD did not undergo neonatal cardiac intervention, by virtue of parental choice determined either prenatally or after birth. An additional eight babies were diagnosed with CCHD in the neonatal period, such that the prenatal detection rate for CCHD was 92% (96/104, 95% CI = 84%-96%). Survival at 1-year for infants deemed suitable for CCHD surgery was 85%. CONCLUSION: In a large unselected population, optimal rates of prenatal detection of critical congenital heart disease can be achieved by a protocolised approach to mid-trimester fetal anatomy ultrasound, underpinned by a programme of sonographer education and training. The cardiac abnormalities most likely to evade prenatal detection are left-sided obstructive lesions.

How well do we truly understand clitoral anatomy? An Irish maternity hospital's perspective.

BACKGROUND: The clitoris has long been ignored in medical literature and teaching, with the first paper describing its anatomy only published in 1993. It is essential that those working in women's health understand the anatomy of this important female sexual organ. AIMS: The aim of this study was to assess the level of knowledge of the anatomy of the clitoris across five cohorts in a busy teaching maternity hospital in Ireland. MATERIALS AND METHODS: A questionnaire was devised, asking ten consultants, ten non-consultant hospital doctors (NCHDs), ten midwives, ten midwifery students and ten medical students to name the anatomical parts of a 3D model of the clitoris. RESULTS: None of the 50 respondents could name all five labelled parts of the clitoris, and 38 of respondents could not correctly name one anatomical part. CONCLUSIONS: It is evident that there is a lack of knowledge of the anatomical parts of the clitoris among the medical staff included in this study. This reflects a lack of adequate teaching in this area in both past and recent years. Despite this, we acknowledge that women's health and sexual health awareness is improving all of the time, and we hope that this study can highlight the need for reformed teaching in this area.

A randomised open-label trial to assess outpatient induction of labour (HOMEIND) and compare efficacy of Propess vs Dilapan-S® for induction of labour at 39 weeks' gestation in normal risk nulliparous women: study protocol for a randomised controlled trial.

BACKGROUND: Induction of labour (IOL) at 39 weeks has been shown to decrease maternal and neonatal adverse outcomes. Given the growing demand for 39-week IOL, it is imperative that effective methods be assessed for induction in the outpatient setting. The aim of this study is to answer the clinical question as to whether Dilapan-S® vs Propess® as a method of cervical ripening is non-inferior in the outpatient setting at 39 weeks and to ascertain whether Dilapan-S® 12 h is non-inferior to Dilapan-S® 24 h. METHODS: This study is an open-label parallel group single-centre randomised trial. Participants are normal risk nulliparous women who have no pregnancy-related or medical contraindication to IOL. Women will be randomised to one of three induction groups-Dilapan-S® (12-h insertion or 24-h insertion) or Propess. Induction will be initiated between 39+0 and 39+4 weeks' gestation and participants will return home for either 12 or 24 h. They will be readmitted 12/24 h later in order to continue with induction of labour. Patient recruitment will take place over 30 months within a single centre. The study will recruit a maximum 109 women for each study arm. Total duration of participants' involvement in the trial will be 8 weeks to allow for postpartum follow-up. DISCUSSION: This study will definitively answer whether Dilapan-S is non-inferior to Propess® as a method of induction of labour in the outpatient setting and whether cervical ripening with Dilapan-S over a 12-h timeframe is non-inferior to cervical ripening with Dilapan-S over a 24-h timeframe. TRIAL REGISTRATION: EudraCT Number 2019-004697-25 Registered 14 September 2020.

Antitumor copper(II) complexes with hydroxyanthraquinones and N,N-heterocyclic ligands.

Five ternary copper(II) complexes, [Cu2(phen)2(L1)(ClO4)2] (1), [Cu2(phen)2(L1)(DMSO)2](PF6)2 (2), [Cu2(bpy)2(L1)(ClO4)2(H2O)2] (3), [Cu2(dmp)2(L1)(ClO4)2(H2O)2] (4), and [Cu(phen)(L2)]2(ClO4)2 (5), in which phen = 1,10-phenanthroline, bpy = 2,2'-bipyridine, dmp = 2,9-dimethyl-1,10-phenanthroline, H2L1 = 1,4-dihydroxyanthracene-9,10-dione and HL2 = 1-hydroxyanthracene-9,10-dione, DMSO = dimethylsulfoxide, were synthesized and fully characterized. Complex 2 was obtained through the substitution of perchlorate for DMSO. When two hydroxyquinone groups are present, L1 makes a bridge between two Cu(II) ions, which also bind two nitrogens of the respective diimine ligand. The compounds bind to calf thymus DNA and oxidatively cleave pUC19 DNA according to the following order of activity 1 > 4-5 > 3. Furthermore, complexes 1, 3, 4 and 5 inhibit topoisomerase-I activity and the growth of myelogenous leukemia cells with the IC50 values of 1.13, 10.60, 0.078, and 1.84 µmol L-1, respectively. Complexes 1 and 4 are the most active in cancer cells and in DNA cleavage.

Smartphone Apps for Surveillance of Gestational Diabetes: Scoping Review.

BACKGROUND: Developments and evolutions in the information and communication technology sector have provided a solid foundation for the emergence of mobile health (mHealth) in recent years. The cornerstone to management of gestational diabetes mellitus (GDM) is the self-management of glycemic indices, dietary intake, and lifestyle adaptations. Given this, it is readily adaptable to incorporation of remote monitoring strategies involving mHealth solutions. OBJECTIVE: We sought to examine and assess the available smartphone apps which enable self-monitoring and remote surveillance of GDM with a particular emphasis on the generation of individualized patient feedback. METHODS: Five databases were searched systematically for any studies evaluating mHealth-supported smartphone solutions for GDM management from study inception until January 2022. The studies were screened and assessed for eligibility of inclusion by 2 independent reviewers. Ultimately, 17 studies were included involving 1871 patients across 11 different countries. The PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews) conceptual framework was adhered to for data extraction and categorization purposes. RESULTS: All studies analyzed as part of this review facilitated direct uploading of data from the handheld glucometer to the downloaded patient-facing smartphone app. Glycemic data were captured by all studies and were reassuringly found to be either improved or noninferior to extant models of hospital-based care. Feedback was delivered in either an automated fashion through in-app communication from the health care team or facilitated through bidirectional communication with the app and hospital portal. Although resource utilization and cost-effective analyses were reported in some studies, the results were disparate and require more robust analysis. Where patient and staff satisfaction levels were evaluated, the response was overwhelmingly positive for mHealth smartphone-delivered care strategies. Emergency cesarean section rates were reduced; however, elective cesarean sections were comparatively increased among studies where the mode of delivery was assessed. Most reviewed studies did not identify any differences in maternal, perinatal, or neonatal health when app-based care was compared with usual in-person review. CONCLUSIONS: This comprehensive scoping review highlights the feasibility, reliability, and acceptability of app-assisted health care for the management of GDM. Although further exploration of the economic benefit is required prior to implementation in a real-world clinical setting, the prospect of smartphone-assisted health care for GDM is hugely promising.

Copper(II) and silver(I)-1,10-phenanthroline-5,6-dione complexes interact with double-stranded DNA: further evidence of their apparent multi-modal activity towards Pseudomonas aeruginosa.

Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (- 7.9 kcal/mol) or minor (- 7.2 kcal/mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M-1) than Ag-phendione (Kapp = 2.79 × 105 M-1) and phendione (Kapp = 1.33 × 105 M-1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa.

A Click Chemistry Approach to Targeted DNA Crosslinking with cis-Platinum(II)-Modified Triplex-Forming Oligonucleotides.

Limitations of clinical platinum(II) therapeutics include systemic toxicity and inherent resistance. Modern approaches, therefore, seek new ways to deliver active platinum(II) to discrete nucleic acid targets. In the field of antigene therapy, triplex-forming oligonucleotides (TFOs) have attracted interest for their ability to specifically recognise extended duplex DNA targets. Here, we report a click chemistry based approach that combines alkyne-modified TFOs with azide-bearing cis-platinum(II) complexes-based on cisplatin, oxaliplatin, and carboplatin motifs-to generate a library of PtII -TFO hybrids. These constructs can be assembled modularly and enable directed platinum(II) crosslinking to purine nucleobases on the target sequence under the guidance of the TFO. By covalently incorporating modifications of thiazole orange-a known DNA-intercalating fluorophore-into PtII -TFOs constructs, enhanced target binding and discrimination between target and off-target sequences was achieved.

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.

Development of Gene-Targeted Polypyridyl Triplex-Forming Oligonucleotide Hybrids.

In the field of nucleic acid therapy there is major interest in the development of libraries of DNA-reactive small molecules which are tethered to vectors that recognize and bind specific genes. This approach mimics enzymatic gene editors, such as ZFNs, TALENs and CRISPR-Cas, but overcomes the limitations imposed by the delivery of a large protein endonuclease which is required for DNA cleavage. Here, we introduce a chemistry-based DNA-cleavage system comprising an artificial metallo-nuclease (AMN) that oxidatively cuts DNA, and a triplex-forming oligonucleotide (TFO) that sequence-specifically recognises duplex DNA. The AMN-TFO hybrids coordinate CuII ions to form chimeric catalytic complexes that are programmable - based on the TFO sequence employed - to bind and cut specific DNA sequences. Use of the alkyne-azide cycloaddition click reaction allows scalable and high-throughput generation of hybrid libraries that can be tuned for specific reactivity and gene-of-interest knockout. As a first approach, we demonstrate targeted cleavage of purine-rich sequences, optimisation of the hybrid system to enhance stability, and discrimination between target and off-target sequences. Our results highlight the potential of this approach where the cutting unit, which mimics the endonuclease cleavage machinery, is directly bound to a TFO guide by click chemistry.

Cu(ii) phenanthroline-phenazine complexes dysregulate mitochondrial function and stimulate apoptosis.

Herein we report an in-depth study on the cytotoxic mechanism of action of four developmental cytotoxic copper(ii) complexes: [Cu(phen)2]2+ (Cu-Phen); [Cu(DPQ)(Phen)]2+ (Cu-DPQ-Phen); [Cu(DPPZ)(Phen)]2+; and [Cu(DPPN)(Phen)]2+ (where Phen = 1,10-phenanthroline, DPQ = dipyrido[3,2-f:2',3'-h]quinoxaline, DPPZ = dipyrido[3,2-a:2',3'-c]phenazine, and DPPN = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine). This complex class is known for its DNA intercalative properties and recent evidence-derived from an in vivo proteomic study-supports the potential targeting of mitochondrial function. Therefore, we focused on mitochondrial-mediated apoptosis related to cytotoxic activity and the potential impact these agents have on mitochondrial function. The Cu(ii) complexes demonstrated superior activity regardless of aromatic extension within the phenazine ligand to the previously demonstrated activity of cisplatin. Unique toxicity mechanisms were also identified in prior demonstrated cisplatin sensitive and resistant cell lines. Double strand breaks in genomic DNA, quantified by γH2AX foci formation, were then coupled with apoptotic gene expression to elucidate the mechanisms of cell death. These results indicate that while DNA damage-induced apoptosis by BAX, XIAP and caspase-9 and -3 expression is moderate for the Cu(ii) complexes when compared to cisplatin, protein targets independent of DNA exert a multimodal mechanistic effect. Significantly, mitochondrial gene expression of oxidative stress, protease expression, and fission/fusion processes-upregulated HMOX, DRP1 and LON, respectively-indicated an increased oxidative damage associated with compromised mitochondrial health upon exposure to these agents. These data support a unique mode of action by these complexes and provide valuable evidence of the developmental potential of these therapeutic inorganic complexes.

Copper bis-Dipyridoquinoxaline Is a Potent DNA Intercalator that Induces Superoxide-Mediated Cleavage via the Minor Groove.

Herein, we report the synthesis, characterisation, X-ray crystallography, and oxidative DNA binding interactions of the copper artificial metallo-nuclease [Cu(DPQ)2(NO3)](NO3), where DPQ = dipyrido[3,2-f:2',3'-h]quinoxaline. The cation [Cu(DPQ)2]2+ (Cu-DPQ), is a high-affinity binder of duplex DNA and presents an intercalative profile in topoisomerase unwinding and viscosity experiments. Artificial metallo-nuclease activity occurs in the absence of exogenous reductant but is greatly enhanced by the presence of the reductant Na-L-ascorbate. Mechanistically, oxidative DNA damage occurs in the minor groove, is mediated aerobically by the Cu(I) complex and is dependent on both superoxide and hydroxyl radical generation. To corroborate cleavage at the minor groove, DNA oxidation of a cytosine-guanine (5'-CCGG-3')-rich oligomer was examined in tandem with a 5-methylcytosine (5'-C5mCGG-3') derivative where 5mC served to sterically block the major groove and direct damage to the minor groove. Overall, both the DNA binding affinity and cleavage mechanism of Cu-DPQ depart from Sigman's reagent [Cu(1,10-phenanthroline)2]2+; however, both complexes are potent oxidants of the minor groove.

Assessment of DNA Topoisomerase I Unwinding Activity, Radical Scavenging Capacity, and Inhibition of Breast Cancer Cell Viability of N-alkyl-acridones and N,N'-dialkyl-9,9'-biacridylidenes.

The anticancer activity of acridone derivatives has attracted increasing interest, therefore, a variety of substituted analogs belonging to this family have been developed and evaluated for their anti-cancer properties. A series of N-alkyl-acridones 1-6 and N,N'-dialkyl-9,9'-biacridylidenes 7-12 with variable alkyl chains were examined for their topoisomerase I activity at neutral and acidic conditions as well as for their binding capacity to calf thymus and possible radical trapping antioxidant activity. It was found that at a neutral pH, topoisomerase I activity of both classes of compounds was similar, while under acidic conditions, enhanced intercalation was observed. N-alkyl-acridone derivatives 1-6 exhibited stronger, dose-dependent, cytotoxic activity against MCF-7 human breast epithelial cancer cells than N,N'-dialkyl-9,9'-biacridylidenes 7-12, revealing that conjugation of the heteroaromatic system plays a significant role on the effective distribution of the compound in the intracellular environment. Cellular investigation of long alkyl derivatives against cell migration exhibited 40-50% wound healing effects and cytoplasm diffusion, while compounds with shorter alkyl chains were accumulated both in the nucleus and cytoplasm. All N,N'-dialkyl-9,9'-biacridylidenes showed unexpected high scavenging activity towards DPPH or ABTS radicals which may be explained by higher stabilization of radical cations by the extended conjugation of heteroaromatic ring system.

Molecular methods for assessment of non-covalent metallodrug-DNA interactions.

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.

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'-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.

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.

In-vivo evaluation of the response of Galleria mellonella larvae to novel copper(II) phenanthroline-phenazine complexes.

Herein we report the in-vivo characterisation and metabolic changes in Galleria mellonella larvae to a series of bis-chelate copper(II) phenanthroline-phenazine cationic complexes of [Cu(phen)2]2+ (Cu-Phen), [Cu(DPQ)(Phen)]2+ (Cu-DPQ-Phen) and [Cu(DPPZ)(Phen)]2+ (Cu-DPPZ-Phen) (where phen = 1,10-phenanthroline, DPQ = dipyrido[3,2-ƒ:2',3'-h]quinoxaline and DPPZ = dipyrido[3,2-a:2',3'-c]phenazine). Our aim was to investigate the influence of the systematic extension of the ligated phenazine ligand in the G. mellonella model as a first step towards assessing the in-vivo tolerance and mode of action of the complex series with respect to the well-studied oxidative chemical nuclease, Cu-Phen. The Lethal Dose50 (LD50) values were established over dose ranges of 2 - 30 µg at 4-, 24-, 48- and 72 h by mortality assessment, with Cu-Phen eliciting the highest mortality at 4 h (Cu-Phen, 12.62 µg < Cu-DPQ-Phen, 21.53 µg < Cu-DPPZ-Phen, 26.07 µg). At other timepoints, a similar profile was observed as the phenazine π-backbone within the complex scaffold was extended. Assessment of both cellular response and related gene expression demonstrated that the complexes did not initiate an immune response. However, Label-Free Quantification proteomic data indicated the larval response was associated with upregulation of key proteins such as Glutathione S-transferase, purine synthesis and glycolysis/gluconeogenesis (e.g. fructose-bisphosphate aldolase and glyceraldehyde-3-phosphate). Both Cu-Phen and Cu-DPQ-Phen elicited a similar in-vivo response in contrast to Cu-DPPZ-Phen, which displayed a substantial increase in nitrogen detoxification proteins and proteins with calcium binding sites. Overall, the response of G. mellonella larvae exposure to the complex series is dominated by detoxification and metabolic proteome response mechanisms.

Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination.

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 (O2•-) 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.

Triggering autophagic cell death with a di-manganese(II) developmental therapeutic.

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.

C 3-symmetric opioid scaffolds are pH-responsive DNA condensation agents.

Herein we report the synthesis of tripodal C3-symmetric opioid scaffolds as high-affinity condensation agents of duplex DNA. Condensation was achieved on both supercoiled and canonical B-DNA structures and identified by agarose electrophoresis, viscosity, turbidity and atomic force microscopy (AFM) measurements. Structurally, the requirement of a tris-opioid scaffold for condensation is demonstrated as both di- (C2-symmetric) and mono-substituted (C1-symmetric) mesitylene-linked opioid derivatives poorly coordinate dsDNA. Condensation, observed by toroidal and globule AFM aggregation, arises from surface-binding ionic interactions between protonated, cationic, tertiary amine groups on the opioid skeleton and the phosphate nucleic acid backbone. Indeed, by converting the 6-hydroxyl group of C3-morphine ( MC3: ) to methoxy substituents in C3-heterocodeine ( HC3: ) and C3-oripavine ( OC3: ) molecules, dsDNA compaction is retained thus negating the possibility of phosphate-hydroxyl surface-binding. Tripodal opioid condensation was identified as pH dependent and strongly influenced by ionic strength with further evidence of cationic amine-phosphate backbone coordination arising from thermal melting analysis and circular dichroism spectroscopy, with compaction also witnessed on synthetic dsDNA co-polymers poly[d(A-T)2] and poly[d(G-C)2]. On-chip microfluidic analysis of DNA condensed by C3-agents provided concentration-dependent protection (inhibition) to site-selective excision by type II restriction enzymes: BamHI, HindIII, SalI and EcoRI, but not to the endonuclease DNase I.

Radical-induced purine lesion formation is dependent on DNA helical topology.

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO•) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5',8-cyclo-2'-deoxyguanosine (5',8-cdG) and 5',8-cyclo-2'-deoxyadenosine (5',8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]4 (TG4T), d[AGGG(TTAGGG)3] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)3A] (mutTel24) were exposed to HO• radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ≫ SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (∼50% solution folded) and mutTel24 (∼90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33 Šwith evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.