Redox-active Agents Research Articles

Nitrogen and phosphorus enriched nanoporous carbonaceous material for gas sorption and supercapacitor applications

Nitrogen and phosphorous-enriched heteroatoms-based nanoporous carbonaceous material (designated as HPHMC900) possesses a high surface area of 1668 m2 g−1 was synthesized. HPHMC900 captures 5.6 and 3 mmol g−1 of CO2 and CH4 at 273 K and 1 bar, respectively. Further, the material has 11.95 mmol g−1 of H2 adsorption capability at 77 K and 1 bar. HPHMC900 has been investigated for supercapacitor application. Further, a symmetric supercapacitor device (SSD) fabricated using the HPHMC900, has a specific capacitance (Csp) of 233 F g−1, energy (E) of 46.6 Wh kg−1, and power (P) of 289 W kg−1, respectively. The high Csp and high energy were achieved at low-concentrated aqueous electrolyte (0.5 M KOH), without using any redox-active agents. Further, it shows 100 % coulombic efficiency and initial capacitance retention (94.9 %) after 10,000 cycles. This research elucidates the correlation between high surface area and heteroatom enrichment with enhanced charge storage and gas adsorption efficiency.

Redox-Active Thiocarbonyl Auxiliaries in Ni-Catalyzed Cross-Couplings of Aliphatic Alcohols.

ConspectusThe Barton-McCombie deoxygenation reaction first established the use of O-alkyl thiocarbonyl derivatives as powerful redox-active agents for C(sp3)-O reduction. In recent years, first-row transition metals capable of engaging with alkyl radical intermediates generated from O-alkyl thiocarbonyl derivatives using alternative stoichiometric radical precursors have been developed. Given the ability of select Ni catalysts to both participate in single-electron oxidative addition pathways and intercept alkyl radical intermediates, our group has investigated the use of O-alkyl thiocarbonyl derivatives as electrophiles in novel cross-coupling reactions. After describing related work in this area, this Account will first summarize our entry point into this field. Here, we used the cyclopropane ring as a reporter of leaving group reactivity to aid in the design and optimization of a novel redox-active O-thiocarbamate leaving group for C(sp3)-O arylation. Motivation for this pursuit was driven by the propensity of the cyclopropane ring to undergo ring opening under polar (2e) oxidative addition pathways or to be maintained under single-electron (1e) conditions. Using these guiding principles, we developed a method for the deoxygenative arylation of cyclopropanol derivatives using a Ni catalyst without the need for a stoichiometric external reductant or photocatalyst. We next summarize our evaluation of an alternative redox-active O-thiocarbonyl imidazole auxiliary in a related deoxygenative cross-coupling. This work demonstrated an extension of our initial approach to the deoxygenative arylation of primary and secondary aliphatic alcohol derivatives. A brief mechanistic investigation revealed that this reaction likely proceeds via a distinct mechanism involving direct homolytic C(sp3)-O bond cleavage. We conclude this Account with a summary of work aimed toward a unique approach for thiocarboxylic acid derivative synthesis. This project was inspired by the efficiency of thionoester generation under most of the reaction conditions evaluated in our prior investigations. Using alcohol, amine, or thiol starting materials, which were activated with convenient thiocarbonyl sources in a single step, we optimized for a Ni-catalyzed cross-coupling capable of providing access to a range of thionoester, thioamide, or dithioester products. In summary, our work has revealed the potential of redox-active thiocarbonyl auxiliaries in Ni-catalyzed cross-couplings with C(sp3)-O electrophiles. We anticipate that the continued investigation of aliphatic thiocarbonyl derivatives as radical precursors with alternative single-electron inputs will be an area of continued growth in the years to come.

Effects of redox active agents on cyanocobalamin stability in heated milk

SummaryReducing agents and oxidizing agents have been identified as contributors to vitamin B12 losses associated with food processing and/or storage. We evaluated the effects of redox active agents on cyanocobalamin stability in heat‐treated non‐fat milk and neutral pH buffers. The largest average cyanocobalamin losses occurred in the presence of an added thiol (glutathione, cysteine, dithiothreitol and the thiamine decomposition product 2‐methyl‐3‐furanthiol) or dithionite, whereas cyanocobalamin recoveries generally increased in the presence of an added oxidizing agent. Cyanocobalamin instability in the heat‐treated, non‐fat milk (~40% loss after 90 min at 100 °C) was attributed to a combination of heat, cystine/cysteine, oxygen and a low‐level presence of transition metals, a combination also found in many other foods for which B12 instability has been reported. The proposed cyanocobalamin decomposition pathway – complexation with heat‐exposed cysteine thiolate, cobalt reduction, hydrolysis of the complex, conversion to hydroxocobalamin and irreversible oxidation – follows the sequence described by others.

Phenolic compounds as redox-active exfoliation agents for group VI transition metal dichalcogenides

Simple, cheap, and green exfoliation methods for the mass production of two-dimensional (2D) nanomaterials are still required for their widespread adoption. In this context, this work presents a systematic study regarding the exfoliation of group VI transition metal dichalcogenides (TMDs) using 14 different food-derived phenolic compounds of different structural complexity. Phenolic compounds have demonstrated to successfully assist the liquid phase exfoliation conferring unique redox functionalities to the TMDs nanosheets. The most performant exfoliating agents have been carefully optimized and demonstrated to produce stable 2D nanoflakes dispersions with peculiar electrochemical properties. This work proves how properly selected natural stabilizing agents allow an effective TMDs exfoliation, giving rise to hybrid organic/inorganic nanomaterials with functional electrochemical and additional catalytic features. The reported approach opens new paths for the green production of exfoliated 2D functional nanomaterials for sensing, biosensing, and energy applications.

The ArsH Protein Product of the Paracoccus denitrificans ars Operon Has an Activity of Organoarsenic Reductase and Is Regulated by a Redox-Responsive Repressor.

Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.

Predicting pKa Values of Quinols and Related Aromatic Compounds with Multiple OH Groups

Quinonoid compounds play central roles as redox-active agents in photosynthesis and respiration and are also promising replacements for inorganic materials currently used in batteries. To design new quinonoid compounds and predict their state of protonation and redox behavior under various conditions, their pKa values must be known. Methods that can predict the pKa values of simple phenols cannot reliably handle complex analogues in which multiple OH groups are present and may form intramolecular hydrogen bonds. We have therefore developed a straightforward method based on a linear relationship between experimental pKa values and calculated differences in energy between quinols and their deprotonated forms. Simple adjustments allow reliable predictions of pKa values when intramolecular hydrogen bonds are present. Our approach has been validated by showing that predicted and experimental values for over 100 quinols and related compounds differ by an average of only 0.3 units. This accuracy makes it possible to select proper pKa values when experimental data vary, predict the acidity of quinols and related compounds before they are made, and determine the sites and orders of deprotonation in complex structures with multiple OH groups.

Utilization of redox modulating small molecules that selectively act as pro-oxidants in cancer cells to open a therapeutic window for improving cancer therapy

There is a rapidly growing body of literature supporting the notion that differential oxidative metabolism in cancer versus normal cells represents a metabolic frailty that can be exploited to open a therapeutic window into cancer therapy. These cancer cell-specific metabolic frailties may be amenable to manipulation with non-toxic small molecule redox active compounds traditionally thought to be antioxidants. In this review we describe the potential mechanisms and clinical applicability in cancer therapy of four small molecule redox active agents: melatonin, vitamin E, selenium, and vitamin C. Each has shown the potential to have pro-oxidant effects in cancer cells while retaining antioxidant activity in normal cells. This dichotomy can be exploited to improve responses to radiation and chemotherapy by opening a therapeutic window based on a testable biochemical rationale amenable to confirmation with biomarker studies during clinical trials. Thus, the unique pro-oxidant/antioxidant properties of melatonin, vitamin E, selenium, and vitamin C have the potential to act as effective adjuvants to traditional cancer therapies, thereby improving cancer patient outcomes.

Metal Complexes as Promising Agents for Biomedical Applications.

In this review article, a brief overview of novel metallotherapeutic agents (with an emphasis on the complexes of essential biometals) promising for medical application is presented. We have also focused on the recent work carried out by our research team, specifically the development of redox-active antimicrobial complexes of sterically hindered diphenols with some essential biometals (copper, zinc, nickel). The complexes of essential metals (manganese, iron, cobalt, nickel, copper, zinc) described in the review show diverse in vitro biological activities, ranging from antimicrobial and antiinflammatory to antiproliferative and enzyme inhibitory. It is necessary to emphasize that the type of organic ligands in these metal complexes seems to be responsible for their pharmacological activities. In the last decades, there has been a significant interest in synthesis and biological evaluation of metal complexes with redox-active ligands. A substantial step in the development of these redox-active agents is the study of their physicochemical and biological properties, including investigations in vitro of model enzyme systems, which can provide evidence on a plausible mechanism underlying the pharmacological activity. When considering the peculiarities of the pharmacological activity of the sterically hindered diphenol derivatives and their nickel(II), copper(II) and zinc(II) complexes synthesized, we took into account the following: (i) all these compounds are potential antioxidants and (ii) their antimicrobial activity possibly results from their ability to affect the electron-transport chain. We obtained novel data demonstrating that the level of antibacterial and antifungal activity in the series of the above-mentioned metal-based antimicrobials depends not only on the nature of the phenolic ligands and complexing metal ions, but also on the lipophilicity and reducing ability of the ligands and metal complexes, specifically regarding the potential biotargets of their antimicrobial action - ferricytochrome c and the superoxide anion radical. The combination of antibacterial, antifungal and antioxidant activity allows one to consider these compounds as promising substances for developing therapeutic agents with a broad spectrum of activities.

Pro- and anti-oxidant properties of near-infrared (NIR) light responsive carbon nanoparticles

Elemental carbon nanomaterials (ECNMs) are redox active agents that can be exploited to purposely modify the redox balance of cells. Both pro- or antioxidant properties have been reported. However, to the best of our knowledge, there are not comprehensive studies exploring both properties on the same material in view of a potential application in medicine. At the same time, the effect of the bulk structure on the pro/antioxidant properties is poorly known. Here, carbon nanoparticles (CNPs) derived by glucose with definite size and shape have been prepared, and their redox properties evaluated in cell free systems in the dark or following activation with a Near Infrared (NIR) laser beam (945 nm, 1.3 W/cm2). We found that, when irradiated with NIR, CNPs efficiently generate heat and singlet oxygen (1O2), a property that can be exploited for dual photo-thermal (PT)/photodynamic (PD) therapy in cancer. On the other hand, in the absence of photo-activation, CNPs react with both oxidant (hydroxyl radicals) and antioxidant (glutathione) species. When tested on a murine macrophages cell line (RAW 264.7) CNPs were clearly antioxidant. Furthermore, albeit efficiently internalized, CNPs do not exert cytotoxic effect up to 80 µg/ml and do not exacerbate TNF-α-mediated inflammation. Overall, the results reported herein suggest that CNPs may represent a new class of safe nanomaterials with potential applications in medicine.

Hierarchical Assemblies of Supramolecular Coordination Complexes.

Hierarchical self-assembly (HAS) is a multilevel organization process that first assembles elementary molecular units into ordered secondary structures via noncovalent interactions, which further act as the building blocks to form more complex multifunctional superstructures at the next level(s). The HAS strategy has been used as a versatile method for the preparation of soft-matter nanoarchitectures of defined size and morphologies, tunable luminescence, and biological importance. However, such preparation can be greatly simplified if well-defined dynamic structures are employed as the cores that upon linking form the desired nanoarchitectures. Discrete supramolecular coordination complexes (SCCs) with well-defined shapes, sizes, and internal cavities have been widely employed to construct hierarchical systems with functional diversity. This Account summarizes the prevailing strategies used in recent years in the preparation of SCC-based HASs and illustrates how the combination of dynamic metal-ligand coordination with other interactions was used to obtain hierarchical systems with interesting properties. HASs with dual orthogonal interactions involving coordination-driven self-assembly and hydrogen bonding/host-guest interaction generally result in robust and flexible supramolecular gels. Likewise, hybridization of SCCs with a suitable dynamic covalent network via a hierarchical strategy is useful to prepare materials with self-healing properties. The intrinsic positive charges of the SCCs also make them suitable precursors for the construction of HASs via electrostatic interactions with negatively charged biological/abiological molecules. Furthermore, the interplay between the hydrophilic and lipophilic characters of HASs by varying the number and spacial orientation of alkyl/oxyethylene chains of the SCC is a simple yet controllable approach to prepare ordered and tunable nanostructures. Certain SCC-cored hierarchical systems exhibit reversible polymorphism, typically between micellar, nanofiber, and vesicular phases, in response to various external perturbations: heat, photoirradiation, pH-variance, redox-active agents, etc. At the same time, multiple noncovalent interaction mediated HASs are growing in numbers and are promising candidates for obtaining functionally diverse materials. The photophysical properties of SCC-based HASs have been used in many analytical applications. For example, embedding tetraphenylethene (TPE)-based pyridyl ligands within metallo-supramolecular structures partially restricts the molecular rotations of its phenyl rings, endowing the resultant SCCs with weak emissions. Further aggregation of such HASs in suitable solvents results in a marked enhancement in emission intensity along with quantum yields. They act as sensitive sensors for different analytes, including pathogens, drugs, etc. HASs are also useful to develop multidrug systems with cooperative chemotherapeutic effects. Hence, the use of HASs with theranostic SCCs combining cell-imaging agents and chemotherapeutic scaffolds is a promising drug delivery strategy for cancer theranostics. At the same time, their responsiveness to stimuli, oftentimes due to the dynamic nature of the metal-ligand interactions, play an important role in drug release via a disassembly mechanism.

Abstract 3219: Sensitizing hypoxic small cell lung cancer cells to radiation and hydrogen peroxide-producing agents using CuATSM

Abstract Cancer cells have increased steady state levels of reactive oxygen species (ROS; O2•- and H2O2) compared to normal cells. It has been proposed that using redox active agents that further increase ROS levels will result is selective cancer cell death. This metabolic frailty can be targeted using drugs deemed safe for human use, ascorbate (ASC) and disulfiram (DSF), via a mechanism of H2O2 production. CuATSM is a drug being used in clinical trials to treat ALS disease. Imaging studies have shown that CuATSM preferentially concentrates in hypoxic tissues, releasing its Cu after entering cells. Copper (Cu) can participate in oxidation reactions that result in highly toxic hydroxyl radicals. Tumors often have areas of hypoxic tissue that exhibit resistance to ionizing radiation (IR). Our hypothesis is that CuATSM can be used to sensitize hypoxic regions of tumors to IR, ASC and/or DSF. Method: H2O2 flux after addition of ASC or DSF was measured in small cell lung cancer (SCLC) cells DMS53 using the 3-aminotriazole method. The Cu uptake of these cells was measured in normoxia and hypoxia after treatment with CuATSM. DMS53 and DMS273 (SCLC lines) were treated with ASC, CuATSM, and/or DSF+CuSO4 at varying oxygen tensions with and without IR and clonogenic assays were performed. Results: Pharmacologically relevant dosing of DSF and ASC resulted in significantly higher fluxes of H2O2 compared to control. Both DSF and ASC enhanced IR clonogenic cell death in SCLC lines. Treatment with CuATSM resulted in increased intracellular Cu and enhanced cell death from ASC and IR in hypoxic conditions. Conclusion: These observations support the hypothesis that the differences in steady-state level of ROS in small cell lung cancer cells can be exploited to develop effective therapies using ASC and DSF. Furthermore CuATSM can enhance responses in hypoxic tumor tissues to both IR and ASC. (Supported by P50 CA174521, T32 CA078586, P30 CA086862 and Carver Research Program of Excellence in Redox Biology and Medicine.) Citation Format: Sebastian Sciegienka, Samuel Rodman, Ann Tomanek-Chalkley, Kelly C. Falls, Douglas R. Spitz, Melissa A. Fath. Sensitizing hypoxic small cell lung cancer cells to radiation and hydrogen peroxide-producing agents using CuATSM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3219.

Abstract 1342: Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells

Abstract Mitochondria-targeted drugs are designed to accumulate in the mitochondrion to modulate metabolism. Triphenylphosphonium (TPP) conjugated agents are a class of mitochondria-targeted drugs that induce mitochondrial dysfunction in cancer cells. Mitochondrial dysfunction is known factor that facilitates the onset of mitophagy. However, the activation of mitophagy to remove drug-induced mitochondrial damage is unknown. Here, we subjected breast MDA-MB-231 cancer cells and MCF-12A noncancerous cells to a series of mitochondria-targeted redox-active agents (MTA) that contain TPP to investigate the onset and activation of autophagy, mitophagy and mitochondrial dysfunction. Collectively, we identified three different MTAs from the series that sequentially damage the mitochondria, enhance autophagic flux, and activate mitophagy selectively in MDA-MB-231 cells. Furthermore, we generated stably expressing mt-mKeima MDA-MB-231 and MCF-12A cell lines to establish a mitophagy flux assay for screening new agents. FACS analysis of mt-mKeima, a pH-sensitive fluorophore, revealed MTAs induced lysosomal dependent mitochondrial degradation and the presence of bafilomycin, a lysosomal inhibitor, suppressed MTA-induced mitophagy. To confirm MTAs induced mitochondrial autophagy, we identified mitochondria located within autophagosomes using confocal microscopy, an endogenous autophagy-related protein complex by coimmunoprecipitation and mitochondrial PINK1 accumulation using immunoblot. These in vitro studies were translated into an in vivo rat SST-2 xenograph breast cancer model to demonstrate that mitochondrial PINK1 accumulates in rat tumor tissue following MitoQ treatment. These data suggest that mitochondria-targeted agents selectively caused mitochondrial depolarization, PINK1 accumulation and mitophagy in MDA-MB-231 cancer cells as compared to MCF-12A noncancerous cells. Citation Format: Thomas Biel, Ashutosh Rao. Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1342.

Targeting Oncogenic Nuclear Factor Kappa B Signaling with Redox-Active Agents for Cancer Treatment.

Nuclear factor kappa B (NF-κB) signaling is essential under physiologically relevant conditions. However, aberrant activation of this pathway plays a pertinent role in tumorigenesis and contributes to resistance. Recent Advances: The importance of the NF-κB pathway means that its targeting must be specific to avoid side effects. For many currently used therapeutics and those under development, the ability to generate reactive oxygen species (ROS) is a promising strategy. As cancer cells exhibit greater ROS levels than their normal counterparts, they are more sensitive to additional ROS, which may be a potential therapeutic niche. It is known that ROS are involved in (i) the activation of NF-κB signaling, when in sublethal amounts; and (ii) high levels induce cytotoxicity resulting in apoptosis. Indeed, ROS-induced cytotoxicity is valuable for its capabilities in killing cancer cells, but establishing the potency of ROS for effective inhibition of NF-κB signaling is necessary. Indeed, some cancer treatments, currently used, activate NF-κB and may stimulate oncogenesis and confer resistance. Thus, combinatorial approaches using ROS-generating agents alongside conventional therapeutics may prove an effective tactic to reduce NF-κB activity to kill cancer cells. One strategy is the use of thiosemicarbazones, which form redox-active metal complexes that generate high ROS levels to deliver potent antitumor activity. These agents also upregulate the metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), which functions as an NF-κB signaling inhibitor. It is proposed that targeting NF-κB signaling may proffer a new therapeutic niche to improve the efficacy of anticancer regimens.

Abstract 1505: Breast cancer cells treated with mitochondria targeted redox active agents induce mitophagy

Abstract Triphenylphosphonium (TPP) conjugated agents induce mitochondrial dysfunction in cancer cells. However, the onset of mitophagy to remove the dysfunctional mitochondria is unknown. Here, a series of mitochondria targeted redox active agents (MTA) that contain TPP were used to investigate mitochondrial dysfunction and mitophagy in MDA-MB-231 cells as compared to MCF-12A cells. Three different MTAs were identified to induced mitochondrial depolarization and enhanced autophagic flux selectively in MDA-MB-231 cells. Mitochondrial reactive oxygen species generation and respiration indicated that MDA-MB-231 cells had a heighten sensitivity to MTA treatments. Using stably expressing mt-mKeima MDA-MB-231 and MCF-12A cell lines, a non-cell type selective decline in mitochondrial alkalinity and altered mitochondrial morphology was detected. Furthermore, FACS analysis of mt-mKeima revealed MTAs induced lysosomal dependent mitochondrial degradation in the presence of Bafilomycin, a lysosomal inhibitor. To confirm MTAs induced mitochondrial autophagy in MDA-MB-231 cells, MitoTracker Red preloaded GFP-LC3 expressing MDA-MB-231 cells were used to identify autophagosomes containing mitochondria using confocal microscopy, in addition to coimmunoprecipitation for the detection of an endogenous autophagy-related protein complex, and immunoblot to for mitochondrial PINK1 accumulation. To translate these in vitro studies to an in vivo rat SST-2 xenograph breast cancer model, tumor mitochondrial extracts from rats treated with MitoQ demonstrated an accumulation of mitochondrial PINK1. Collectively, these data suggest that mitochondrial agents selectively caused mitochondrial depolarization, PINK1 accumulation and mitophagy in MDA-MB-231 cancer cells as compared to MCF-12A healthy cells. Citation Format: Thomas Biel, Ashutosh Rao. Breast cancer cells treated with mitochondria targeted redox active agents induce mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2017-1505

P 106 - Targeting of O2●-/NO ratio as a strategy to improve energy metabolism in diabetes

Redox homeostasis disturbance, mainly caused by increased O 2 •– level and/or decreased NO bioavailability, represents an important contributing factor in the (ethio)pathology of diabetes. We examined whether targeting of O 2 •– /NO ratio by two redox-modulating compounds, L-arginine (substrate for NO synthases) and Mn(II) pentaazamacrocyclic mimics of SOD, could improve diabetic state and associated impairment of energy metabolism. Multiple beneficial effects of L-arginine and SOD mimics in alloxan-induced diabetes were observed. First of all, acting directly on pancreas, L-arginine and SOD mimic induce β-cells regeneration. In skeletal muscle, those treatments restore diabetes-induced impairment in mitochondrial energy metabolism (OXPHOS) and glucose transport (GLUT4) by targeting AMPKα signaling. Similarly, SOD mimic improves energy metabolism in hippocampus of diabetic rats. L-arginine and SOD mimic stabilize diabetes-induced redox disbalance in diabetic skin acting on NO producing (NOS) and O 2 •– /H 2 O 2 removing (MnSOD and GPx) systems. The data suggest that fine settings of O 2 •– /NO ratio by L-arginine and SOD mimic could have beneficial implications for several pathological hallmarks of diabetes: impaired insulin synthesis and sensitivity as well as accompanying metabolic complications and speak in favor of therapeutic potential of these redox-active agents in diabetic conditions.

Determination of glutathione redox potential and pH value in subcellular compartments of malaria parasites

The malaria parasite Plasmodium falciparum is exposed to multiple sources of oxidative challenge during its complex life cycle in the Anopheles vector and its human host. In order to further elucidate redox-based parasite host cell interactions and mechanisms of drug action, we targeted the genetically encoded glutathione redox sensor roGFP2 coupled to human glutaredoxin 1 (roGFP2-hGrx1) as well as the ratiometric pH sensor pHluorin to the apicoplast and the mitochondrion of P. falciparum. Using live cell imaging, this allowed for the first time the determination of the pH values of the apicoplast (7.12±0.40) and mitochondrion (7.37±0.09) in the intraerythrocytic asexual stages of the parasite. Based on the roGFP2-hGrx1 signals, glutathione-dependent redox potentials of −267mV and −328mV, respectively, were obtained. Employing these novel tools, initial studies on the effects of redox-active agents and clinically employed antimalarial drugs were carried out on both organelles.

Mimicking Photosynthesis with Electrode-Supported Lipid Nanoassemblies.

The grand scale, ultimate efficiency, and sustainability of natural photosynthesis have inspired generations of researchers in biomimetic light energy utilization. As an essential and ubiquitous component in all photosynthetic machinery, lipids and their assemblies have long been recognized as powerful molecular scaffolds in building artificial photosynthetic systems. Model lipid bilayers, such as black lipid membranes and liposomes (vesicles), have been extensively used to host natural as well as synthetic photo- and redox-active species, thereby enabling key photosynthetic processes, such as energy transfer and photoinduced electron transfer, to be examined in well-defined, natural-like membrane settings. Despite their long history, these lipid models remain highly relevant and still enjoy wide practice today. In this Account, we share with the reader our recent effort of introducing electrode-supported lipid nanoassemblies as new lipid models into photosynthesis biomimicking. This line of research builds off several solid-supported lipid bilayer architectures established relatively recently by workers in membrane biophysics and reveals important new features that match and sometimes exceed what earlier lipid models are capable of offering. Here, our eight-year exploration unfolds in three sections: (1) New photosynthetic mimics based on solid-supported lipid bilayers. This systematic effort has brought three solid-supported bilayers into artificial photosynthesis research: lipid bilayers supported on indium tin oxide electrodes, hybrid bilayers, and tethered lipid bilayers formed on gold. Quantitative on-electrode deposition of various photo- and redox-active agents, including fullerene, Ru(bpy)32+, and porphyrin, is realized via liposomal hosts. Vectorial electron transfer across single lipid-bilayer leaflets is achieved between electron donor/acceptor directionally organized therein, taking advantage of multiple incorporation sites offered by these bilayers as well as their sequential formation on electrodes. Supported on electrodes, these bilayers uniformly afford reliable photocurrent generation and modular system design. (2) Gold-supported hybrid bilayers as a powerful model platform for probing biomembrane-associated photoelectrochemical processes. These hybrid nanostructures consist of one alkanethiol (or substituted alkanethiol) and one lipid monolayer, whose chemical identity and makeup can be separately controlled and modified. Such precise molecular organization and flexible formation, in turn, enable a series of physicochemical parameters key to photosynthetic processes to be explicitly examined and cross-compared. A few such examples, based on donor/acceptor distance and loading, interfacial dipole, and redox level, are included here to illustrate the usefulness and versatility of this system. (3) Mimicking photosynthesis with supercomplexed lipid nanoassemblies. This research effort was motivated to address the low light absorption suffered by single-bilayer based photosynthetic mimics and has yielded a new lipid-based approach to mimicking Nature's way of organizing multiple photosynthetic subunits. Rhodamine and fullerene assembled within these lipid supercomplexes display robust electronic communication. The remarkable possibility of using lipid matrix to further improve photoconversion efficiency is revealed by cholesterol, whose addition triggers exciton formation that promotes faster energy and electron transfer in these lipid nanoassemblies.

Biological Relevance of Free Radicals and Nitroxides.

Nitroxides are stable, kinetically-persistent free radicals which have been successfully used in the study and intervention of oxidative stress, a critical issue pertaining to cellular health which results from an imbalance in the levels of damaging free radicals and redox-active species in the cellular environment. This review gives an overview of some of the biological processes that produce radicals and other reactive oxygen species with relevance to oxidative stress, and then discusses interactions of nitroxides with these species in terms of the use of nitroxides as redox-sensitive probes and redox-active therapeutic agents.

Abstract 3005: The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity

Abstract Reactive oxygen species (ROS) are natural byproducts of mitochondrial oxidative phosphorylation. Dysfunction in electron shuttling leads to enhanced ROS production that is notorious for inflicting damage to macromolecules. Mitochondrial DNA (mtDNA) is particularly sensitive to ROS as it is located in close proximity to the respiratory chain. Additionally, mtDNA lack histones which are known to provide protection from ROS. Mitochondria also have limited DNA repair mechanisms making damage to mtDNA more detrimental to mitochondrial physiology. Recent evidence suggests that oxidative insult, leads to mtDNA degradation to prevent the accumulation of mutagenic base lesions, which is enabled by the high redundancy of mtDNA. The primary objectives of this study were to determine the impact of redox-active mitochondrial-targeted chemotherapeutic agents on mitochondrial physiology and mtDNA integrity. All experiments were performed in two cancer cell lines [MDA-MB-231 (breast) and H23 (lung)] that were treated with 2 uM of the mitochondrial-targeted redox-active compounds: MitoTempol [C4 (MT4)], MitoQuinone (MQ) and MitoChromanol-Acetate (MCA) for 1 or 24 hrs. Mitochondrial status was determined by measuring mitochondrial membrane potential, superoxide production, oxygen consumption rate (OCR) and protein levels of the respiratory chain complexes using the JC-1 and MitoSOX assays, Seahorse XF analysis, and immunoblotting, respectively. Next, mtDNA integrity was analyzed by quantifying the degree of mtDNA fragmentation using PCR amplification of a 10kb fragment compared to a short 100bp fragment. Moreover, changes in the mtDNA replication machinery were assessed using immunoblotting. MQ and MCA induced a loss in mitochondrial function and mtDNA replication machinery as indicated by a reduction in membrane potential, OCR and protein levels. Furthermore, increases in superoxide levels and mtDNA fragmentation were observed with MQ and MCA treatment. Finally, we assessed mitochondrial content using two methods. The first method being the ratio of mitochondrial to nuclear DNA, determined by qPCR of short (∼100bp) nuclear and mitochondrial sequences. The second method was the ratio of mitochondrial outer membrane proteins to cytoplasmic housekeeping proteins, determined by immunoblotting. Both mitochondria DNA and outer membrane proteins decreased with MQ and MCA treatment suggesting that redox-active mitochondrial-targeted agents may reduce the mitochondrial content in cancer cells. Citation Format: Kaytee L. Pokrzywinski, Thomas G. Biel, Dmitry Kryndushkin, V. Ashutosh Rao. The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3005.

Abstract 1090: microRNA regulation of Nrf2 and the antioxidant response in breast cancer cells following redox therapy

Abstract Mitoquinone (MitoQ), a redox-active mitochondrially targeted chemotherapy, activates autophagy through the production of reactive oxygen species (ROS) in breast cancer cells. Studies have also shown a concomitant activation of Nrf2 and the antioxidant response with MitoQ treatment. Nrf2 (nuclear factor (erythroid-derived 2)-like 2) is a transcription factor that regulates the antioxidant response element. Drugs that stimulate Nrf2 are ideal for the treatment of diseases induced by oxidative stress; however, over-activation of Nrf2 may promote tumor growth and diminish the effects of redox active chemotherapies. Therefore, understanding the relationship between autophagy and oxidative stress is crucial for determining the fate of cancer cells exposed to redox-active oncology agents. The aim of this study was to evaluate microRNA (miRNA) as a link between oxidative stress and autophagy through modulation of Nrf2 activity. microRNA are endogenous small (18-22 base pairs long) double stranded RNA that bind to the 3’ UTR of target mRNA. miRNA function through translational repression via mRNA recycling and degradation. To this aim we set out on finding miRNA that suppress the antioxidant response, modulated by Nrf2, in cancer cells after redox therapy with the goal of inducing a death response. After 24 hours of non-target pool or Nrf2 silencing, using small interfering RNAs, MDA-MB-231 cells were treated with DMSO or MitoQ for 18 hours. RNA was extracted and miRNAs were identified using Illumina RNA sequencing. DEseq revealed 97 miRNAs that were differentially expressed at α < 0.05. Biologically significant miRNAs were then distinguished by using a Log2 fold change cut off of -1.0 < and < 1.0, which identified 39 microRNAs. Since miRNA should be high when their target is low we were predominantly interested in the 15 miRNAs that were down-regulated (DMSO:MitoQ). 7 of the 15 down-regulated miRNAs were also down-regulated in the presence of Nrf2 silencing in both the DMSO and MitoQ treatments. We are currently investigating these 7 miRNAs in terms of the specific effect they have on the Nrf2 pathway during redox therapy. Citation Format: Francesca Mascia, Kaytee L. Pokrzywinski, V. Ashutosh Rao. microRNA regulation of Nrf2 and the antioxidant response in breast cancer cells following redox therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1090.