Micael Hardy

HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements.

Methods for the detection and quantitation of hydroethidine (HE) and its oxidation products by HPLC analysis are described. Synthetic methods for preparation of authentic standards (2-hydroxyethidium and diethidium) are provided. Potential applications of the HPLC methods to chemical and biological systems are discussed. Specific examples of chromatograms obtained using UV-Vis absorption, fluorescence, electrochemical, and mass spectrometry detectors are provided. The development of a dual electrochemical and fluorescence detection methodology and its applications are described. The HPLC-based method enables analyses of HE and its oxidation products such as ethidium and the dimeric products of HE. The ramifications of HPLC measurement of HE and its oxidation products in the detection and quantitation of 2-hydroxyethidium, the diagnostic marker product of superoxide and HE, in the intracellular milieu are discussed. Similarly, mitochondria-targeted HE conjugated to a triphenylphosphonium group (Mito-HE or Mito-SOX) also forms oxidation products (dimers of Mito-HE and Mito-E+) that can affect the detection and quantitation of 2-hydroxy-mito-ethidium, the diagnostic marker product of Mito-HE and superoxide in mitochondria.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems HIGH-THROUGHPUT REAL-TIME ANALYSES

Background: Recently, new “targeted” fluorescent probes that react selectively with reactive oxygen and nitrogen species to yield specific products have been discovered. Results: High-throughput fluorescence and HPLC-based methodology for global profiling of ROS/RNS is described. Conclusion: This methodology enables real-time monitoring of multiple oxidants in cellular systems. Significance: The global profiling approach using different ROS/RNS-specific fluorescent probes will help establish the identity of oxidants in redox regulation and signaling. Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.

Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides

Boronates, a group of organic compounds, are emerging as one of the most effective probes for detecting and quantifying peroxynitrite, hypochlorous acid, and hydrogen peroxide. Boronates react with peroxynitrite nearly a million times faster than with hydrogen peroxide. Boronate-containing fluorogenic compounds have been used to monitor real time generation of peroxynitrite in cells and for imaging hydrogen peroxide in living animals. This perspective highlights potential applications of boronates and other fluorescent probes to high-throughput analyses of peroxynitrite and hydroperoxides in toxicological studies.

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps.

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Mitochondria-Targeted Analogues of Metformin Exhibit Enhanced Antiproliferative and Radiosensitizing Effects in Pancreatic Cancer Cells

Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogues (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP(+)). In particular, the analogue Mito-Met10, synthesized by attaching TPP(+) to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells, Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in nontransformed control cells. Moreover, Mito-Met10 potently triggered G1 cell-cycle phase arrest in PDAC cells, enhanced their radiosensitivity, and more potently abrogated PDAC growth in preclinical mouse models, compared with Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including aggressive cancers like PDAC in great need of more effective therapeutic options. Cancer Res; 76(13); 3904-15. ©2016 AACR.

Improving the Trapping of Superoxide Radical with a β-Cyclodextrin– 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) Conjugate

During the last decade, the detection of superoxide radical in biological systems continued to receive increasing attention. The superoxide radical is positioned upstream in many free radical cascades leading to reactive oxygen species (ROS). These species are assumed to act as mediators in various physiological and pathological processes, such as signal transduction, aging, inflammatoryand age-related diseases. The search for reliable tools to study ROS in biological systems is important not only for unraveling their complex contributions but also for the development of diagnostic and therapeutic applications. In recent years, hydroethidine derivatives and other fluorescent probes have been developed successfully as ROS probes. However, the complexity of the dye chemistry in biological systems could lead potentially to misinterpretations concerning the ROS implication, and so there is a need to develop complementary techniques. In the late 70s, the coupled spin trapping/EPR technique (ST/EPR) has been developed for the detection and characterization of free radicals. This technique is based on the trapping of free radicals by diamagnetic spin traps leading to the formation of persistent spin adducts exhibiting EPR patterns characteristic of the radical trapped. A large number of studies using the ST/EPR technique has been devoted to the characterization of superoxide generated by various chemical and biological systems. Until the late 90s, almost all these studies reported the use of the wellknown 5,5-dimethyl-pyrroline-N-oxide (DMPO) as spin trap. However, in biological systems, the short half-lifetime ( 1 min) of the DMPO–superoxide spin adduct and its spontaneous decomposition to the DMPO–hydroxyl spin adduct severely limited the characterization of superoxide by ST/EPR. In the meantime, spin traps such as 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) and (Mito-DEPMPO), which exhibit improved performances compared with those of DMPO, have been developed. However, in biological systems the characterization of superoxide by ST/EPR is still hampered by the readily reduction of the spin adducts to EPR silent compounds by bioreductants and the relatively low trapping rate of superoxide radical (from 0.1 to 85 mol 1 L s , depending of the spin trap) compared with its disproportionation rate (3 10 mol 1 L s 1 at pH 7.3) and its reaction with SOD enzymes (5 10 mol 1 L s ). A few years ago, we showed that the addition of six equivalents of b-cyclodextrin derivatives during spin-trapping experiments resulted in an improved superoxide radical detection, due to the increase of the half-life time of the corresponding spin adduct and to its partial protection to reduction by ascorbate and glutathione/glutathione peroxidase. These results were attributed to the selective inclusion of the superoxide spin adduct (Kspinadduct > Knitrone) preventing further interactions with its immediate surrounding. Aiming at expanding these features to biological systems, we and others recently turned our attention to cyclodextrin functionalized nitrone spin traps. In the recent [a] Dr. M. Hardy, Dr. D. Bardelang, Dr. H. Karoui, Dr. J.-P. Finet, Dr. O. Ouari, Prof. P. Tordo UMR 6264, Laboratoire Chimie Provence, Equipe SREP Universit s d’Aix-Marseille 1, 2, 3 et CNRS Avenue Escadrille Normandie Niemen 13397 Marseille Cedex 20 (France) Fax: (+33) 491-288-758 E-mail : paul.tordo@univ-provence.fr olivier.ouari@univ-provence.fr [b] Prof. A. Rockenbauer Chemical Research Center, Institute for Structural Chemistry 1525 Budapest, PO Box 17 (Hungary) [c] Dr. L. Jicsinszky Cyclolab Ltd, 1525 Budapest, PO Box 435 (Hungary) [d] R. Rosas Spectropole, Universit Paul C zanne Centre Scientifique de Saint-J r me 13397 Marseille cedex 20 (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901342.

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2̇̄), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO− formation in activated macrophages. A new diagnostic marker product for ONOO− is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO− formed from Nox2-derived O2̇̄ and nitric oxide synthase-derived nitric oxide.

Hydropropidine: A novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide

Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr(+)), the reduction product of propidium iodide, for detecting extracellular superoxide (O(2)(•-)). HPr(+) is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O(2)(•-). We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr(+) and allow for exclusive detection of extracellular O(2)(•-). Our results indicate that O(2)(•-) reacts with HPr(+) (k=1.2×10(4) M(-1) s(-1)) to form exclusively 2-hydroxypropidium (2-OH-Pr(2+)) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O(2)(•-) (Zhao et al., Free Radic. Biol. Med.34:1359-1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O(2)(•-) with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O(2)(•-) and HPr(+) is slightly higher than that of HE and O(2)(•-) and is closer to that of Mito-HE and O(2)(•-). Similar to HE, HPr(+) undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fentons reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr(2+)) and the dimeric products (e.g., Pr(2+)-Pr(2+)). In contrast to HE, there was very little intracellular uptake of HPr(+). We conclude that HPr(+) is a useful probe for detecting O(2)(•-) and other one-electron oxidizing species in an extracellular milieu.

Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O2-) and hydrogen peroxide (H2O2) are discussed. Over the last decade, significant progress has been made in O2- and H2O2 detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O2-. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O2- and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H2O2, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O2- and H2O2 in different cellular compartments will improve our understanding of their role in redox signaling.