Development of novel fluorogenic antioxidants for imaging reactive oxygen species in live cells

This thesis describes our advances in the preparation of lipophilic fluorogenic probes for the specific imaging of reactive oxygen species (ROS) in the lipid membrane of live cells. The work revolves around a simple design involving the preparation of a two segment receptor-reporter type free radical scavenger-fluorophore probe (an off-on fluorogenic antioxidant indicator). The receptor segment in the probe mimics the structure and activity of the naturally occurring antioxidant α-tocopherol. A covalently tethered lipophilic fluorophore serves the purpose of reporting, via emission enhancement, structural changes at the receptor end following its reaction with free radicals.In this thesis we initially explore the off/on molecular switch mechanism operating in the first generation fluorogenic probe we prepared, a Bodipy-Trolox adduct that we named B-TOH. Through mechanistic studies including oxygen uptake, laser flash photolysis, electrochemistry, and DFT calculations we were able to establish that photoinduced electron transfer (PeT) occurs from the receptor to the reporter segment. Molecular imaging of lipid peroxyl radicals in model lipid membranes and in live cells are next reported and we lay out the potential and drawbacks for utilizing B-TOH towards monitoring lipid peroxyl radicals in the lipid membrane of live cells. The information gained in these two chapters allowed us to determine the reporter (fluorophore) electrochemical and spectroscopic requirements that need to be met in order to modulate the sensitivity of B-TOH. We subsequently describe the preparation, spectroscopic, and electrochemical characterization of a family of 16 new Bodipy dyes with tuneable redox potentials and versatile functional groups. Electron withdrawing or donating groups (Et, H, Cl or CN) at positions C2 and C6 enabled tuning the redox potentials within a ca. 0.7 eV window without significantly affecting either the HOMO-LUMO gap or the absorption and emission spectra. Hydroxymethyl or formyl groups at the meso (C8) position in turn provided functionality for covalent tethering to receptors and biomolecules of interest. The dyes can thus be coupled to both electrophiles and nucleophiles. We subsequently generate two new fluorogenic probes (second generation) where we exploit the new Bodipy chromophores synthesized. We further optimize the linker moiety between receptor and reporter segment in order to control the chemical reactivity of the probes. The development of a high-throughput fluorescence assay for monitoring kinetics of peroxyl radical reactions in liposomes is described where the evolution of the fluorescence intensity over time provides a rapid facile method to conduct competitive kinetic studies in the presence of α-tocopherol and analogues We additionally explore the selective targeting of cell organelles, specifically mitochondria (third generation probes). A chapter is devoted to describe the synthesis and mechanistic studies of mitochondria-targeting probes.We conclude this thesis with a chapter dedicated to Conclusions and New directions. The new probes here described will allow a non-invasive spatial and temporal monitoring of the oxidative state in live cell on an organelle level. We may foresee that imaging studies with specific sensors will ultimately enable us to better understand vital links between the chemistry and the biology of ROS.Cette thèse discute de la préparation et du développement de sondes fluorescentes pour l'imagerie spécifique des espèces réactives de l'oxygène (ERO) dans les membranes de cellules vivantes. Le travail est centré sur une conception simple impliquant la préparation d'une sonde piégeuse de radicaux libres en deux segments récepteur-reporteur (un indicateur antioxydant fluorescent arrêt-marche). Le segment récepteur de la sonde mimique la structure et l'activité de l'antioxydant naturel α-tocophérol. Un fluorophore lipophile lié par un lien covalent rapporte, à l'aide d'une augmentation de la fluorescence, les changements structurels du côtè récepteur suite à sa réaction avec des radicaux libres.Cette thèse explore initialement le mécanisme de fonctionnement de l'interrupteur arrêt / marche moléculaire dans la sonde de première génération que nous avons préparée, un produit d'addition Bodipy-Trolox que nous avons nommé B-TOH. Par l'entremise d'études mécanistiques incluant l'absorption d'oxygène, la photolyse éclair au laser, électrochimie et calculs sur la théorie de la fonctionnelle de la densité, nous étions capable de déterminer que le transfert d'électron photoinduit se produit du segment récepteur au segment reporteur. L'imagerie moléculaire de radicaux peroxydes lipidiques dans les membranes lipides modèles et dans les cellules vivantes sont ensuite rapportées et nous mentionnons les inconvénients potentiels reliés à l'utilisation de B-TOH à l'évaluation de radicaux peroxydes lipidiques dans la membrane lipide de cellules vivantes. L'information acquise durant ces deux chapitres nous a permis de déterminer les exigences électrochimiques et spectroscopiques du reporteur (fluorophore) qui doivent être remplies afin de moduler la sensibilité de B-TOH. Par la suite, nous décrivons la préparation ainsi que la caractérisation spectroscopique et électrochimique de 16 nouvelles molécules Bodipy avec des potentiels d'oxydoréduction variables et groupes fonctionnels polyvalents. Des groupes donneur ou accepteur d'électrons (Et, H, Cl ou CN) aux positions C2 et C6 permettent de changer le potentiel d'oxydoréduction dans une fenêtre de ca. 0.7 eV sans changer significativement ni l'écart HOMO-LUMO ni les spectres d'absorption ou d'émission. Des groupes hydroxymethyl ou formyl à la position méso (C8) quant à eux fournissent la fonctionnalité requise pour la formation de liens covalents avec des récepteurs et molécules biologiques d'intérêts. Ces molécules fluorescentes peuvent donc être couplées non seulement aux électrophiles mais, également aux nucléophiles. Nous générons ensuite deux nouvelles sondes fluorescentes (deuxième génération) en exploitant les nouveaux chromophores Bodipy synthétisés. Nous optimisons davantage le groupe caractéristique lieur entre les segments récepteurs et reporteur afin de contrôler la réactivité chimique des sondes. Le développement d'une analyse à haut débit qui suit la cinétique de la réaction des radicaux peroxydes dans des liposomes est décrite où l'évolution de l'intensité de la fluorescence avec le temps nous fournie une méthode rapide et facile pour mener des études cinétiques compétitives dans la présence de α-tocophérol et ses analogues.Nous explorons additionnellement le ciblage sélectif d'organelle cellulaire, spécifiquement des mitochondries (sonde de troisième génération). Un chapitre est dévoué à la description de la synthèse et aux études mécanistiques de sondes cibleuses de mitochondrie. Nous concluons cette thèse par un chapitre dédié aux conclusions et aux nouvelles directions. Les nouvelles sondes décrites ici permettront l'évaluation spatiotemporelle et non-invasive de l'état d'oxydation de cellules vivantes au niveau des organelles. Nous pouvons prévoir que des études d'imageries avec des sondes spécifiques vont éventuellement nous permettre de mieux comprendre des liens vitaux entre la chimie et la biologie des ERO

Fluorogenic α‑Tocopherol Analogue for Monitoring the Antioxidant Status within the Inner Mitochondrial Membrane of Live Cells

We report here the preparation of a lipophilic fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the inner mitochondrial lipid membrane (IMM) and is sensitive to the presence of lipid peroxyl radicals, effective chain carriers in the lipid chain autoxidation. Mito-Bodipy-TOH enables monitoring of the antioxidant status, i.e., the antioxidant load and ability to prevent lipid chain autoxidation, within the inner mitochondrial membrane of live cells. The new probe consists of 3 segments: a receptor, a reporter, and a mitochondria-targeting element, constructed, respectively, from an α-tocopherol-like chromanol moiety, a BODIPY fluorophore, and a triphenylphosphonium cation (TPP). The chromanol moiety ensures reactivity akin to that of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant, while the BODIPY fluorophore and TPP ensure partitioning within the inner mitochondrial membrane. Mechanistic studies conducted either in homogeneous solution or in liposomes and in the presence of free radical initiators show that the antioxidant activity of Mito-Bodipy-TOH is on par with that of α-tocopherol. Studies conducted on live fibroblast cells further show the antioxidant depletion in the presence of methyl viologen (paraquat), a known agent of oxidative stress and source of superoxide radical anion (and indirectly, a causative of lipid peroxidation) within the mitochondria matrix. We recorded a ca. 8-fold emission enhancement with Mito-Bodipy-TOH in cells stressed with methyl viologen, whereas no enhancement was observed in control studies with untreated cells. Our findings underscore the potential of the new fluorogenic antioxidant Mito-Bodipy-TOH to study the chemical link between antioxidant load, lipid peroxidation and mitochondrial physiology

Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on <i>meso</i>-Formyl BODIPY Dyes

We describe herein a fluorescence-based assay to characterize and report on nucleophilic addition to carbonyl moieties and highlight the advantages a fluorescence-based assay and multiplex analysis can offer. The assay relies on the fluorogenic properties of <i>meso</i>-formyl boron-dipyrromethene (BODIPY) dyes that become emissive following nucleophilic addition. A reactivity palette is assembled based on the increasing electrophilic character of five <i>meso</i>-formyl BODIPY compounds tested. We show that increasing rates of emission enhancement correlate with the decreasing electrophilic character of BODIPY dyes in the presence of an acid catalyst and a nucleophile. These results are consistent with the rate-limiting step involving activation of the electrophile. Increasing product formation is shown to correlate with the increasing electrophilic character of the BODIPY dyes, as expected based on thermodynamics. In addition to providing rates of reaction, analysis of the fluorescence parameters for the reaction mixtures, including emission quantum yields and fluorescence lifetimes, enables us to determine the extent of reactant conversion at equilibrium (in our case the estimated yield of a transient species) and the presence of different products, without the need for isolation. We anticipate that our reactivity palette approach, combined with the in-depth fluorescence analysis discussed herein, will provide guidelines toward developing fluorogenic assays of reactivity offering multiplex information, beyond fluorescence intensity

Abstract 2458: Targeting membrane fluidity as a therapeutic strategy in cancer using BPM 31510

Abstract Cancer cells membranes are relatively more fluid compared to healthy cells. Higher fluidity in cancer cells closely relate to their invasive potential, proliferation, and metastatic ability. Pharmacological modulation of membrane fluidity as a novel therapeutic strategy for potential treatment of cancer is investigated in this study. BPM 31510, a proprietary CoQ10 based liposomal formulation currently in clinical trials affects cell membrane fluidity to influence cancer cell survival. To study the effect of BPM 31510 on biophysical parameters of membrane structure in cancer cells, the CoQ10 concentration in the liposomes was systematically increased and the membrane rigidity (Fluorescence Anisotropy) as function of temperature was measured. A progressive (significant, p&amp;lt;0.05) increase in rigidity of liposomal membranes was observed with increase in CoQ10 concentration to a maxima, after which any further increase in CoQ10 concentration resulted in decreases in membrane rigidity. Interestingly, the local maxima in rigidity matched with the concentration of CoQ10 in BPM 31510 formulation. A spectrum of cell lines including cancer of breast, pancreas, liver, prostate, ovarian cell lines and corresponding healthy non-disease control cell lines including fibroblast, smooth muscles, primary prostate were treated with BPM 31510. For all cell lines, there was a temporal increase in cell membrane rigidity between 0-8 hours following treatment with BPM 31510. The magnitude of increase in membrane rigidity was higher for cells with higher initial membrane fluidity for cancer cells compared to healthy cells. Further, magnitude and rate of this reorganization of cell lipid and protein profile, bioenergetics and metabolic state as a maladaptive response of cells to BPM 31510 correlated with the magnitude of change in cell membrane rigidity. Finally, decrease in cell viability was higher for cells with higher membrane fluidity compared to cells with rigid membrane after 48 hours of treatment of BPM 31510. In in vivo preclinical animal model of cancer, there was a strong correlation between BPM 31510 in-plane rigidity measurements to observed bio-distribution and efficacy. This work provides profound insight into CoQ10 effect on cell membrane dynamics and mechanism of action of BPM 31510. The study provides compelling evidence in support of targeting membrane fluidity characteristics in cancer cells as a novel modality with therapeutic potential in the treatment of cancer. Citation Format: Sumit Garg, Sirisha Dhavala, Katerina Krumova, Vivek K. Vishnudas, Joaquin J. Jimenez, Michael Kiebish, Rangaprasad Sarangarajan, Niven R. Narain. Targeting membrane fluidity as a therapeutic strategy in cancer using BPM 31510. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2458. doi:10.1158/1538-7445.AM2015-2458</jats:p