Academic literature on the topic 'Anionophore'

Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis. However, so far, the measurement of bicarbonate concentration changes in living cells has only been possible by measuring intracellular pH changes. In this article, we report the development of a genetically encoded pH-independent fluorescence-based single-use sensory cellular test system for monitoring intracellular bicarbonate concentration changes in living cells. We describe the usefulness of the developed biosensor in characterizing the bicarbonate transport activities of anionophores—small molecules capable of facilitating the membrane permeation of this anion. We also demonstrate the ability of the bicarbonate sensory cellular test system to measure intracellular bicarbonate concentration changes in response to activation and specific inhibition of wild-type human CFTR protein when co-expressed with the bicarbonate sensing and reporting units in living cells. A valuable benefit of the bicarbonate sensory cellular test system could be the screening of novel anionophore library compounds for bicarbonate transport activity with efficiencies close to the natural anion channel CFTR, which is not functional in the respiratory epithelia of cystic fibrosis patients.

Oral and lung cancer are included in the most prevalent respiratory diseases, being the latter one of the main causes of mortality worldwide. Despite new advances in diagnosis and clinical care, success of conventional treatments is still limited since patients end up developing resistances and presenting recurrences. Due to the therapeutic limitations to address these pathologies, it is necessary to identify new compounds, with different mechanisms of action and greater efficiency to overcome these neoplasms. Cancer cells acquire a series of characteristics during carcinogenesis, as a reversed pH gradient compared to normal cells, which favors cancer progression promoting proliferation and evasion of apoptosis. Recently, a new therapeutic strategy against cancer, which involves the modulation of intracellular pH has been proposed. Accordingly, our research group is focused on analyzing the potential of anion transporter compounds as new chemotherapeutic agents, since they possess the ability to selectively lower the intracellular pH. In particular, this doctoral thesis is focused on the anticancer effect characterization, at the cellular and molecular level, of novel anionophores, derived from natural compounds known as tambjamines. Firstly, the effect of the synthetic analogues on cell viability in oral and pulmonary cancer cell lines, as well as in cancer stem cells derived from patients’ tumors, has been determined, proving to be potent cytotoxic agents. Likewise, the implication of ion homeostasis disruption driven by these compounds has been studied at the cellular level. In this regard, it has been characterized how the compounds induce lysosomal alkalization and cause a massive vacuolization in the cytoplasm, which is consistent with mitochondrial swelling. These two phenomena led to the loss of function of both organelles. At the same time, the mechanisms of action linked to the osmotic imbalance caused by the compounds have been studied in detail. On one hand, it has been observed how these anionophores increase the activity of proteins related to cellular stress response, and how the apoptotic pathway is triggered, although this is not directly responsible for the death of the entire cell population. Likewise, an accumulation of autophagosomes, has been detected after treatment with these compounds, which might be related to the blockade of autophagy, consequence of the lysosomal failure after treatment with these compounds. Moreover, it has also been observed how the cells treated with these anionophores lose the integrity of the plasma membrane, indicating that the cytotoxic process culminates in necrosis in the vast majority of the cell population. Besides this, in this doctoral thesis, AKT protein has been identified as a potential molecular target of one of our compounds, using in silico docking experiments. In turn, it has been corroborated by surface plasmon resonance that the binding affinity between the selected tambjamine analogue and AKT is high, in the micromolar range. Likewise, a decrease in protein activity and in the total amount of AKT protein has been detected after treatment. Furthermore, the main signaling pathways involved in the cytotoxic process have been elucidated studying the modifications of miRNA expression patterns after the treatment with tambjamine, being the most affected PI3K / AKT, apoptosis and autophagy. Finally, to complete the preclinical studies, the toxicity and efficacy of these compounds in murine models of lung cancer have been evaluated in in vivo preliminary studies, showing a potent antitumor capacity in both ectopic and orthotopic models. Overall, these synthetic analogues of tambjamine may be considered a good tool to induce death in cancer cells through a new therapeutic strategy that modifies the intracellular pH and these compounds may become a good therapeutic option for apoptosis-resistant tumors.
El cáncer de cavidad oral y de pulmón se engloban dentro de las enfermedades de vías respiratorias más comunes, siendo este último una de las principales causas de mortalidad en el mundo. A pesar de los nuevos avances en el diagnóstico y la atención clínica, el éxito de los tratamientos convencionales es todavía limitado, ya que los pacientes acaban desarrollando resistencias y presentando recidivas. Debido a las limitaciones terapéuticas para abordar estas patologías, es necesario identificar nuevos compuestos con diferentes mecanismos de acción y mayor eficacia para combatir este tipo de neoplasias. Las células cancerosas adquieren una serie de características durante la carcinogénesis, como un gradiente de pH invertido en comparación con las células normales, lo cual favorece la progresión del cáncer mediante el aumento de la proliferación y la evasión de la apoptosis. Recientemente se ha propuesto una nueva estrategia terapéutica contra el cáncer la cual implica la modulación del pH intracelular. Es por esto que nuestro grupo de investigación estudia el potencial de compuestos transportadores de aniones como nuevos agentes quimioterapéuticos, ya que poseen la capacidad de disminuir el pH intracelular selectivamente. En concreto, este trabajo de tesis se ha centrado en caracterizar el efecto anticanceroso de anionóforos, compuestos transportadores de aniones, derivados de moléculas naturales llamadas tambjaminas, tanto a nivel celular como molecular. En primer lugar, se ha determinado el efecto de los análogos sintéticos de tambjamina sobre la viabilidad celular en líneas de cáncer oral y pulmonar, así como en células madre cancerosas derivadas de tumores de pacientes, demostrando ser potentes agentes citotóxicos. A su vez, se ha estudiado qué implicación tiene la pérdida de la homeostasis iónica impulsada por estos compuestos a nivel celular. En este sentido, se ha caracterizado cómo los compuestos inducen la alcalinización de los lisosomas, y provocan una vacuolización masiva en el citoplasma que se corresponde con el hinchamiento de la mitocondrias. Estos dos fenómenos conllevan la pérdida de función de ambos orgánulos. Al mismo tiempo, se han estudiado en detalle los mecanismos de acción ligados al desequilibrio osmótico provocado por los compuestos. Por una parte, se ha observado cómo estos anionóforos estimulan un aumento en la actividad de proteínas relacionadas con respuesta a estrés celular, y cómo provocan la activación de la vía apoptótica, sin que ésta sea la responsable directa de la muerte de toda la población celular. Conjuntamente, se ha detectado una acumulación de autofagosomas, relacionado con el bloqueo de la autofagia, consecuencia del fallo lisosomal tras el tratamiento con estos compuestos. A su vez, se ha podido observar cómo las células tratadas con estos anionóforos pierden la integridad de la membrana plasmática, indicando que el proceso citotóxico culmina en necrosis en una gran mayoría de la población celular. Por otro lado, en esta tesis doctoral, mediante experimentos computacionales in silico, se ha identificado la proteína AKT como una posible diana molecular de uno de nuestros compuestos. A su vez, se ha corroborado por espectroscopia mediante resonancia de plasmones superficiales, que la afinidad de unión entre el análogo de tambjamina y AKT es elevada, situada en un rango micromolar. Asimismo, se ha detectado una disminución tanto de la fosforilación, como de la proteína total AKT tras el tratamiento con tambjamina. Igualmente, gracias al estudio de la modificación de los patrones de expresión de miRNA tras el tratamiento con el compuesto, se han dilucidado las principales rutas de señalización involucradas en el proceso citotóxico, siendo las más afectadas PI3K/AKT, apoptosis y autofagia. Por último, para completar los estudios preclínicos, se ha evaluado la toxicidad y eficacia de estos compuestos in vivo en modelos murinos de cáncer de pulmón en estudios preliminares, mostrando una potente capacidad antitumoral tanto en el modelo ectópico como ortotópico. Por todo esto, los análogos sintéticos de tambjamina pueden ser considerados una buena herramienta para inducir muerte en células cancerosas mediante una nueva estrategia terapéutica que modifica el pH intracelular y podrían llegar a ser buenos fármacos para abordar el tratamiento de tumores resistentes a la apoptosis.

The utility of fluorophores for sensing applications in the current state of the art of biological imaging hardly needs to be stated. The use of fluorophores in exploring and determining the internal structure and active dynamics of cellular processes has been pivotal, allowing us to explore areas of study inaccessible through other means. A simple search of fluorophores in Scifinder© demonstrates their popularity, as the number of hits increases year after year, until the year of 2015 when there were 1400+ journal articles published with the phrase. Fluorophore applications range far and wide, from sensing applications related to environmental concerns, to public health, to clinical usage. Fluorophores have been developed to detect explosive residues, to monitor environmental pollutants, and even to detect illicit substances. In cellular applications, a fluorophore needs to be well suited to examining the relevant processes, including participating in the cellular milieu and actively signifying the phenomena that are desired. Chapter I examines the usage of alkynes in fluorescent sensing scaffolds and gives a survey of their applicability in the field. Chapter II demonstrates the utility of disulfide-based macrocyclic scaffolds in the design of supramolecular hosts for chloride anions and their use as solid-state sensors for these anions. Chapter III explores the synthesis and application of an alkyne-based scaffold in the reversible detection of dithiol/disulfide redox flux and a new mode of quantification of dithiol-disulfide redox couples, a classically difficult area of study. Chapter IV focuses on methods utilized to improve the disulfide-based redox sensing capabilities. Chapters V and VI explore the properties of a new fluorophore scaffold discovered during research into another sensing scaffold, demonstrating a new reaction which yields a heretofore underexplored heterocycle with novel photophysical and supramolecular behaviors. This dissertation contains both previously published and unpublished co-authored material.

L’éclosion de bactéries résistantes aux antibiotiques constitue un problème sérieux auquel fait face notre système de santé. L’une des stratégies récemment proposées afin de s’attaquer efficacement et irréversiblement à ces microorganismes multi-résistants est de cibler directement leur membrane via l’action de molécules induisant un débalancement électrolytique de part et d’autre de cette dernière. Parallèlement, ces mêmes agents peuvent aussi avoir des applications dans le traitement de maladies originant des dysfonctions du transport ionique, comme la fibrose kystique. À cet égard, nous présentons dans cette thèse différents sels d’imidazolium et benzimidazolium N,N-disubstitués possédant un potentiel à la fois antimicrobien et ionophore. Notre approche se résume d’abord en un volet mécanistique où une série de modifications structurelles ont été apportées à des sels d’imidazolium et benzimidazolium afin d’observer comment ces changements modulent l’efficacité du transport d’anions dans la membrane artificielle d’un liposome. Nous avons à ce titre pu conclure que l’espèce formée de deux bras aromatiques phényléthynylbenzyl, disposées symétriquement de part et d’autre d’un cation imidazolium, induisait le meilleur transport des anions chlorures, au travers d’une membrane de liposomes, à des concentrations de l’ordre du micromolaire. En outre, les monocations imidazolium et benzimidazolium flanqués d’un contre-anion bis(trifluorométhylsulfonyl)amide ont conduit à une activité ionophore plus rapide. Qui plus est, en s’appuyant sur ces résultats, nous avons présenté le premier exemple, à notre connaissance, d’un transporteur d’anions et de cations, contenant le cation benzimidazolium et capable d’agir aussi bien dans des liposomes que dans des bactéries. Dans un second temps, les meilleurs agents ionophores ont été étudiés dans les membranes plus complexes des bactéries et des globules rouges humains pour vérifier leur effet bactéricide et leur innocuité. Le design de nos transporteurs formés d’un espaceur luthidine a ainsi permis d’obtenir un agent antimicrobien efficace dans des bactéries gram positives et négatives (B. thuringiensis et E. coli) avec une toxicité limitée de l’ordre de 10% sur les globules rouges humains à ses concentrations bactéricides.
The emergence of antibiotic resistant bacteria is a serious problem that our health system faces. One recently proposed strategy to effectively and irreversibly kill these multi-resistant microorganisms is to directly target the integrity of their membrane, using small molecules able to induce an electrolyte imbalance. Moreover, the same molecules may find applications in the treatement od diseases originating from the dysfunction of ion transport, such as cystic fibrosis. Herein we present different imidazolium and benzimidazolium salts N,N-disubstituted with both antimicrobial and ionophoric potential. We first performed mechanistic studies where different structural changes have been made to the imidazolium and benzimidazolium salts to observe how these modifications modulate the efficiency of the anion transport in artificial membrane liposomes. We were able to conclude that the species formed of two aromatic arms phenylethynylbenzyl arranged symmetrically on either side of an imidazolium cation, induced a better transport of chloride anions, through a membrane of liposomes at the micromolar range. In addition, monocations imidazolium and benzimidazolium flanked with an bis(trifluorométhylsulfonyl)amide anion led to faster ionophore activity. Moreover, based on these results we presented the first example, to our knowledge, for an anions and cations benzimidazolium-based transporter, acting as well in liposomes as in bacteria. Secondly, the best anionophore agents were analyzed in more complex bacterias and human red blood cells membranes to study their bactericidal potential and innocuity. Among all the benzimidazolium salts studied, we identified one compound, which presents interesting antibacterial properties as a result of its ability to induce an electrolytic imbalance and to disrupt the integrity and the potential of the bacterial membranes. At the same time this antibacterial agent presented a low toxicity to human cells in bacteriostatic range concentrations.