Small molecule anion carriers facilitate lactate transport in model liposomes and cells.

An excessive production of lactate by cancer cells fosters tumor growth and metastasis. Therefore, targeting lactate metabolism and transport offers a new therapeutic strategy against cancer, based on dependency of some cancer cells for lactate as energy fuel or as oncogenic signal. Herein we present a family of anionophores based on the structure of click-tambjamines that have proved to be extremely active lactate carriers across phospholipid membranes. Compound 1, the most potent lactate transmembrane carrier, was studied in HeLa cells. The use of a monocarboxylate transporters (MCTs) inhibitor proved that 1 is an active lactate transporter in living cells, confirming the results obtained in phospholipid vesicles. Moreover, an additive effect of compound 1 with cisplatin was observed in HeLa cells. Identification of active lactate anionophores working in living cells opens up ways to exploit this class of compounds as molecular tools and drugs addressing dysregulated lactate metabolism.

Synthesis and evaluation of 2,9-disubstituted-1,10-phenanthroline derivatives as G-quadruplex binders.

G-quadruplex (G4) structures are non-canonical DNA/RNA secondary structures able to form within guanine rich nucleic acids sequences. They are present in several regions of the human genome including gene promoters, untranslated sequences, and telomeres. Due to their biological relevance G4 structures are considered important drug targets, in particular for anticancer therapies, leading to the development of G4 stabilizing small molecules. Telomeric regions have received special attention in this field since they can fold into several distinct intramolecular G-quadruplexes topologies. Herein, we report the synthesis of 2,9-disubstituted-1,10-phenanthroline derivatives and their ability to stabilize different intramolecular telomeric G4 sequences. We evaluated ligand-induced stabilization, selectivity and specificity of ligands using Förster Resonance Energy Transfer (FRET) melting experiments and circular dichroism (CD). In addition, we assessed the cytotoxicity of ligands against two cancer cell lines (A549 and H1299) and one healthy cell line (NHDF).

A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer.

Overcoming resistance is one of the most challenging features in current anticancer therapy. Autophagy is a cellular process that confers resistance in some advanced tumors, since it enables cancer cells to adapt to stressful situations, such as anticancer treatments. Hence, the inhibition of this cytoprotective autophagy leads to tumor cells sensitization and death. In this regard, we designed a novel potent anionophore compound that specifically targets lysosomes, called LAI-1 (late-stage autophagy inhibitor-1), and evaluated its role in blocking autophagy and its potential anticancer effects in three lung cancer cell lines from different histological subtypes. Compared to other autophagy inhibitors, such as chloroquine and 3-Methyladenine, the LAI-1 treatment induced more potent anticancer effects in all tested cancer cells. LAI-1 was able to efficiently target and deacidify lysosomes, while acidifying cytoplasmic pH. Consequently, LAI-1 efficiently blocked autophagy, indicated by the increased LC3-II/I ratio and p62/SQSTM1 levels. Moreover, no colocalization was observed between autophagosomes, marked with LC3 or p62/SQSTM1, and lysosomes, stained with LAMP-1, after the LAI-1 treatment, indicating the blockage of autophagolysosome formation. Furthermore, LAI-1 induced cell death by activating apoptosis (enhancing the cleavage of caspase-3 and PARP) or necrosis, depending on the cancer cell line. Finally, LAI-1 sensitized cancer cells to the first-line chemotherapeutic agent cisplatin. Altogether, LAI-1 is a new late-stage autophagy inhibitor that causes lysosomal dysfunction and the blockage of autophagolysosome formation, as well as potently induces cancer cell death and sensitization to conventional treatments at lower concentrations than other known autophagy inhibitors, appearing as a potential new therapeutic approach to overcome cancer resistance.

Rational design of mitochondria targeted thiabendazole-based Ir(III) biscyclometalated complexes for a multimodal photodynamic therapy of cancer.

Despite their outstanding properties as potential photosensitizers for photodynamic therapy (PDT), Ir(III) biscyclometalated complexes need both further developments to overcome remaining limitations and in-depth investigations into their mechanisms of action to reach clinic application in the treatment of cancer. This work describes the synthesis of a family of Ir(III) complexes of general formula [Ir(C^N)2(N^N')]Cl (N^N' = thiabendazole-based ligands; C^N = ppy (2-phenylpyridinate) (Series A), or dfppy (2-(2,4-difluorophenyl)pyridinate) (Series B)) and their evaluation as potential PDT agents. These complexes are partially soluble in water and exhibit cytotoxic activity in the absence of light irradiation versus several cancer cell lines. Furthermore, the cytotoxic activity of derivatives of Series A is enhanced upon irradiation, particularly for complexes [1a]Cl and [3a]Cl, which show phototoxicity indexes (PI) above 20. Endocytosis was established as the uptake mechanism for [1a]Cl and [3a]Cl in prostate cancer cells by flow cytometry. These derivatives mainly accumulate in the mitochondria as shown by colocalization confocal microscopy experiments. Presumably, [1a]Cl and [3a]Cl induce death on cancer cells under irradiation through apoptosis triggered by a multimodal mechanism of action, which likely involves damage over mitochondrial DNA and mitochondrial membrane depolarization. Both processes seem to be the result of photocatalytic oxidation processes.

Antimetastatic Properties of Prodigiosin and the BH3-Mimetic Obatoclax (GX15-070) in Melanoma.

Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a reduction in tumor size, but allows cancer cells to disseminate, which ultimately causes patient death. Therefore, novel anticancer compounds with both effects need to be developed. In this work, we analyze the antimetastatic properties of prodigiosin and obatoclax (GX15-070), anticancer drugs of the Prodiginines (PGs) family. We studied PGs' effects on cellular adhesion and morphology in the human primary and metastatic melanoma cell lines, SK-MEL-28 and SK-MEL-5, and in the murine melanoma cell line, B16F10A. Cell adhesion sharply decreased in the treated cells, and this was accompanied by a reduction in filopodia protrusions and a significant decrease in the number of focal-adhesion structures. Moreover, cell migration was assessed through the wound-healing assay and cell motility was severely inhibited after 24 h of treatment. To elucidate the molecular mechanisms involved, changes in metastasis-related genes were analyzed through a gene-expression array. Key genes related to cellular invasion, migration and chemoresistance were significantly down-regulated. Finally, an in vivo model of melanoma-induced lung metastasis was established and significant differences in lung tumors were observed in the obatoclax-treated mice. Altogether, these results describe, in depth, PGs' cellular antimetastatic effects and identify in vivo antimetastatic properties of Obatoclax.

Structure-antitumor activity relationships of tripodal imidazolium-amino acid based salts. Effect of the nature of the amino acid, amide substitution and anion.

The antitumor activity of imidazolium salts is highly dependent upon their lipophilicity that can be tuned by the introduction of different hydrophobic substituents on the nitrogen atoms of the imidazolium ring of the molecule. Taking this into consideration, we have synthesized and characterized a series of tripodal imidazolium salts derived from L-valine and L-phenylalanine containing different hydrophobic groups and tested them against four cancer cell lines at physiological and acidic pH. At acidic pH (6.2) the anticancer activity of some of the tripodal compounds changes dramatically, and this parameter is crucial to control their cytotoxicity and selectivity. Moreover, several of these compounds displayed selectivity against the control healthy cell line higher than four. The transmembrane anion transport studies revealed moderate transport abilities suggesting that the observed biological activity is likely not the result of just their transport activity. The observed trends in biological activity at acidic pH agree well with the results for the CF leakage assay. These results strongly suggest that this class of compounds can serve as potential chemotherapeutic agents.

Multi-Smart and Scalable Bioligands-Free Nanomedical Platform for Intratumorally Targeted Tambjamine Delivery, a Difficult to Administrate Highly Cytotoxic Drug.

Cancer is one of the leading causes of mortality worldwide due, in part, to limited success of some current therapeutic approaches. The clinical potential of many promising drugs is restricted by their systemic toxicity and lack of selectivity towards cancer cells, leading to insufficient drug concentration at the tumor site. To overcome these hurdles, we developed a novel drug delivery system based on polyurea/polyurethane nanocapsules (NCs) showing pH-synchronized amphoteric properties that facilitate their accumulation and selectivity into acidic tissues, such as tumor microenvironment. We have demonstrated that the anticancer drug used in this study, a hydrophobic anionophore named T21, increases its cytotoxic activity in acidic conditions when nanoencapsulated, which correlates with a more efficient cellular internalization. A biodistribution assay performed in mice has shown that the NCs are able to reach the tumor and the observed systemic toxicity of the free drug is significantly reduced in vivo when nanoencapsulated. Additionally, T21 antitumor activity is preserved, accompanied by tumor mass reduction compared to control mice. Altogether, this work shows these NCs as a potential drug delivery system able to reach the tumor microenvironment, reducing the undesired systemic toxic effects. Moreover, these nanosystems are prepared under scalable methodologies and straightforward process, and provide tumor selectivity through a smart mechanism independent of targeting ligands.

Toxicological assessment of commercial monolayer tungsten disulfide nanomaterials aqueous suspensions using human A549 cells and the model fungus Saccharomyces cerevisiae.

The utilization of tungsten disulfide (WS2) nanomaterials in distinct applications is raising due to their unique physico-chemical properties, such as low friction coefficient and high strength, which highlights the necessity to study their potential toxicological effects, due to the potential increase of environmental and human exposure. The aim of this work was to analyze commercially available aqueous dispersions of monolayer tungsten disulfide (2D WS2) nanomaterials with distinct lateral size employing a portfolio of physico-chemical and toxicological evaluations. The structure and stoichiometry of monolayer tungsten disulfide (WS2-ACS-M) and nano size monolayer tungsten disulfide (WS2-ACS-N) was analyzed by Raman spectroscopy, whereas a more quantitative approach to study the nature of formed oxidized species was undertaken employing X-ray photoelectron spectroscopy. Adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the ecotoxicology model Saccharomyces cerevisiae were selected as unicellular eukaryotic systems to assess the cytotoxicity of the nanomaterials. Cell viability and reactive oxygen species (ROS) determinations demonstrated different toxicity levels depending on the cellular model used. While both 2D WS2 suspensions showed very low toxicity towards the A549 cells, a comparable concentration (160 mg L-1) reduced the viability of yeast cells. The toxicity of a nano size 2D WS2 commercialized in dry form from the same provider was also assessed, showing ability to reduce yeast cells viability as well. Overall, the presented data reveal the physico-chemical properties and the potential toxicity of commercial 2D WS2 aqueous suspensions when interacting with distinct eukaryotic organisms, showing differences in function of the biological system exposed.

Assessment of Physico-Chemical and Toxicological Properties of Commercial 2D Boron Nitride Nanopowder and Nanoplatelets.

Boron nitride (BN) nanomaterials have been increasingly explored for potential applications in chemistry and biology fields (e.g., biomedical, pharmaceutical, and energy industries) due to their unique physico-chemical properties. However, their safe utilization requires a profound knowledge on their potential toxicological and environmental impact. To date, BN nanoparticles have been considered to have a high biocompatibility degree, but in some cases, contradictory results on their potential toxicity have been reported. Therefore, in the present study, we assessed two commercial 2D BN samples, namely BN-nanopowder (BN-PW) and BN-nanoplatelet (BN-PL), with the objective to identify whether distinct physico-chemical features may have an influence on the biological responses of exposed cellular models. Morphological, structural, and composition analyses showed that the most remarkable difference between both commercial samples was the diameter of their disk-like shape, which was of 200-300 nm for BN-PL and 100-150 nm for BN-PW. Their potential toxicity was investigated using adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the unicellular fungus Saccharomycescerevisiae, as human and environmental eukaryotic models respectively, employing in vitro assays. In both cases, cellular viability assays and reactive oxygen species (ROS) determinations where performed. The impact of the selected nanomaterials in the viability of both unicellular models was very low, with only a slight reduction of S. cerevisiae colony forming units being observed after a long exposure period (24 h) to high concentrations (800 mg/L) of both nanomaterials. Similarly, BN-PW and BN-PL showed a low capacity to induce the formation of reactive oxygen species in the studied conditions. Even at the highest concentration and exposure times, no major cytotoxicity indicators were observed in human cells and yeast. The results obtained in the present study provide novel insights into the safety of 2D BN nanomaterials, indicating no significant differences in the toxicological potential of similar commercial products with a distinct lateral size, which showed to be safe products in the concentrations and exposure conditions tested.

Fate assessment of commercial 2D MoS2 aqueous dispersions at physicochemical and toxicological level.

The physicochemical properties and the toxicological potential of commercially available MoS2 nanoparticles with different lateral size and degradation stage were studied in the present research work. To achieve this, the structure and stoichiometry of fresh and old aqueous suspensions of micro-MoS2 and nano-MoS2 was analyzed by Raman, while x-ray photoelectron spectroscopy allowed to identify more quantitatively the nature of the formed oxidized species. A, the toxicological impact of the nanomaterials under analysis was studied using adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the unicellular fungus S. cerevisiae as biological models. Cell viability assays and reactive oxygen species (ROS) determinations demonstrated different toxicity levels depending on the cellular model used and in function of the degradation state of the selected commercial nanoproducts. Both MoS2 nanoparticle types induced sublethal damage on the A549 cells though the increase of intracellular ROS levels, while comparable concentrations reduced the viability of yeast cells. In addition, the old MoS2 nanoparticles suspensions exhibited a higher toxicity for both human and yeast cells than the fresh ones. Our findings demonstrate that the fate assessment of nanomaterials is a critical aspect to increase the understanding on their characteristics and on their potential impact on biological systems along their life cycle.

Advances in anion transport and supramolecular medicinal chemistry.

Advances in anion transport by synthetic supramolecular systems are discussed in this article. Developments in the design of discrete molecular carriers for anions and supramolecular anion channels are reviewed followed by an overview of the use of these systems in biological systems as putative treatments for diseases such as cystic fibrosis and cancer.

Small Molecule Anion Carriers Correct Abnormal Airway Surface Liquid Properties in Cystic Fibrosis Airway Epithelia.

Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.

Click-tambjamines as efficient and tunable bioactive anion transporters.

A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design.

Alternative chloride transport pathways as pharmacological targets for the treatment of cystic fibrosis.

Cystic fibrosis is a hereditary disease that originates from mutations in the epithelial chloride channel CFTR. Whereas established therapies for the treatment of cystic fibrosis target CFTR to repair its function, alternative therapeutic strategies aim for the restoration of chloride transport by the activation of other chloride transport proteins such as TMEM16A or SLC26A9 or by the application of synthetic anionophores. TMEM16A is an anion-selective channel that is activated by the binding of Ca2+ from the cytoplasm. Pharmacological efforts aim for the increase of its open probability at resting Ca2+ concentrations. SLC26 is an uncoupled chloride transporter, which shuttles chloride across the membrane by an alternate-access mechanism. Its activation requires its mobilization from intracellular stores. Finally, anionophores are small synthetic molecules that bind chloride to form lipid-soluble complexes, which shuttle the anion across the membrane. All three approaches are currently pursued and have provided promising initial results.

Targeting Autophagy for Cancer Treatment and Tumor Chemosensitization.

Autophagy is a tightly regulated catabolic process that facilitates nutrient recycling from damaged organelles and other cellular components through lysosomal degradation. Deregulation of this process has been associated with the development of several pathophysiological processes, such as cancer and neurodegenerative diseases. In cancer, autophagy has opposing roles, being either cytoprotective or cytotoxic. Thus, deciphering the role of autophagy in each tumor context is crucial. Moreover, autophagy has been shown to contribute to chemoresistance in some patients. In this regard, autophagy modulation has recently emerged as a promising therapeutic strategy for the treatment and chemosensitization of tumors, and has already demonstrated positive clinical results in patients. In this review, the dual role of autophagy during carcinogenesis is discussed and current therapeutic strategies aimed at targeting autophagy for the treatment of cancer, both under preclinical and clinical development, are presented. The use of autophagy modulators in combination therapies, in order to overcome drug resistance during cancer treatment, is also discussed as well as the potential challenges and limitations for the use of these novel therapeutic strategies in the clinic.

The Natural-Based Antitumor Compound T21 Decreases Survivin Levels through Potent STAT3 Inhibition in Lung Cancer Models.

Lung cancer is the leading cause of cancer-related deaths worldwide; hence novel treatments for this malignancy are eagerly needed. Since natural-based compounds represent a rich source of novel chemical entities in drug discovery, we have focused our attention on tambjamines, natural compounds isolated from marine invertebrates that have shown diverse pharmacological activities. Based on these structures, we have recently identified the novel indole-based tambjamine analog 21 (T21) as a promising antitumor agent, which modulates the expression of apoptotic proteins such as survivin. This antiapoptotic protein plays an important role in carcinogenesis and chemoresistance. In this work, we have elucidated the molecular mechanism by which the anticancer compound T21 exerts survivin inhibition and have validated this protein as a therapeutic target in different lung cancer models. T21 was able to reduce survivin protein levels in vitro by repressing its gene expression through the blockade of Janus kinase/Signal Transducer and Activator of Transcription-3 (JAK/STAT3)/survivin signaling pathway. Interestingly, this occurred even when the pathway was overstimulated with its ligand interleukin 6 (IL-6), which is frequently overexpressed in lung cancer patients who show poor clinical outcomes. Altogether, these results show T21 as a potent anticancer compound that effectively decreases survivin levels through STAT3 inhibition in lung cancer, appearing as a promising therapeutic drug for cancer treatment.

pH-Dependent Chloride Transport by Pseudopeptidic Cages for the Selective Killing of Cancer Cells in Acidic Microenvironments.

Acidic microenvironments in solid tumors are a hallmark of cancer. Inspired by that, we designed a family of pseudopeptidic cage-like anionophores displaying pH-dependent activity. When protonated, they efficiently bind chloride anions. They also transport chloride through lipid bilayers, with their anionophoric properties improving at acidic pH, suggesting an H+ /Cl- symport mechanism. NMR studies in DPC micelles demonstrate that the cages bind chloride within the lipid phase. The chloride affinity and the chloride-exchange rate with the aqueous bulk solution are improved when the pH is lowered. This increases cytotoxicity towards lung adenocarcinoma cells at the pH of the microenvironment of a solid tumor. These properties depend on the nature of the amino-acid side chains of the cages, which modulate their lipophilicity and interactions with the cell membrane. This paves the way towards using pH as a parameter to control the selectivity of cytotoxic ionophores as anticancer drugs.

Small molecule-facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease that originates from the defective function of the CF transmembrane conductance regulator (CFTR) protein, a cAMP-dependent anion channel involved in fluid transport across epithelium. Because small synthetic transmembrane anion transporters (anionophores) can replace the biological anion transport mechanisms, independent of genetic mutations in the CFTR, such anionophores are candidates as new potential treatments for CF. EXPERIMENTAL APPROACH: In order to assess their effects on cell physiology, we have analysed the transport properties of five anionophore compounds, three prodigiosines and two tambjamines. Chloride efflux was measured in large uni-lamellar vesicles and in HEK293 cells with chloride-sensitive electrodes. Iodide influx was evaluated in FRT cells transfected with iodide-sensitive YFP. Transport of bicarbonate was assessed by changes of pH after a NH4 + pre-pulse using the BCECF fluorescent probe. Assays were also carried out in FRT cells permanently transfected with wild type and mutant human CFTR. KEY RESULTS: All studied compounds are capable of transporting halides and bicarbonate across the cell membrane, with a higher transport capacity at acidic pH. Interestingly, the presence of these anionophores did not interfere with the activation of CFTR and did not modify the action of lumacaftor (a CFTR corrector) or ivacaftor (a CFTR potentiator). CONCLUSION AND IMPLICATIONS: These anionophores, at low concentrations, transported chloride and bicarbonate across cell membranes, without affecting CFTR function. They therefore provide promising starting points for the development of novel treatments for CF.

Therapeutic strategies involving survivin inhibition in cancer.

Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.

Anion-Transport Mechanism of a Triazole-Bearing Derivative of Prodigiosine: A Candidate for Cystic Fibrosis Therapy.

Cystic fibrosis (CF) is a genetic lethal disease, originated from the defective function of the CFTR protein, a chloride and bicarbonate permeable transmembrane channel. CF mutations affect CFTR protein through a variety of molecular mechanisms which result in different functional defects. Current therapeutic approaches are targeted to specific groups of patients that share a common functional defect. We seek to develop an innovative therapeutic approach for the treatment of CF using anionophores, small molecules that facilitate the transmembrane transport of anions. We have characterized the anion transport mechanism of a synthetic molecule based on the structure of prodigiosine, a red pigment produced by bacteria. Anionophore-driven chloride efflux from large unilamellar vesicles is consistent with activity of an uniporter carrier that facilitates the transport of anions through lipid membranes down the electrochemical gradient. There are no evidences of transport coupling with protons. The selectivity sequence of the prodigiosin inspired EH160 ionophore is formate > acetate > nitrate > chloride > bicarbonate. Sulfate, phosphate, aspartate, isothionate, and gluconate are not significantly transported by these anionophores. Protonation at acidic pH is important for the transport capacity of the anionophore. This prodigiosin derived ionophore induces anion transport in living cells. Its low toxicity and capacity to transport chloride and bicarbonate, when applied at low concentration, constitute a promising starting point for the development of drug candidates for CF therapy.