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.

The different anion transport capability of prodiginine- and tambjamine-like molecules.

Prodiginines and tambjamines are anion-selective ionophores capable of facilitating the transport of anions across the plasma membrane in mammalian cells. One of the potential applications of these anionophores is the possibility of employing them as a substitutive therapy for pathologies involving anion channels, as in cystic fibrosis. We have studied the interaction of a large anion as gluconate with three prodiginine- and two tambjamine-like compounds. Apparent dissociation constants for the chloride, iodide and gluconate complexes were estimated from iodide influx experiments in mammalian cells exposed to different extracellular anion combinations. Our experiments indicate that gluconate is not transported by the prodiginines, leaving the anionophores free to transport chloride and iodide. Conversely, gluconate would be transported to some extent by the tambjamines, competing with halides for the anionophores, and consequently reducing their flux. This might be related to the different structural features of both families of compounds. These data have important implications for the selection of impermeable anions in the analysis of the anionophore mechanism.

A Sandwich Azobenzene-Diamide Dimer for Photoregulated Chloride Transport.

There has been a tremendous evolution for artificial ion transport systems, especially gated synthetic systems, which closely mimic their natural congeners. Herein, we demonstrate a trans-azobenzene-based photoregulatory anionophoric system that transports chloride by forming a sandwich dimeric complex. Further studies confirmed a carrier-mediated chloride-anion antiport mechanism, and the supramolecular interactions involved in chloride recognition within the sandwich complex were revealed from theoretical studies. Reversible trans-cis photoisomerization of the azobenzene was achieved without any significant contribution from the thermal cis→trans isomerization at room temperature. Photoregulatory transport activity across the lipid bilayer membrane inferred an outstanding off-on response of the azobenzene photoswitch.

A Novel Genetically Encoded Single Use Sensory Cellular Test System Measures Bicarbonate Concentration Changes in Living Cells.

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.

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.

Tripodal squaramide conjugates as highly effective transmembrane anion transporters.

Two tripodal squaramide conjugates having 4-(trifluoromethyl)phenyl and 3,5-bis(trifluoromethyl)phenyl substituents were synthesized and found to exhibit highly efficient transmembrane anion transport with the EC50 values being 0.14 and 0.75mol%, respectively. Though one of them has been reported to act as a strong anion receptor, in particular for sulfate anions, these two compounds exhibit no significant selectivity with respect to the tested monovalent anions and a very low level of activity in the presence of sulfate anions.

2,6-Bis(benzimidazol-2-yl)pyridine as a potent transmembrane anion transporter.

2,6-Bis(benzimidazol-2-yl)pyridine was shown to exhibit potent anionophoric activity via a process of both Cl(-)/NO3(-) antiport and H(+)/Cl(-) symport. This is in sharp contrast to the finding that its corresponding N-methylated analog exhibited negligible activity and reveals the importance of the imidazolyl-NH fragments in the anion-transport process.

Synthesis and transmembrane anion/cation symport activity of a rigid bis(choloyl) conjugate functionalized with guanidino groups.

A rigid bis(choloyl) conjugate functionalized with guanidino groups was synthesized and fully characterized on the basis of NMR ((1)H and (13)C) and ESI MS (LR and HR) data. Its transmembrane ionophoric activity across egg-yolk l-α-phosphatidylcholine-based liposomal membranes was investigated by means of chloride ion selective electrode technique and pH discharge assay. The data indicate that under the assay conditions, this conjugate was capable of promoting the transport of anions, presumably via a cation/anion symport process. A Hill analysis reveals that two molecules of this compound are assembled into the transport-active species.