Anion selective ionophores, anionophores, are small molecules capable of facilitating the transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores in living cells was studied and chloride efflux and iodine influx from living cells mediated by these derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results support the viability of developing small molecule anionophores as anion channel protein surrogates with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the malfunction of natural anion transport mechanisms.

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.

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.

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.