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

Abstract

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 unilamellar 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 NH 4 + 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. 1 | INTRODUCTION Cystic fibrosis (CF) is the most common, autosomal recessive, lethal genetic disease in the Caucasian population (Bobadilla, Macek, Fine, & Farrell, 2002; Strausbaugh & Davis, 2007). The major cause of CF morbidity and mortality is lung dysfunction, characterised by infection, inflammation, and airway damage, leading to respiratory failure. More than 2,000 mutations in the CF transmembrane

Anion carriers as potential treatments for cystic fibrosis: transport in cystic fibrosis cells, and additivity to channel-targeting drugs

“…54 Encouragingly, the present results and ref. 41 demonstrate that the action of anionophores is additive to the clinically-licensed CFTR modulators lumacaor and ivacaor, raising the possibility that combination therapy with anionophores and CFTR modulators might be used to treat CF patients. While ivacaor and lumacaor are orally bioavailable drugs, 39,40 we envisage that anionophores would be delivered to the lungs of CF patients by aerosolization as for inhaled antibiotics and gene therapy vectors.…”

“…They also support a variety of potential therapeutic applications for anionophores, some as anticancer and antimicrobial agents, [45][46][47] but others as CF therapeutics. 29,41,48 If anionophores are to be used as research tools, it is important to establish their sensitivity to small molecules that inhibit endogenous anion transport systems. We therefore studied the effects of CaCC inh -A01 (active against CaCC) and CFTR inh -172 (active against CFTR), 36,37 on transport by 11, 12, 15 and 19.…”

“…Of note, similar results were recently reported using F508del-CFTR-YFP-FRT cells treated with lumacaor, ivacaor and anionophores derived from prodigiosin and tambjamine. 41 Thus, anionophores mediate anion transport in cells expressing wild-type and F508del-CFTR and their effects are additive to the action of drugs that rescue F508del-CFTR.…”

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

“…The anionophore-facilitated halide transport was then expressed as the initial quenching rate of the Yellow Fluorescent Protein (YFP) upon an extracellular application of iodide. We already demonstrated that MM3 facilitates a significant iodide transport in FRT cells, requiring 2.8 ± 0.6 µM to induce half of the maximum transport rate [22]. Instead, the halide transport capacity of MM34 was not previously assayed in mammalian cells.…”

“…Previous studies have identified several anionophores inspired by natural products such as prodiginines and tambjamines, able to transport halides in mammalian cells at non-toxic concentrations [18][19][20][21]. Transmembrane transport activities comparable to that of the native CFTR were measured for the best tambjamines candidates [22]. Although promising, these results were not obtained in a physiologically relevant disease model.…”