Background and Purpose Pharmacological inhibitors of TMEM16A (ANO1), a Ca2+‐activated Cl− channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh‐A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+. Experimental Approach We recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging. Key Results Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCCinh‐A01. Acute addition of niclosamide directly increased intracellular Ca2+, an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh‐A01 did not elevate intracellular Ca2+, thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors. Conclusions and Implications Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca2+ handling, which may in part preclude their use as TMEM16A research tools.

The fluid covering the surface of airway epithelia represents a first barrier against pathogens.The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, two cytokines with a relevant role in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, anti-bacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC sodium channel, ATP12A proton pump, ADRB2 beta-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNFα treatment was hyperviscosity of the airway surface as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a betaadrenergic agonist switched airway surface to a low viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state thus perpetuating the proinflammatory airway surface condition.

F508del, the most frequent mutation in cystic fibrosis (CF), impairs the stability and folding of the CFTR chloride channel, thus resulting in intracellular retention and CFTR degradation. The F508del defect can be targeted with pharmacological correctors, such as VX-809 and VX-445, that stabilize CFTR and improve its trafficking to plasma membrane. Using a functional test to evaluate a panel of chemical compounds, we have identified tricyclic pyrrolo-quinolines as novel F508del correctors with high efficacy on primary airway epithelial cells from CF patients. The most effective compound, PP028, showed synergy when combined with VX-809 and VX-661 but not with VX-445. By testing the ability of correctors to stabilize CFTR fragments of different length, we found that VX-809 is effective on the amino-terminal portion of the protein that includes the first membrane-spanning domain (amino acids 1–387). Instead, PP028 and VX-445 only show a stabilizing effect when the second membrane-spanning domain is included (amino acids 1–1181). Our results indicate that tricyclic pyrrolo-quinolines are a novel class of CFTR correctors that, similarly to VX-445, interact with CFTR at a site different from that of VX-809. Tricyclic pirrolo-quinolines may represent novel CFTR correctors suitable for combinatorial pharmacological treatments to treat the basic defect in CF.

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Pharmacological modulators of the Ca2+ signaling cascade are important research tools and may translate into novel therapeutic strategies for a series of human diseases. We carried out a screening of a maximally diverse chemical library using the Ca2+-sensitive Cl− channel TMEM16A as a functional readout. We found compounds able to potentiate UTP-dependent TMEM16A activation. Mechanism of action of these compounds was investigated by a panel of assays that looked at intracellular Ca2+ mobilization triggered by extracellular agonists or by caged-IP3 photolysis, PIP2 breakdown by phospholipase C, and ion channel activity on nuclear membrane. One compound appears as selective potentiator of inositol triphosphate receptor type 1 (ITPR1) with a possible application for some forms of spinocerebellar ataxia. A second compound is instead a potentiator of the P2RY2 purinergic receptor, an activity that could promote fluid secretion in dry eye and chronic obstructive respiratory diseases.