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Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air–liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure–activity relationship studies led to the discovery of enantiomerically pure 39endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitrodruglike profile. The in vivocharacterization of γ-carboline 39showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

Functional expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuates expression and secretion of the proinflammatory cytokines IL-6, IL-8, and CXCL1/2 in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport may contribute to lung inflammation in cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The mutations G551D and G1349D, which affect the nucleotide-binding domains (NBDs) of CFTR protein, reduce channel activity. This defect can be corrected pharmacologically by small molecules called potentiators. CF mutations residing in the intracellular loops (ICLs), connecting the transmembrane segments of CFTR, may also reduce channel activity. We have investigated the extent of loss of function caused by ICL mutations and the sensitivity to pharmacological stimulation. We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. The 1,4-dihydropyridine felodipine and the phenylglycine PG-01 [2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide] were strongly effective on the three CFTR mutants. The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Our results indicate that CFTR potentiators correct the basic defect caused by CF mutations residing in different CFTR domains. However, there are differences among potentiators, with felodipine and PG-01 having a wider pharmacological activity, and SF-01 being more mutation specific. Our observations are useful in the prioritization and development of drugs targeting the CF basic defect.

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. In a recent study, several highly polar alpha-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (J Pharmacol Exp Ther 322:1023-1035, 2007), more than 10,000-fold better than that of thiazolidinone and glycine hydrazide CFTR inhibitors previously identified by high-throughput screening. In this study, we resynthesized and evaluated the alpha-aminoazaheterocyclic-methylglyoxal adducts reported to have high CFTR inhibition potency (compounds 5, 7, and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 microM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp experiments, and yellow fluorescence protein-fluorescence quenching. As positive controls, approximately 100% of CFTR inhibition was found by thiazolidinone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by alpha-aminoazaheterocyclic-methylglyoxal adducts.

Mutations occurring in the CFTR gene, encoding for the cystic fibrosis transmembrane conductance regulator chloride channel, cause cystic fibrosis (CF). Mutations belonging to class II, such as ΔPhe508, give rise to a protein with both a defective maturation and altered channel gating. Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. We have previously identified antihypertensive 1,4-dihydropyridines (DHPs), a class of drugs that block voltage-dependent Ca2+channels, as effective potentiators of CFTR gating, able to correct the defective activity of CFTR mutants (Mol Pharmacol 68:1736-1746, 2005). However, optimization of potency for CFTR versus Ca2+channels is required to design selective compounds for CFTR pharmacotherapy. In the present study, we have established DHP structure-activity relationship for both CFTR potentiation and Ca2+channel inhibition using cell-based assays for both types of channels. A panel of 333 felodipine analogs was studied to understand the effect of various substitutions and modifications in the DHP scaffold. Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca2+channels and strong effect as potentiators on the ΔPhe508, G551D, and G1349D CFTR mutants.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel gene. CF mutations like ΔF508 cause both a mistrafficking of the protein and a gating defect. Other mutations, like G551D, cause only a gating defect. Our aim was to find chemical compounds able to stimulate the activity of CFTR mutant proteins by screening a library containing approved drugs. Two thousand compounds were tested on Fischer rat thyroid cells coexpressing ΔF508-CFTR and a halide-sensitive yellow fluorescent protein (YFP) after correction of the trafficking defect by low-temperature incubation. The YFP-based screening allowed the identification of the antihypertensive 1,4-dihydropyridines (DHPs) nifedipine, nicardipine, nimodipine, isradipine, nitrendipine, felodipine, and niguldipine as compounds able to activate ΔF508-CFTR. This effect was not derived from the inhibition of voltage-dependent Ca2+ channels, the pharmacological target of antihypertensive DHPs. Indeed, methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl)pyridine-5-carboxylate (BayK-8644), a DHP that is effective as an activator of such channels, also stimulated CFTR activity. DHPs were also effective on the G551D-CFTR mutant by inducing a 16- to 45-fold increase of the CFTR Cl- currents. DHP activity was confirmed in airway epithelial cells from patients with CF. DHPs may represent a novel class of therapeutic agents able to correct the defect caused by a set of CF mutations.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The ΔF508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 µM (with forskolin, 20 µM) by an iodide uptake assay in epithelial cells coexpressing ΔF508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after ΔF508-CFTR rescue by 24-h culture at 27°C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective ΔF508-CFTR gating ("potentiators") with nanomolar potency that were active in human ΔF508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2–1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated ΔF508-CFTR in the presence of forskolin with Ka ∼ 70 nM and also activated the CFTR gating mutants G551D and G1349D with Ka values of ∼1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had Ka ∼ 20 nM for activation of ΔF508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by gating mutants and possibly for a subset of ΔF508 subjects with significant ΔF508-CFTR plasma-membrane expression.

A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca2+-activated Cl– transport, whereas the swelling-activated Cl– transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca2+,Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.

Activation of Cl- and K+ conductances by nucleotide receptor-operated mobilization of intracellular Ca2+ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl- ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at -100 mV with 100 µmol/l of either ATP or UTP. The second current was carried by K+ ions and was blocked by Cs+. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at -20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca2+ concentration ([Ca2+]i) that was similar in the presence or absence of extracellular Ca2+. The ED50 for UTP was 24 umol/l and that for ATP was 94 µmol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by thapsigargin prevented both the nucleotide-induced [Ca2+]i increase and the activation of membrane currents. Addition of 2 mmol/l Ca2+ to thapsigargin-treated cells produced a sustained increase of Cl- and K+ currents, which was reversed by Ca2+ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca2+]i that stimulates Ca2+-dependent Cl- and K+ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca2+ stores.

Patch-clamp experiments on human cultured keratinocytes revealed the presence of three types of ion channel. The first type was a Cl--selective channel, the current/voltage relationship of which showed outward rectification, the mean conductance at positive and negative membrane potentials being 66 pS and 16 pS respectively. The second type of channel showed almost equal permeability to alkali ions but was impermeable to Cl- and to the large organic cation N-methyl-d-glucamine. Its current/voltage relationship was linear with a mean unitary conductance of 18 pS in symmetrical 140 mmol/l NaCl. Finally, the third type was a large-conductance cation channel, which had in physiological ionic conditions a peculiar rectifying current/voltage relationship, the shape of which was strongly dependent on the concentration of divalent cations on both sides of the membrane. Lowering of Ca2+ and/or Mg2+ on either side of the patch led to a marked increase of the single-channel current. With identical solutions without Ca2+ and Mg2+ on both sides of the patch the current/voltage relationship became ohmic and reached a conductance of 150–200 pS. In addition, channel activity was reversibly affected by changes of the external Ca2+ concentration. In particular, open-channel probability strongly increased at negative membrane potentials when the external Ca2+ was lowered from millimolar to micromolar values. Whole-cell experiments confirm the role of the extracellular Ca2+ as a modulator of the cation conductance.

1Chromones (sodium cromoglycate and sodium nedocromil) block cell swelling‐activated Cl−channels in NIH‐3T3 fibroblasts and endothelial cells. This has led to hypothesize that cell volume regulation might be involved in asthma pathogenesis.2Using whole‐cell patch‐clamp experiments, we studied the effect of chromones on volume‐sensitive Cl−currents in transformed human tracheal epithelial cells (9HTEo‐) and in primary cultures of human bronchial epithelial cells (BE).3Cl−currents activated by hypotonic shock were poorly blocked by extracellular nedocromil or cromoglycate. The block was voltage‐dependent since it was observed only at positive membrane potentials. At the concentration of 5 mm, the current inhibition by both chromones at +80 mV was about 40% for 9HTEo‐ and only 20% for BE.4Intracellular application of chromones elicited a voltage‐independent inhibition in 9HTEo‐ cells. Under this condition, volume‐sensitive Cl−currents were reduced at all membrane potentials (60 and 45% inhibition by 2 mmnedocromil and cromoglycate respectively). In contrast intracellular chromones were ineffective in BE cells.5The relative refractoriness to chromones, in contrast with the high sensitivity shown by other Cl−channels, suggests that the epithelial volume‐sensitive Cl−channel is not involved in asthma.

The patch clamp technique was used to reveal single channel activity in the membrane of human cultured fibroblasts. The most frequently detected ion channel type was a Ca2+-dependent K+ channel with a conductance of 287±38 pS in symmetrical 130 mM KCl. The channel showed a peculiar low Ca2+-sensitivity compared to that of similar channels in other preparations. In fact micromolar values of internal Ca2+ were not effective in the channel activation, except at high depolarizing membrane potentials. The activity was highly increased only when the channel was exposed to relatively high internal Ca2+ concentrations (0.2–2.0 mM).

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