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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 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed 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 advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors—elexacaftor/tezacaftor—and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients’ responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants.

In genetic diseases, the most prevalent mechanism of pathogenicity is an altered expression of dosage-sensitive genes. Drugs that restore physiological levels of these genes should be effective in treating the associated conditions. We developed a screening strategy, based on a bicistronic dual-reporter vector, for identifying compounds that modulate proteins levels, and used it in a pharmacological screening approach. To provide a proof-of-principle, we chose Autosomal Dominant LeukoDystrophy (ADLD), an ultra-rare adult-onset neurodegenerative disorder caused by lamin B1 (LMNB1) overexpression. We used a stable CHO cell line simultaneously expresses an AcGFP reporter fused to LMNB1 and a Ds-Red normalizer. Using high-content imaging analysis, we screened a library of 717 biologically active compounds and approved drugs, and identified alvespimycin, an HSP90 inhibitor, as a positive hit. We confirmed that alvespimycin can reduce LMNB1 levels by 30-80% in five different cell lines (fibroblasts, NIH3T3, CHO, COS-7 and rat primary glial cells). In ADLD fibroblasts, alvespimycin reduced cytoplasmic LMNB1 by about 50%. We propose this approach for effectively identifying potential drugs for treating genetic diseases associated with deletions/duplications, and paving the way towards phase II clinical trials. This article is protected by copyright. All rights reserved.

Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this mutation still face several disease-related symptoms. Thus, more potent compound combinations are still needed. Here, we investigated the mechanism of action (MoA) of RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by RDR01752 was assessed by biochemical, immunofluorescence microscopy and functional assays in cell lines and in intestinal organoids. To determine the MoA of RDR01752, we assessed its additive effects to those of genetic revertants of F508del-CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature. Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752 produced lower functional rescue. However, in functional assays using intestinal organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy. RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W, as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and function compared to each corrector alone. The lack of additivity of RDR01752 with the genetic revertant R1070W suggests that this compound has the same effect as the insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of F508del-CFTR.

Defective epithelial ion transport is the hallmark of the common life-limiting genetic disease cystic fibrosis (CF). CF is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette transporter, which functions as an epithelial anion channel regulated by phosphorylation and cycles of ATP binding and hydrolysis. Here, we review selectively the structure and function of CFTR, including how the membrane-spanning domains form an anion-selective pore and the roles of the regulatory domain and nucleotide-binding domains in channel gating. We discuss the development of combination therapy for CF mutations, including CFTR correctors, which rescue misfolding to transport mutant CFTR to the plasma membrane and CFTR potentiators, which increase CFTR activity by enhancing channel gating. Finally, we highlight studies of CFTR processing and membrane trafficking, which identify proteostasis regulators that have the potential to lead to innovative new therapies for CF. Thus, understanding of CFTR has driven new insights into epithelial ion transport and transformed the treatment of CF.

We identified a small molecule that, at picomolar concentrations, rescues mutant CFTR chloride channel from protein degradation., F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

TMEM16A is a plasma membrane protein with voltage- and calcium-dependent chloride channel activity. The role of the various TMEM16A domains in expression and function is poorly known. In a previous study, we found that replacing the first ATG of the TMEM16A coding sequence with a nonsense codon (M1X mutation), to force translation from the second ATG localized at position 117, only had minor functional consequences. Therefore, we concluded that this region is dispensable for TMEM16A processing and channel activity. We have now removed the first 116 codons from the TMEM16A coding sequence. Surprisingly, the expression of the resulting mutant, Δ(1–116), resulted in complete loss of activity. We hypothesized that, in the mutant M1X, translation may start at a position before the second ATG, using a non-canonical start codon. Therefore, we placed an HA-epitope at position 89 in the M1X mutant. We found, by western blot analysis, that the HA-epitope can be detected, thus demonstrating that translation starts from an upstream non-ATG codon. We truncated the N-terminus of TMEM16A at different sites while keeping the HA-epitope. We found that stepwise shortening of TMEM16A caused an in parallel stepwise decrease in TMEM16A expression and function. Our results indicate that indeed the N-terminus of TMEM16A is important for its activity. The use of an alternative start codon appears to occur in a naturally-occurring TMEM16A isoform that is particularly expressed in human testis. Future experiments will need to address the role of normal and alternative amino-terminus in TMEM16A structure and function., Graphical abstract, Highlights • TMEM16A is a membrane protein with chloride channel activity. • Two ATG codons are present at positions 1 and 117 of the TMEM16A coding sequence. • Deletion of the first ATG, in contrast to removal of the first 116 codons, does not abolish TMEM16A function. • Without the first ATG, TMEM16A translation begins from a non-canonical start codon.

The Ca(2+)-activated Cl(-) channels (CaCCs) are involved in a variety of physiological functions, such as transepithelial anion transport, smooth muscle contraction and olfaction. Recently, the question of the molecular identity of CaCCs has apparently been resolved with the identification of TMEM16A protein (also known as anoctamin-1). Expression of TMEM16A is associated with the appearance of Ca(2+)- and voltage-dependent Cl(-) currents with properties similar to those of native CaCCs. The putative structure of TMEM16A consists of eight transmembrane domains, with both the amino- and the carboxy-terminus protruding in the cytosol. TMEM16A is also characterized by the existence of different protein variants generated by alternative splicing. A close paralogue of TMEM16A, TMEM16B (anoctamin-2), is also associated with CaCC activity, although with different properties. The TMEM16B-dependent channels require higher intracellular Ca(2+) concentrations and have faster activation and deactivation kinetics. Expression of other anoctamins is devoid of detectable channel activity. These proteins, such as TMEM16F (anoctamin-6), may have different functions.

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue.

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins.

Background Deletion of phenylalanine 508 is the most frequent mutation causing cystic fibrosis. It causes multiple defects: 1) misfolding of the protein causing retention at the ER (processing defect); 2) reduced channel activity (gating defect); 3) reduced plasma membrane residency time due to increased internalization rate and defective recycling. Methods Druggability of F508del-CFTR was demonstrated by several studies. Correctors are molecules able to improve maturation and trafficking to the membrane of F508del- CFTR. Correctors could act as pharmacological chaperones or as proteostasis regulators. Pharmacological chaperones act directly on mutant CFTR, while proteostasis regulators modify the proteostasis environment leading to beneficial effects on CFTR maturation. Results The use of a single compound is not sufficient to promote a therapeutically relevant F508del-CFTR rescue. Drug therapy for CF will require combinations of correctors exploiting different mechanisms of action, i.e. pharmacological chaperones combined together or with a proteostasis regulator. Conclusion Development of more effective CF drugs could therefore rely on a better understanding of the molecular events underlying CFTR processing/degradation. This review will focus on most promising pathways and related targets for the development of novel CF pharmacotherapies.

The correction of premature termination codons (PTCs) by agents that promote readthrough represents a promising emerging tool for the treatment of many genetic diseases. The efficiency of the treatment, however, varies depending on the stop codon itself and the amount of correctible transcripts related to the efficiency of nonsense-mediated decay. In the current study, a screen by in vitro minigene assay of all six PTCs described in exon 15 of the CFTR gene demonstrated alternative splicing to differing degrees for five of them. Of the five, PTC mutations c.2537G>A (p.Trp846*(UAG) ) and c.2551C>T (p.Arg851*) cause the greatest proportion of transcripts lacking exon 15; both mutations altering exonic splicing regulatory elements. In order to increase the amount of full-length transcripts, different pharmacological treatments were performed showing both negative and positive effects on exon inclusion for the same mutation. Therefore, the total amount of transcripts together with the splicing profile should be assessed to anticipate and improve efficacy of readthrough therapy.

The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27°C) or with pharmacological correctors. However, the efficacy of correctors on the mutant protein appears to be dependent on the cell expression system. We have used a bronchial epithelial cell line, CFBE41o−, to determine the efficacy of various known treatments and to discover new correctors. Compared with other cell types, CFBE41o− shows the largest response to low temperature and the lowest one to correctors such as corr-4a and VRT-325. A screening of a small-molecule library identified 9-aminoacridine and ciclopirox, which were significantly more effective than corr-4a and VRT-325. Analysis with microarrays revealed that 9-aminoacridine, ciclopirox, and low temperature, in contrast to corr-4a, cause a profound change in cell transcriptome. These data suggest that 9-aminoacridine and ciclopirox act on F508del-CFTR maturation as proteostasis regulators, a mechanism already proposed for the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). However, we found that 9-aminoacridine, ciclopirox, and SAHA, in contrast to corr-4a, VRT-325, and low temperature, do not increase chloride secretion in primary bronchial epithelial cells from CF patients. These conflicting data appeared to be correlated with different gene expression signatures generated by these treatments in the cell line and in primary bronchial epithelial cells. Our results suggest that F508del-CFTR correctors acting by altering the cell transcriptome may be particularly active in heterologous expression systems but markedly less effective in native epithelial cells.

SCN− (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN− is oxidized by H2O2, a reaction catalyzed by lactoperoxidase, to produce OSCN− (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN− in the airway surface fluid, we studied transepithelial SCN− transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN− flux. cAMP and Ca2+ regulatory pathways controlled SCN− transport through cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl− channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN−/Cl− exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN− transport. Measurements of H2O2 production at the apical surface of bronchial cells indicated that the extent of SCN− transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN− transport mechanisms under resting and stimulated conditions. Defects in SCN− transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.