Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by more than 2100 mutations in the CF transmembrane conductance regulator (CFTR) gene, generating variability in disease severity among individuals with CF sharing the same CFTR genotype. Systems biology can assist in the collection and visualization of CF data to extract additional biological significance and find novel therapeutic targets. Here, we present the CyFi-MAP-a disease map repository of CFTR molecular mechanisms and pathways involved in CF. Specifically, we represented the wild-type (wt-CFTR) and the F508del associated processes (F508del-CFTR) in separate submaps, with pathways related to protein biosynthesis, endoplasmic reticulum retention, export, activation/inactivation of channel function, and recycling/degradation after endocytosis. CyFi-MAP is an open-access resource with specific, curated and continuously updated information on CFTR-related pathways available online at https://cysticfibrosismap.github.io/ . This tool was developed as a reference CF pathway data repository to be continuously updated and used worldwide in CF research.

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

Most cases of Cystic Fibrosis (CF) are diagnosed early in life. However, people with atypical CF forms pose diagnosis dilemmas, requiring laboratory support for diagnosis confirmation/exclusion. Ex vivo analysis of fresh rectal biopsies by Ussing chamber has been the best discriminant biomarker for CF diagnosis/prognosis so far. Here we aimed to evaluate different electrophysiological parameters from Ussing chamber analysis of rectal biopsies from people with CF (PwCF) to establish the one with highest correlations with clinical features as the best CF diagnosis/prognosis biomarker. We analyzed measurements of CFTR-mediated Cl- secretion in rectal biopsies from 143 individuals (∼592 biopsies), the largest cohort so far analyzed by this approach. New parameters were analyzed and compared with the previous biomarker, i.e., the IBMX (I)/Forskolin (F)/Carbachol (C)-stimulated short-circuit current (I'sc-I/F/C). Correlations with clinical features showed that the best parameter corresponded to voltage measurements of the I/F + (I/F/CCH) response (VI/F+I/F/C), with higher correlations vs. I'sc-I/F/C for: sweat chloride (59 vs. 52%), fecal elastase (69 vs. 55%) and lung function, measured by FEV1 (27 vs. 20%). Altogether data show that VI/F+I/F/C is the most sensitive, reproducible, and robust predictive biomarker for CF diagnosis/prognosis effectively discriminating classical, atypical CF and non-CF groups.

For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine.

Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases.

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such "mutation-agnostic" therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs - ADRA2C and CXCR3 - increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the secretory chloride/bicarbonate channel in airways and intestine that is activated through ATP binding and phosphorylation by protein kinase A, but fails to operate in cystic fibrosis (CF). TMEM16A (also known as anoctamin 1, ANO1) is thought to function as the Ca2+ activated secretory chloride channel independent of CFTR. Here we report that tissue specific knockout of the TMEM16A gene in mouse intestine and airways not only eliminates Ca2+-activated Cl- currents, but unexpectedly also abrogates CFTR-mediated Cl- secretion and completely abolishes cAMP-activated whole cell currents. The data demonstrate fundamentally new roles of TMEM16A in differentiated epithelial cells: TMEM16A provides a mechanism for enhanced ER Ca2+ store release, possibly engaging Store Operated cAMP Signaling (SOcAMPS) and activating Ca2+ regulated adenylyl cyclases. TMEM16A is shown to be essential for proper activation and membrane expression of CFTR. This intimate regulatory relationship is the cause for the functional overlap of CFTR and Ca2+-dependent chloride transport.

In autosomal dominant polycystic kidney disease (ADPKD) multiple bilateral renal cysts gradually enlarge, leading to a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) are known to drive cyst enlargement. Here we demonstrate that loss of Pkd1 increased expression of TMEM16A and CFTR and Cl- secretion in murine kidneys, with TMEM16A essentially contributing to cyst growth. Upregulated TMEM16A enhanced intracellular Ca2+ signaling and proliferation of Pkd1-deficient renal epithelial cells. In contrast, increase in Ca2+ signaling, cell proliferation and CFTR expression was not observed in Pkd1/Tmem16a double knockout mice. Knockout of Tmem16a or inhibition of TMEM16A in vivo by the FDA-approved drugs niclosamide and benzbromarone, as well as the TMEM16A-specific inhibitor Ani9 largely reduced cyst enlargement and abnormal cyst cell proliferation. The present data establish a therapeutic concept for the treatment of ADPKD.

Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.

In polycystic kidney disease (PKD) multiple bilateral renal cysts gradually enlarge causing a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) drive cyst enlargement. We demonstrated recently that a loss of PKD1 increases expression and function of TMEM16A in murine kidneys and in mouse M1 collecting duct cells. The data demonstrated that TMEM16A contributes essentially to cyst growth by upregulating intracellular Ca2+ signaling. Enhanced expression of TMEM16A and Ca2+ signaling increased both cell proliferation and fluid secretion, which suggested inhibition of TMEM16A as a novel therapy in ADPKD. About 15 % of all ADPKD cases are caused by mutations in PKD2. To analyze the effects of loss of function of PKD2 on Ca2+ signaling, we knocked-down Pkd2 in mouse primary renal epithelial cells in the present study, using viral transfection of shRNA. Unlike in Pkd1-/- cells, knockdown of PKD2 lowered basal Ca2+ and augmented store-operated Ca2+ entry, which was both independent of TMEM16A. However, disease causing purinergic Ca2+ store release was enhanced, similar to that observed in Pkd1-/- renal epithelial cells. The present data suggest pharmacological inhibition of TMEM16A as a treatment in ADPKD caused by mutations in both PKD1 and PKD2.

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca2+-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization. Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic. We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density. This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.

Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

Cystic Fibrosis (CF) is caused by >2000 mutations in the CF transmembrane conductance regulator (CFTR) gene, but one mutation-F508del-occurs in ~80% of patients worldwide. Besides its main function as an anion channel, the CFTR protein has been implicated in epithelial differentiation, tissue regeneration, and, when dysfunctional, cancer. However, the mechanisms that regulate such relationships are not fully elucidated. Krüppel-like factors (KLFs) are a family of transcription factors (TFs) playing central roles in development, stem cell differentiation, and proliferation. Herein, we hypothesized that these TFs might have an impact on CFTR expression and function, being its missing link to differentiation. Our results indicate that KLF4 (but not KLF2 nor KLF5) is upregulated in CF vs. non-CF cells and that it negatively regulates wt-CFTR expression and function. Of note, F508del-CFTR expressing cells are insensitive to KLF4 modulation. Next, we investigated which KLF4-related pathways have an effect on CFTR. Our data also show that KLF4 modulates wt-CFTR (but not F508del-CFTR) via both the serine/threonine kinase AKT1 (AKT) and glycogen synthase kinase 3 beta (GSK3β) signaling. While AKT acts positively, GSK3β is a negative regulator of CFTR. This crosstalk between wt-CFTR and KLF4 via AKT/ GSK3β signaling, which is disrupted in CF, constitutes a novel mechanism linking CFTR to the epithelial differentiation.

Activation of the Ca2+ activated Cl- channel TMEM16A is proposed as a treatment in inflammatory airway disease. It is assumed that activation of TMEM16A will induce electrolyte secretion, and thus reduce airway mucus plugging and improve mucociliary clearance. A benefit of activation of TMEM16A was shown in vitro and in studies in sheep, but others reported an increase in mucus production and airway contraction by activation of TMEM16A. We analyzed expression of TMEM16A in healthy and inflamed human and mouse airways and examined the consequences of activation or inhibition of TMEM16A in asthmatic mice. TMEM16A was found to be upregulated in the lungs of patients with asthma or cystic fibrosis, as well as in the airways of asthmatic mice. Activation or potentiation of TMEM16A by the compounds Eact or brevenal, respectively, induced acute mucus release from airway goblet cells and induced bronchoconstriction in mice in vivo. In contrast, niclosamide, an inhibitor of TMEM16A, blocked mucus production and mucus secretion in vivo and in vitro. Treatment of airway epithelial cells with niclosamide strongly inhibited expression of the essential transcription factor of Th2-dependent inflammation and goblet cell differentiation, SAM pointed domain-containing ETS-like factor (SPDEF). Activation of TMEM16A in people with inflammatory airway diseases is likely to induce mucus secretion along with airway constriction. In contrast, inhibitors of TMEM16A may suppress pulmonary Th2 inflammation, goblet cell metaplasia, mucus production, and bronchoconstriction, partially by inhibiting expression of SPDEF.

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.

Previous analysis of CFTR-knockout (CFTR-/-) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR-/- piglets). Tracheas of CFTR-/- piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR-/- airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR-/- piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αβγENaC were unchanged in CFTR-/- airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR-/- airways and intestine. Small intestinal epithelium of CFTR-/- piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR-/- animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR-/- piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR-/- piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR-/- piglets, along with a compromised CFTR- and TMEM16A-dependent Cl- transport.