Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator (CFTR) correctors VX-445 and VX-121.

Cystic fibrosis (CF) results from mutations in the CFTR anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The BKCa potassium channel is also critical for maintaining lung ASL volume. Here, we show the CFTR corrector, VX-445 (Elexacaftor), a component of Trikafta, induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121 - a corrector under clinical evaluation. Whole-cell patch-clamp recordings confirmed potentiated channel activity from CFTR correctors on the BKCa α-subunit, and excised patch-clamp recordings demonstrated a significant increase in open probability. In mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced action potential firing frequency in primary hippocampal and cortical neurons. VX-445 effects were observed at low micomolar concentrations (1-10 µM) - within the range reported in plasma and tissues from CF patients. We raise the possibilities that CFTR correctors gain additional clinical benefit by activation of BKCa in the lung, yet may lead to adverse events through BKCa activation, elsewhere.

In vitro modulator responsiveness of 655 CFTR variants found in people with cystic fibrosis.

BACKGROUND: In 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. METHODS: Plasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 h of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variant's forskolin/cAMP-induced baseline Cl- transport activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. RESULTS AND CONCLUSIONS: 253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10 % of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by ≥10 % of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by ≥10 percentage points for an additional 140 unapproved variants with ≥10 % but <50 % of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.

KCa3.1 potentiation stimulates Cl- secretion in F508del and G551D CFTR-corrected primary human bronchial epithelial cells.

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Cl- secretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl- secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl- equivalent current (ICl) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (Ra) and basolateral membrane (BLM; Rb) resistances. In WT CFTR HBEs, DCEBIO stimulated ICl, which was increased by forskolin. Similarly, forskolin stimulated ICl, and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited ICl. DCEBIO decreased Rb, whereas TRAM-34 increased Rb, consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate ICl, although the subsequent addition of forskolin + VX-770 increased ICl. Importantly, following stimulation of ICl with forskolin + VX-770, DCEBIO induced a further significant increase in ICl. As above, DCEBIO reduced Rb, whereas TRAM-34 increased Rb, consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on ICl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate ICl. However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled ICl. Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl- secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl- secretion across CF airway.

CFTR bearing variant p.Phe312del exhibits function inconsistent with phenotype and negligible response to ivacaftor.

The chloride channel dysfunction caused by deleterious cystic fibrosis transmembrane conductance regulator (CFTR) variants generally correlates with severity of cystic fibrosis (CF). However, 3 adults bearing the common severe variant p.Phe508del (legacy: F508del) and a deletion variant in an ivacaftor binding region of CFTR (p.Phe312del; legacy: F312del) manifested only elevated sweat chloride concentration (sw[Cl-]; 87-105 mEq/L). A database review of 25 individuals with F312del and a CF-causing variant revealed elevated sw[Cl-] (75-123 mEq/L) and variable CF features. F312del occurs at a higher-than-expected frequency in the general population, confirming that individuals with F312del and a CF-causing variant do not consistently develop overt CF features. In primary nasal cells, CFTR bearing F312del and F508del generated substantial chloride transport (66.0% ± 4.5% of WT-CFTR) but did not respond to ivacaftor. Single-channel analysis demonstrated that F312del did not affect current flow through CFTR, minimally altered gating, and ablated the ivacaftor response. When expressed stably in CF bronchial epithelial (CFBE41o-) cells, F312del-CFTR demonstrated residual function (50.9% ± 3.3% WT-CFTR) and a subtle decrease in forskolin response compared with WT-CFTR. F312del provides an exception to the established correlation between CFTR chloride transport and CF phenotype and informs our molecular understanding of ivacaftor response.

Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis.

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

Translating in vitro CFTR rescue into small molecule correctors for cystic fibrosis using the Library of Integrated Network-based Cellular Signatures drug discovery platform.

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The common ΔF508-CFTR mutation results in protein misfolding and proteasomal degradation. If ΔF508-CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508-CFTR trafficking and function, including low-temperature, small molecules, overexpression of miR-138, or knockdown of SIN3A. The challenge remains to translate such interventions into therapies and to understand their mechanisms. One approach for connecting such interventions to small molecule therapies that has previously succeeded for CF and other diseases is via mRNA expression profiling and iterative searches of small molecules with similar expression signatures. Here, we query the Library of Integrated Network-based Cellular Signatures using transcriptomic signatures from previously generated CF expression data, including RNAi- and low temperature-based rescue signatures. This LINCS in silico screen prioritized 135 small molecules that mimicked our rescue interventions based on their genomewide transcriptional perturbations. Functional screens of these small molecules identified eight compounds that partially restored ΔF508-CFTR function, as assessed by cAMP-activated chloride conductance. Of these, XL147 rescued ΔF508-CFTR function in primary CF airway epithelia, while also showing cooperativity when administered with C18. Improved CF corrector therapies are needed and this integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508-CFTR function and identify gene networks underlying such rescue.

Mechanistic analysis and significance of sphingomyelinase-mediated decreases in transepithelial CFTR currents in nHBEs.

Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508-CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib-1, which also decreased CFTR function. The SMase-mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5' adenosine monophosphate-activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non-CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508-CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid-SMase protein than nHBEs. Lastly, we determined that airway-like neutrophils, which are increased in the CF lung, secrete acid-SMase. Identifying the mechanism of SMase-mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid-SMase from cell types relevant to CF.

Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR-mediated apical conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes cystic fibrosis (CF). The loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus build-up, inflammation, and bacterial infections that lead to lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF relevant airway bacteria which degrades sphingomyelin into ceramide and phosphocholine, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR-mediated transepithelial anion secretion in both primary bronchial and tracheal epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Focusing on primary cells, we took a holistic ion homeostasis approach to determine a cause for reduced anion secretion following SMase treatment. Using impedance analysis, we determined that basolateral SMase inhibits apical and basolateral conductance in non-CF primary cells without affecting paracellular permeability. In CF primary airway cells, correction with clinically relevant CFTR modulators did not prevent SMase-mediated inhibition of CFTR currents. Furthermore, SMase was found to inhibit only apical conductance in these cells. Future work should determine the mechanism for SMase-mediated inhibition of CFTR currents, and further explore the clinical relevance of SMase and sphingolipid imbalances.

Integrative chemogenomic analysis identifies small molecules that partially rescue ΔF508-CFTR for cystic fibrosis.

Rare diseases affect 10% of the first-world population, yet over 95% lack even a single pharmaceutical treatment. In the present age of information, we need ways to leverage our vast data and knowledge to streamline therapeutic development and lessen this gap. Here, we develop and implement an innovative informatic approach to identify therapeutic molecules, using the Connectivity Map and LINCS L1000 databases and disease-associated transcriptional signatures and pathways. We apply this to cystic fibrosis (CF), the most common genetic disease in people of northern European ancestry leading to chronic lung disease and reduced lifespan. We selected and tested 120 small molecules in a CF cell line, finding 8 with activity, and confirmed 3 in primary CF airway epithelia. Although chemically diverse, the transcriptional profiles of the hits suggest a common mechanism associated with the unfolded protein response and/or TNFα signaling. This study highlights the power of informatics to help identify new therapies and reveal mechanistic insights while moving beyond target-centric drug discovery.

Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

CFTR modulator theratyping: Current status, gaps and future directions.

BACKGROUND: New drugs that improve the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with discreet disease-causing variants have been successfully developed for cystic fibrosis (CF) patients. Preclinical model systems have played a critical role in this process, and have the potential to inform researchers and CF healthcare providers regarding the nature of defects in rare CFTR variants, and to potentially support use of modulator therapies in new populations. METHODS: The Cystic Fibrosis Foundation (CFF) assembled a workshop of international experts to discuss the use of preclinical model systems to examine the nature of CF-causing variants in CFTR and the role of in vitro CFTR modulator testing to inform in vivo modulator use. The theme of the workshop was centered on CFTR theratyping, a term that encompasses the use of CFTR modulators to define defects in CFTR in vitro, with application to both common and rare CFTR variants. RESULTS: Several preclinical model systems were identified in various stages of maturity, ranging from the expression of CFTR variant cDNA in stable cell lines to examination of cells derived from CF patients, including the gastrointestinal tract, the respiratory tree, and the blood. Common themes included the ongoing need for standardization, validation, and defining the predictive capacity of data derived from model systems to estimate clinical outcomes from modulator-treated CF patients. CONCLUSIONS: CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.

Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator and Drugs: Insights from Cellular Trafficking.

The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein's absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.

Identification of the amino acids inserted during suppression of CFTR nonsense mutations and determination of their functional consequences.

In-frame premature termination codons (PTCs) account for ∼11% of all disease-associated mutations. PTC suppression therapy utilizes small molecules that suppress translation termination at a PTC to restore synthesis of a full-length protein. PTC suppression is mediated by the base pairing of a near-cognate aminoacyl-tRNA with a PTC and subsequently, the amino acid becomes incorporated into the nascent polypeptide at the site of the PTC. However, little is known about the identity of the amino acid(s) inserted at a PTC during this process in mammalian cells, or how the surrounding sequence context influences amino acid incorporation. Here, we determined the amino acids inserted at the cystic fibrosis transmembrane conductance regulator (CFTR) W1282X PTC (a UGA codon) in the context of its three upstream and downstream CFTR codons during G418-mediated suppression. We found that leucine, cysteine and tryptophan are inserted during W1282X suppression. Interestingly, these amino acids (and their proportions) are significantly different from those recently identified following G418-mediated suppression of the CFTR G542X UGA mutation. These results demonstrate for the first time that local mRNA sequence context plays a key role in near-cognate aminoacyl-tRNA selection during PTC suppression. We also found that some variant CFTR proteins generated by PTC suppression exhibit reduced maturation and activity, indicating the complexity of nonsense suppression therapy. However, both a CFTR corrector and potentiator enhanced activity of protein variants generated by G418-mediated suppression. These results suggest that PTC suppression in combination with CFTR modulators may be beneficial for the treatment of CF patients with PTCs.

Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice.

BACKGROUND & AIMS: Sjögren's syndrome and autoimmune pancreatitis are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. Nonobese diabetic/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren's syndrome and chronic pancreatitis and MRL/Mp mice are models of autoimmune pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a ductal Cl- channel essential for ductal fluid and HCO3- secretion. We used these models to ask the following questions: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function? METHODS: We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal, and acinar cells damage, infiltration, immune cells and function were measured in vivo and in isolated duct/acini. RESULTS: In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular, C18 restored salivation, rescued CFTR expression and localization, and nearly eliminated the inflammation and tissue damage. Transgenic overexpression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca2+ signaling, Orai1, inositol triphosphate receptors, Aquaporin 5 expression, and fluid secretion were restored by rescuing ductal CFTR. CONCLUSIONS: Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren's syndrome and pancreatitis.

Fatty Acid Cysteamine Conjugates as Novel and Potent Autophagy Activators That Enhance the Correction of Misfolded F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

A depressed autophagy has previously been reported in cystic fibrosis patients with the common F508del-CFTR mutation. This report describes the synthesis and preliminary biological characterization of a novel series of autophagy activators involving fatty acid cysteamine conjugates. These molecular entities were synthesized by first covalently linking cysteamine to docosahexaenoic acid. The resulting conjugate 1 synergistically activated autophagy in primary homozygous F508del-CFTR human bronchial epithelial (hBE) cells at submicromolar concentrations. When conjugate 1 was used in combination with the corrector lumacaftor and the potentiator ivacaftor, it showed an additive effect, as measured by the increase in the chloride current in a functional assay. In order to obtain a more stable form for oral dosing, the sulfhydryl group in conjugate 1 was converted into a functionalized disulfide moiety. The resulting conjugate 5 is orally bioavailable in the mouse, rat, and dog and allows a sustained delivery of the biologically active conjugate 1.

Evidence against resveratrol as a viable therapy for the rescue of defective ΔF508 CFTR.

BACKGROUND: Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. METHODS: Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. RESULTS: Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol. CONCLUSIONS: High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. GENERAL SIGNIFICANCE: Taken together, our results do not support the use of resveratrol supplements as a therapy for patients with cystic fibrosis. It is possible that further modifications of the resveratrol backbone would yield a more efficacious compound.

Effect of apical hyperosmotic sodium challenge and amiloride on sodium transport in human bronchial epithelial cells from cystic fibrosis donors.

Hypertonic saline (HS) inhalation therapy benefits cystic fibrosis (CF) patients [Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006; Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan W, Bye PT; the National Hypertonic Saline in Cystic Fibrosis (NHSCF) Study Group. N Engl J Med 354: 229-240, 2006]. Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na(+) channel blocker amiloride (Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: 1) Ussing chambers to measure amiloride-sensitive short-circuit currents (INa); 2) continuous perfusion Ussing chambers; and 3) near "thin-film" conditions in which the airway surface of the inserts was exposed to a small volume (30 µl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure INa under isosmotic conditions (near thin-film experiments; Tarran R, Boucher RC. Methods Mol Med 70: 479-492, 2002). HS solutions (660 mosmol/kgH2O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (ISC), conductance (GT), and capacitance (CT) were measured by transepithelial impedance analysis (Danahay H, Atherton HC, Jackson AD, Kreindler JL, Poll CT, Bridges RJ. Am J Physiol Lung Cell Mol Physiol 290: L558-L569, 2006; Singh AK, Singh S, Devor DC, Frizzell RA, van Driessche W, Bridges RJ. Methods Mol Med 70: 129-142, 2002). Exposure to apical HS inhibited INa, GT, and CT. The INa inhibition required 60 min of reexposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit INa was <2.5 min. Under near thin-film conditions, apical exposure to HS inhibited INa, but as osmotically driven water moved to the apical surface, the aqueous apical volume increased, leading to an amiloride-insensitive decrease in its osmolality and to recovery of INa that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of INa following exposure to HS. Our conclusions are that exposure to HS inhibits hBE INa and that amiloride diminishes this effect.

Mechanisms of bicarbonate secretion: lessons from the airways.

Early studies showed that airway cells secrete HCO(3)(-) in response to cAMP-mediated agonists and HCO(3)(-) secretion was impaired in cystic fibrosis (CF). Studies with Calu-3 cells, an airway serous model with high expression of CFTR, also show the secretion of HCO(3)(-) when cells are stimulated with cAMP-mediated agonists. Activation of basolateral membrane hIK-1 K(+) channels inhibits HCO(3)(-) secretion and stimulates Cl(-) secretion. CFTR mediates the exit of both HCO(3)(-) and Cl(-) across the apical membrane. Entry of HCO(3)(-) on a basolateral membrane NBC or Cl(-) on the NKCC determines which anion is secreted. Switching between these two secreted anions is determined by the activity of hIK-1 K(+) channels.

Requirements for efficient correction of ΔF508 CFTR revealed by analyses of evolved sequences.

Misfolding of ΔF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the ΔF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores ΔF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening.

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.