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In cystic fibrosis, ion-transport abnormalities cause problems in many organs. A small molecule that forms cell-membrane pores allowing ion transport shows therapeutic promise in human cells and a model of the disease. A natural molecule that facilitates ion transport aids lung function., Author(s): David N. Sheppard, Anthony P. Davis Author Affiliations: Pore-forming small molecules offer a promising way to tackle cystic fibrosis In cystic fibrosis, abnormalities in the ion-channel protein CFTR cause [...]

Background and Purpose: Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl− channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP‐628006 with a distinct chemical structure. Experimental Approach Using electrophysiological assays with CFTR‐expressing heterologous cells and CF patient‐derived human bronchial epithelial (hBE) cells, we compared the effects of CP‐628006 with the marketed CFTR potentiator ivacaftor. Key Results: CP‐628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del‐CFTR were larger than those with the gating variant G551D‐CFTR. In excised inside‐out membrane patches, CP‐628006 potentiated wild‐type, F508del‐CFTR, and G551D‐CFTR by increasing the frequency and duration of channel openings. CP‐628006 increased the affinity and efficacy of F508del‐CFTR gating by ATP. In these respects, CP‐628006 behaved like ivacaftor. CP‐628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP‐628006 conferred ATP‐dependent gating on G551D‐CFTR, whereas the action of ivacaftor was ATP‐independent. For G551D‐CFTR, but not F508del‐CFTR, the action of CP‐628006 plus ivacaftor was greater than ivacaftor alone. CP‐628006 delayed, but did not prevent, the deactivation of F508del‐CFTR at the plasma membrane, whereas ivacaftor accentuated F508del‐CFTR deactivation. Conclusions and Implications: CP‐628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility. [ABSTRACT FROM AUTHOR]

Background and Purpose: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects.Experimental Approach: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques.Key Results: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR.Conclusions and Implications: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent. [ABSTRACT FROM AUTHOR]

Background and Purpose Dysfunction of the cystic fibrosis transmembrane conductance regulator ( CFTR) Cl− channel causes the genetic disease cystic fibrosis ( CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561 E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561 E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. Experimental Approach Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques. Key Results Low temperature incubation delivered a small proportion of A561 E- CFTR protein to the cell surface. Like F508del- CFTR, low temperature-rescued A561 E- CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561 E- CFTR also exhibited thermoinstability, losing function more quickly than F508del- CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561 E- CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561 E- and F508del- CFTR Cl− channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches. Conclusions and Implications Like F508del- CFTR, A561 E- CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561 E- CFTR. Transformational drug therapy for A561 E- CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability. [ABSTRACT FROM AUTHOR]

The mutation F508del is the commonest cause of the genetic disease cystic fibrosis (CF). CF disrupts the function of many organs in the body, most notably the lungs, by perturbing salt and water transport across epithelial surfaces. F508del causes harm in two principal ways. First, the mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Second, F508del perturbs the Cl− channel function of CFTR by disrupting channel gating. Here, we discuss the development of rational new therapies for CF that target F508del-CFTR. We highlight how structural studies provide new insight into the role of F508 in the regulation of channel gating by cycles of ATP binding and hydrolysis. We emphasize the use of high-throughput screening to identify lead compounds for therapy development. These compounds include CFTR correctors that restore the expression of F508del-CFTR at the apical membrane of epithelial cells and CFTR potentiators that rescue the F508del-CFTR gating defect. Initial results from clinical trials of CFTR correctors and potentiators augur well for the development of small molecule therapies that target the root cause of CF: mutations in CFTR. [ABSTRACT FROM AUTHOR]

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (Isc) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases Isc, we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased Isc in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (KD) of 134 mM. This THF-induced increase in Isc was enhanced by forskolin (10 µM), inhibited by the PKA inhibitor H-89 (10 µM) and the thiazolidinone CFTRinh-172 (10 µM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase Isc in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl- channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the ▵R-S660A-CFTR Cl- channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR. [ABSTRACT FROM AUTHOR]

The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562l were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wt- and V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F505del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control. [ABSTRACT FROM AUTHOR]

• CFTR correctors rescue the plasma membrane expression of F508del-CFTR. • Extracellular phosphate enhances F508del-CFTR function rescued by CFTR correctors. • Cystic fibrosis airway epithelia express the phosphate transporter SLC34A2. • Extracellular phosphate levels might contribute to variable drug responses. Background : The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors. Methods : With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature. Results : Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl− currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl− currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate. Conclusions : Extracellular phosphate modulates the magnitude of CFTR-mediated Cl− currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) mutations have complex effects on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. They disrupt its processing to and stability at the plasma membrane and function as an ATP-gated Cl − channel. Here, we review therapeutic strategies to overcome defective CFTR processing and stability. Because CF mutations have multiple impacts on the assembly of CFTR protein, combination therapy with several pharmacological chaperones is likely to be required to rescue mutant CFTR expression at the plasma membrane. Alternatively, proteostasis regulators, proteins which regulate the synthesis, intracellular transport and membrane stability of CFTR might be targeted to enhance the plasma membrane expression of mutant CFTR. Finally, we consider an innovative approach to bypass CFTR dysfunction in CF, the delivery of artificial anion transporters to CF epithelia to shuttle Cl − across the apical membrane. The identification of therapies or combinations of therapies, which rescue all CF mutations, is now a priority. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl- channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopicCFTRCl-currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wildtype CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified fulllength F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl- channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl- currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl- channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. [ABSTRACT FROM AUTHOR]

Abstract: With knowledge of the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR), its physiological role and dysfunction in cystic fibrosis (CF), therapeutic strategies are now being developed that target the root cause of CF rather than disease symptoms. Here, we review progress towards the development of rational new therapies for CF. We highlight the discovery of small molecules that rescue the cell surface expression and defective channel gating of CF mutants, termed CFTR correctors and CFTR potentiators, respectively. We draw attention to alternative approaches to restore epithelial ion transport to CF epithelia, including inhibitors of the epithelial Na+ channel (ENaC) and activators of the Ca2+-activated Cl− channel TMEM16A. The expertise required to translate small molecules identified in the laboratory to drugs for CF patients depends on our ability to coordinate drug development at an international level and our ability to provide pertinent biological information using suitable disease models. [Copyright &y& Elsevier]