Rescue of ΔF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

2006 ◽  
Vol 290 (6) ◽  
pp. L1117-L1130 ◽  
Author(s):  
Fredrick Van Goor ◽  
Kimberly S. Straley ◽  
Dong Cao ◽  
Jesús González ◽  
Sabine Hadida ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecules ◽  
Fluid Transport ◽  
Single Channel ◽  
Primary Cultures ◽  
Genetic Defect ◽  
Common Mutation ◽  
Compound Libraries ◽  
Epithelial Cultures ◽  
Δf508 Cftr

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl− channel. The most common mutation results in a deletion of phenylalanine at position 508 (ΔF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of ΔF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores ΔF508-CFTR-mediated Cl− transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of ΔF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.

scholarly journals CFTR: cystic fibrosis and beyond

2014 ◽  
Vol 44 (4) ◽  
pp. 1042-1054 ◽  
Author(s):  
Marcus A. Mall ◽  
Dominik Hartl
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Lessons Learned ◽  
Chronic Obstructive ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Obstructive Pulmonary Disease ◽  
Airway Diseases ◽  
Δf508 Cftr

Cystic fibrosis (CF) remains the most common fatal hereditary lung disease. The discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene 25 years ago set the stage for: 1) unravelling the molecular and cellular basis of CF lung disease; 2) the generation of animal models to study in vivo pathogenesis; and 3) the development of mutation-specific therapies that are now becoming available for a subgroup of patients with CF. This article highlights major advances in our understanding of how CFTR dysfunction causes chronic mucus obstruction, neutrophilic inflammation and bacterial infection in CF airways. Furthermore, we focus on recent breakthroughs and remaining challenges of novel therapies targeting the basic CF defect, and discuss the next steps to be taken to make disease-modifying therapies available to a larger group of patients with CF, including those carrying the most common mutation ΔF508-CFTR. Finally, we will summarise emerging evidence indicating that acquired CFTR dysfunction may be implicated in the pathogenesis of chronic obstructive pulmonary disease, suggesting that lessons learned from CF may be applicable to common airway diseases associated with mucus plugging.

scholarly journals Integrative chemogenomic analysis identifies small molecules that partially rescue ΔF508‐CFTR for cystic fibrosis

2021 ◽  
Author(s):  
Rachel A. Hodos ◽  
Matthew D. Strub ◽  
Shyam Ramachandran ◽  
Ella A. Meleshkevitch ◽  
Dmitri Y. Boudko ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecules ◽  
Δf508 Cftr

Chemical conjugation of ΔF508-CFTR corrector deoxyspergualin to transporter human serum albumin enhances its ability to rescue Cl− channel functions

2008 ◽  
Vol 295 (2) ◽  
pp. L336-L347 ◽  
Author(s):  
Caroline Norez ◽  
Matteo Pasetto ◽  
Maria Cristina Dechecchi ◽  
Erika Barison ◽  
Cristina Anselmi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Degradation Pathway ◽  
Cross Linking ◽  
Common Mutation ◽  
Pharmacological Chaperones ◽  
Δf508 Cftr ◽  
Ubiquitin Proteasome

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein (ΔF508-CFTR) that fails to fold properly, thus mutated ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in ΔF508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide-based cross-linker and its administration to cells carrying ΔF508-CFTR resulted in a greater enhancement of ΔF508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations.

Increased functional cell surface expression of CFTR and ΔF508-CFTR by the anthracycline doxorubicin

2001 ◽  
Vol 280 (5) ◽  
pp. C1031-C1037 ◽  
Author(s):  
Rangan Maitra ◽  
Collin M. Shaw ◽  
Bruce A. Stanton ◽  
Joshua W. Hamilton
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Chloride Secretion ◽  
Cell Surface Expression ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Δf508 Cftr ◽  
Cftr Protein

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, ΔF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, ΔF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 μM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased ΔF508-CFTR cell surface expression and ΔF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of ΔF508-CFTR

2000 ◽  
Vol 278 (2) ◽  
pp. C259-C267 ◽  
Author(s):  
Ronald C. Rubenstein ◽  
Pamela L. Zeitlin
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Ubiquitin Proteasome System ◽  
Common Mutation ◽  
Mrna Levels ◽  
Δf508 Cftr ◽  
Protein Immunoreactivity ◽  
And Function

The most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), ΔF508, is a trafficking mutant that has prolonged associations with molecular chaperones and is rapidly degraded, at least in part by the ubiquitin-proteasome system. Sodium 4-phenylbutyrate (4PBA) improves ΔF508-CFTR trafficking and function in vitro in cystic fibrosis epithelial cells and in vivo. To further understand the mechanism of action of 4PBA, we tested the hypothesis that 4PBA modulates the targeting of ΔF508-CFTR for ubiquitination and degradation by reducing the expression of Hsc70 in cystic fibrosis epithelial cells. IB3-1 cells (genotype ΔF508/W1282X) that were treated with 0.05–5 mM 4PBA for 2 days in culture demonstrated a dose-dependent reduction in Hsc70 protein immunoreactivity and mRNA levels. Immunoprecipitation with Hsc70-specific antiserum demonstrated that Hsc70 and CFTR associated under control conditions and that treatment with 4PBA reduced these complexes. Levels of immunoreactive Hsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBA treatment. These data suggest that 4PBA may improve ΔF508-CFTR trafficking by allowing a greater proportion of mutant CFTR to escape association with Hsc70.

scholarly journals Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures

2010 ◽  
Vol 298 (3) ◽  
pp. L304-L314 ◽  
Author(s):  
Deborah M. Cholon ◽  
Wanda K. O'Neal ◽  
Scott H. Randell ◽  
John R. Riordan ◽  
Martina Gentzsch
Keyword(s):  
Apical Membrane ◽  
Endocytic Trafficking ◽  
Epithelial Cell Line ◽  
Common Mutation ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Wild Type ◽  
Human Airway ◽  
Epithelial Cultures ◽  
Δf508 Cftr

CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, ΔF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in ΔF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of ΔF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of ΔF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.

scholarly journals Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

2005 ◽  
Vol 16 (5) ◽  
pp. 2154-2167 ◽  
Author(s):  
Silvia M. Kreda ◽  
Marcus Mall ◽  
April Mengos ◽  
Lori Rochelle ◽  
James Yankaskas ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Normal Subjects ◽  
Cell Types ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Ciliated Cells ◽  
Metabolic Fate ◽  
Specific Expression ◽  
Δf508 Cftr ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and ΔF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of ΔF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No ΔF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of ΔF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and ΔF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.

Mislocalization of ΔF508 CFTR in cystic fibrosis sweat gland

1992 ◽  
Vol 1 (5) ◽  
pp. 321-327 ◽  
Author(s):  
Norbert Kartner ◽  
Olga Augustinas ◽  
Timothy J. Jensen ◽  
A. Leonard Naismith ◽  
John R. Riordan
Keyword(s):  
Cystic Fibrosis ◽  
Sweat Gland ◽  
Δf508 Cftr

CFTR ΔF508 mutation has minimal effect on the gene expression profile of differentiated human airway epithelia

2005 ◽  
Vol 289 (4) ◽  
pp. L545-L553 ◽  
Author(s):  
Joseph Zabner ◽  
Todd E. Scheetz ◽  
Hakeem G. Almabrazi ◽  
Thomas L. Casavant ◽  
Jian Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Large Scale ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Primary Cultures ◽  
Gene Expression Profiles ◽  
Filter Method ◽  
Tissue Destruction ◽  
Airway Epithelia

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel regulated by phosphorylation. Most of the disease-associated morbidity is the consequence of chronic lung infection with progressive tissue destruction. As an approach to investigate the cellular effects of CFTR mutations, we used large-scale microarray hybridization to contrast the gene expression profiles of well-differentiated primary cultures of human CF and non-CF airway epithelia grown under resting culture conditions. We surveyed the expression profiles for 10 non-CF and 10 ΔF508 homozygote samples. Of the 22,283 genes represented on the Affymetrix U133A GeneChip, we found evidence of significant changes in expression in 24 genes by two-sample t-test ( P < 0.00001). A second, three-filter method of comparative analysis found no significant differences between the groups. The levels of CFTR mRNA were comparable in both groups. There were no significant differences in the gene expression patterns between male and female CF specimens. There were 18 genes with significant increases and 6 genes with decreases in CF relative to non-CF samples. Although the function of many of the differentially expressed genes is unknown, one transcript that was elevated in CF, the KCl cotransporter (KCC4), is a candidate for further study. Overall, the results indicate that CFTR dysfunction has little direct impact on airway epithelial gene expression in samples grown under these conditions.

Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia

2000 ◽  
Vol 279 (2) ◽  
pp. C461-C479 ◽  
Author(s):  
Daniel C. Devor ◽  
Robert J. Bridges ◽  
Joseph M. Pilewski
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pharmacological Agents ◽  
Transport Defect

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and ΔF508 (ΔF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current ( I sc) averaged 27.0 ± 0.6 μA/cm2 ( n = 350). Amiloride reduced this I sc by 13.5 ± 0.5 μA/cm2 ( n = 317). In ΔF-HBE, baseline I sc was 33.8 ± 1.2 μA/cm2 ( n = 200), and amiloride reduced this by 29.6 ± 1.5 μA/cm2 ( n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I sc(Δ I sc = 8.4 ± 0.8 μA/cm2; n = 119). Addition of acetazolamide, 5-( N-ethyl- N-isopropyl)-amiloride, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid further reduced I sc, suggesting forskolin also stimulates HCO3 − secretion. This was confirmed by ion substitution studies. The forskolin-induced I scwas inhibited by 293B, Ba2+, clofilium, and quinine, whereas charybdotoxin was without effect. In ΔF-HBE the forskolin I sc response was reduced to 1.2 ± 0.3 μA/cm2 ( n = 30). In wt HBE, mucosal UTP induced a transient increase in I sc (Δ I sc = 15.5 ± 1.1 μA/cm2; n = 44) followed by a sustained plateau, whereas in ΔF-HBE the increase in I sc was reduced to 5.8 ± 0.7 μA/cm2 ( n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I sc by 11.6 ± 0.9 ( n = 20), 10.8 ± 1.7 ( n = 18), 10.0 ± 1.6 ( n = 5), and 7.9 ± 0.8 μA/cm2( n = 17), respectively. In ΔF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl− secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl−secretory response (2.1 ± 0.3 μA/cm2, n = 21). In ΔF-HBE, genistein alone stimulated Cl− secretion (2.5 ± 0.5 μA/cm2, n = 11). After incubation of ΔF-HBE at 26°C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na+ absorption across ΔF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca2+-, but not cAMP-mediated agonists, stimulated K+ secretion across both wt HBE and ΔF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K+ and apical Cl− conductances directly modulate Cl−secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

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