Nanomolar Affinity Small Molecule Correctors of Defective ΔF508-CFTR Chloride Channel Gating

Yang, Hong; Shelat, Anang A.; Guy, R. Kiplin; Gopinath, Vadiraj S.; Tonghui, A.; Du, Kai; Lukacs, Gergely L.; Taddei, Alessandro; Folli, Chiara; Pedemonte, Nicoletta; Galietta, Luis J. V.; Verkman, A. S.

doi:10.1074/jbc.m303098200

Cited by 195 publications

(225 citation statements)

References 22 publications

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“…(i) This could exacerbate the severity of the disease for ⌬F508 patients especially if these mutant channels can reach the surfaces of a subset of epithelial cell types as argued by some (7), and (ii) therapies that target only the biosynthetic processing defect of this mutant may be ineffective. Our results support the emerging view (14) that the treatment of most CF patients may require combination therapies that include CFTR channel openers.…”

Section: Monolayers Transepithelial Currents Across Monolayers Of Cfsupporting

confidence: 79%

See 1 more Smart Citation

Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs

Wang¹,

Li²,

Clancy

et al. 2005

Journal of Biological Chemistry

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Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ⌬F508-CFTR, which inefficiently traffics to the surfaces of most cells. The ⌬F508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ⌬F508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ⌬F508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ⌬F508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ⌬F508-CFTR channels in excised membrane patches by 10 -15-fold under conditions in which wildtype channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ⌬F508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.

show abstract

Section: Monolayers Transepithelial Currents Across Monolayers Of Cfsupporting

confidence: 79%

“…By using these compounds as functional probes of mutant CFTR gating, we show that (i) the ⌬F508 mutation profoundly decreases the opening rates of surface-localized channels and (ii) this defect can be corrected by such compounds. Our results support the view that effective CF therapies may require the use of CFTR channel openers (9,14).…”

supporting

confidence: 79%

Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs

Wang¹,

Li²,

Clancy

et al. 2005

Journal of Biological Chemistry

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“…High-throughput drug screening efforts have successfully identified many small molecule compounds that partially rescue the trafficking defect of F508del-CFTR, called correctors (9)(10)(11)(12). Thus far, correctors have shown limited ability to improve F508del-CFTR trafficking.…”

Section: Introductionmentioning

confidence: 99%

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

Holleran

Glover

Peters

et al. 2012

Mol Med

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Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.

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“…The folding and maturation of ΔF508 CFTR can be rescued by several treatments in cell culture, although the rescued protein has a reduced efficiency of maturation and a reduced half-life at the plasma membrane [12][13][14][15][16][17] . The functional activity of the 'corrected' protein is also at least partially rescued by these treatments [12][13][14][15]18 , suggesting that the most predominant biophysical manifestations of this mutation affect the protein in a transient manner during biosynthesis. The ability of the mutant protein to be rescued also suggests that the ΔF508 defect is subtle in nature and that a therapeutic strategy to correct this misfolding might potentially be developed that could benefit the vast majority of patients with CF 19,20 .…”

mentioning

confidence: 99%

Side chain and backbone contributions of Phe508 to CFTR folding

Thibodeau

Brautigam

Machius

et al. 2004

Nat Struct Mol Biol

169

236

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Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.CFTR is a 1,480-residue polytopic membrane protein belonging to the ATP-binding cassette (ABC) superfamily of proteins, and is composed of two transmembrane domains (TMDs), two NBDs and a regulatory domain (R) 1,2 . Mutations in CFTR give rise to several diseases, including CF, a disease of abnormal ion secretion across epithelia 1 . More than 1,000 individual mutations have been identified that give rise to a spectrum of differing disease severities and symptoms (http://www.genet.sickkids.on.ca/cftr) 3 . The most common mutation is the deletion of a phenylalanine at position 508 (ΔF508) in the N-terminal NBD (NBD1) 4 . The deletion of Phe508 in CFTR gives rise to a temperature-sensitive folding defect evidenced by failure of the full-length protein to mature, retention in the endoplasmic reticulum (ER) and subsequent degradation by the proteasome 5-11 . © 2005 Nature Publishing GroupCorrespondence should be addressed to P.J.T. (Philip.Thomas@UTSouthwestern.edu). Note added in proof: Crystal structures of the human F508A missense NBD1 (with solublizing mutations F429S and H667R) and the corrected ΔF508 NBD1 (with three known suppressor mutations G550E, R553Q and R555K, and the solublizing mutations F409L, F429S, F433L and H667R) have been reported 51 . Consistent with the current study, neither of these mutant structures differ substantially from the murine wild-type structure outside of the flexible regulatory regions, nor does the ΔF508 mutation measurably alter the ΔG unfolding of the domain. The in vivo yield of soluble ΔF508 protein is decreased relative to both the wild-type and F508A proteins with both solublizing and suppressor mutations, consistent with a decrease in the efficiency of domain folding as described in this study.Note: Supplementary information is available on the Nature Structural & Molecular Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. The folding and maturation of ΔF508 CFTR can be rescued by several treatments in cell culture, although the rescued protein has a reduced efficiency of maturatio...

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Nanomolar Affinity Small Molecule Correctors of Defective ΔF508-CFTR Chloride Channel Gating

Cited by 195 publications

References 22 publications

Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs

Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

Side chain and backbone contributions of Phe508 to CFTR folding

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