Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

Pyle, Louise C.; Ehrhardt, Annette; Mitchell, Lisa High; Fan, Lijuan; Ren, Aiming; Naren, Anjaparavanda P.; Yao, Li; Clancy, John; Bolger, Graeme B.; Sorscher, Eric J.; Rowe, Steven M.

doi:10.1152/ajplung.00465.2010

Cited by 27 publications

(35 citation statements)

References 48 publications

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“…Furthermore, in line with previous findings demonstrating that pharmacological molecules (correctors and potentiators) might increase the susceptibility of the R-domain of F508del CFTR to be phosphorylated (Pyle et al, 2011), we found that both correctors increase both the cAMP and the activated PKA compartmentalization in the sub-plasma-membrane region, allowing the consequent rescue of F508del-CFTR-dependent chloride efflux (Fig. 3).…”

Section: Discussionsupporting

confidence: 92%

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Abbattiscianni

Favia

Mancini

et al. 2016

Journal of Cell Science

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The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP 2 ) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTRdependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.

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Section: Discussionsupporting

confidence: 92%

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Abbattiscianni

Favia

Mancini

et al. 2016

Journal of Cell Science

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“…We have shown previously that this assay is a sensitive measure of the phosphorylation of the CFTR R-R by the cAMP-dependent PKA (20). Roflumilast induced robust phosphorylation of isolated CFTR R-R in a dose-dependent fashion that was sensitive to the PKA inhibitor H89 ( Figure 3B).…”

Section: Roflumilast Activates Cftrdependent Anion Transport By Incrementioning

confidence: 70%

“…R-region phosphorylation was performed in COS7 cells as described (20). COS7 cells were transfected transiently with a plasmid encoding the CF R-region (amino acids 635-836) with an N-terminal HA epitope tag.…”

Section: Voltage Clamp Studies In Ussing Chambersmentioning

confidence: 99%

Cystic Fibrosis Transmembrane Conductance Regulator Activation by Roflumilast Contributes to Therapeutic Benefit in Chronic Bronchitis

Lambert

Raju²,

Tang

et al. 2014

Am J Respir Cell Mol Biol

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Cigarette smoking causes acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction and is associated with delayed mucociliary clearance and chronic bronchitis. Roflumilast is a clinically approved phosphodiesterase 4 inhibitor that improves lung function in patients with chronic bronchitis. We hypothesized that its therapeutic benefit was related in part to activation of CFTR. Primary human bronchial epithelial (HBE) cells, Calu-3, and T84 monolayers were exposed to whole cigarette smoke (WCS) or air with or without roflumilast treatment. CFTRdependent ion transport was measured in modified Ussing chambers. Airway surface liquid (ASL) was determined by confocal microscopy. Intestinal fluid secretion of ligated murine intestine was monitored ex vivo. Roflumilast activated CFTR-dependent anion transport in normal HBE cells with a half maximal effective concentration of 2.9 nM. Roflumilast partially restored CFTR activity in WCS-exposed HBE cells (5.3 6 1.1 mA/cm 2 vs. 1.2 6 0.2 mA/cm 2 [control]; P , 0.05) and was additive with ivacaftor, a specific CFTR potentiator approved for the treatment of CF. Roflumilast improved the depleted ASL depth of HBE monolayers exposed to WCS (9.0 6 3.1 mm vs. 5.6 6 2.0 mm [control]; P , 0.05), achieving 79% of that observed in air controls. CFTR activation by roflumilast also induced CFTR-dependent fluid secretion in murine intestine, increasing the wet:dry ratio and the diameter of ligated murine segments. Roflumilast activates CFTR-mediated anion transport in airway and intestinal epithelia via a cyclic adenosine monophosphate-dependent pathway and partially reverses the deleterious effects of WCS, resulting in augmented ASL depth. Roflumilast may benefit patients with chronic obstructive pulmonary disease with chronic bronchitis by activating CFTR, which may also underlie noninfectious diarrhea caused by roflumilast.

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“…First-or second-passage primary human bronchial epithelial (HBE) cells were derived from lung explants (26,27). Cells were grown for 6-8 weeks until terminally differentiated.…”

Section: Dual Luciferase Assaymentioning

confidence: 99%

“…Short-circuit current (I sc ) was measured under voltage clamp conditions inUssing chambers (Physiologic Instruments, San Diego, CA) (21,26,27). Amiloride (100 mM) was added to block residual epithelial sodium channel current, followed by the CFTR agonist, forskolin, and the inhibitor, CFTR Inh -172 (10 mM).…”

Section: Short-circuit Current Measurementsmentioning

confidence: 99%

Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

Xue

Mutyam²,

Tang³

et al. 2014

Am J Respir Cell Mol Biol

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136

126

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New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/ delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

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Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

Abstract: Pyle LC, Ehrhardt A, Mitchell LH, Fan L, Ren A, Naren AP, Li Y, Clancy JP, Bolger GB, Sorscher EJ, Rowe SM. Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators.

Cited by 27 publications

References 48 publications

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

Cystic Fibrosis Transmembrane Conductance Regulator Activation by Roflumilast Contributes to Therapeutic Benefit in Chronic Bronchitis

Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

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