scholarly journals Emerging Cystic Fibrosis Transmembrane Conductance Regulator Modulators as New Drugs for Cystic Fibrosis: A Portrait of in Vitro Pharmacology and Clinical Translation

2019 ◽  
Vol 3 (1) ◽  
pp. 4-10 ◽  
Author(s):  
Drishti P. Ghelani ◽  
Elena K. Schneider-Futschik
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Translation ◽  
New Drugs ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
In Vitro Pharmacology ◽  
Cystic Fibrosis Transmembrane

scholarly journals An “ex vivo model” contributing to the diagnosis and evaluation of new drugs in cystic fibrosis

2017 ◽  
Vol 37 (3) ◽  
pp. 207-213
Author(s):  
A.M. Di Lullo ◽  
M. Scorza ◽  
F. Amato ◽  
M. Comegna ◽  
V. Raia ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ex Vivo ◽  
Sono State ◽  
New Drugs ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Ex Vivo Model ◽  
Cystic Fibrosis Transmembrane ◽  
Gène Cftr

La fibrosi cistica (FC) è una malattia autosomica recessiva causata da mutazioni nel gene CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Finora sono state descritte circa 2000 mutazioni, ma per la maggior parte di esse è difficile definirne l’effetto senza complesse procedure in vitro. Abbiamo effettuato il campionamento (mediante brushing), la cultura e l’analisi di cellule epiteliali nasali umane (HNEC) utilizzando una serie di tecniche che possono aiutare a testare l’effetto delle mutazioni CFTR. Abbiamo eseguito 50 brushing da pazienti FC e controlli, e in 45 casi si è ottenuta una coltura positiva. Utilizzando cellule in coltura: i) abbiamo dimostrato l’espressione ampiamente eterogenea del CFTR nei pazienti e nei controlli; ii) abbiamo definito l’effetto di splicing di una mutazione sul gene CFTR; iii) abbiamo valutato l’attività di gating di CFTR in pazienti portatori di differenti mutazioni; iv) abbiamo dimostrato che il butirrato migliora in modo significativo l’espressione di CFTR. I dati provenienti dal nostro studio sperimentale dimostrano che l’uso del modello ex-vivo di cellule epiteliali nasali è un importante e valido strumento di ricerca e di diagnosi nella studio della FC e può anche essere mirato alla sperimentazione ed alla verifica di nuovi farmaci. In definitiva, in base ai nostri dati è possibile esprimere le seguenti conclusioni: 1) il prelievo delle cellule epiteliali nasali mediante brushing è applicabile senza alcuna anestesia ed è ben tollerato da tutti i pazienti affetti da FC (bambini e adulti), è scarsamente invasivo e facilmente ripetibile, è anche in grado di ottenere una sufficiente quantità di HNECs rappresentative, ben conservate, idonee allo studio della funzionalità di CFTR; 2) la conservazione delle cellule prelevate è possibile fino a 48 ore prima che si provveda all’allestimento della coltura e ciò permette di avviare studi multicentrici con prelievi in ogni sede e quindi di ottenere una ampia numerosità campionaria; 3) la coltura di cellule epiteliali nasali può essere considerata un modello adatto a studiare l’effetto molecolare di nuove mutazioni del gene CFTR e/o mutazioni specifiche di pazienti “carriers” dal significato incerto; 4) il modello ex-vivo delle HNECs consente inoltre di valutare, prima dell’impiego nell’uomo, l’effetto di farmaci (potenziatori e/o correttori) sulle cellule di pazienti portatori di mutazioni specifiche di CFTR; tali farmaci possono modulare l’espressione genica del canale CFTR aprendo così nuove frontiere terapeutiche e migliori prospettive di vita per pazienti affetti da una patologia cronica come la Fibrosi Cistica; 5) la metodologia da noi istituita risulta essere idonea alla misura quantitativa, mediante fluorescenza, dell’attività di gating del canale CFTR presente nelle membrane delle cellule epiteliali nasali prelevate da pazienti portatori di differenti genotipi; in tal modo è possibile individuare: a) pazienti FC portatori di 2 mutazioni gravi con un’attività < 10% (in rapporto ai controlli -100%), b) soggetti FC portatori contemporaneamente di una mutazione grave e di una lieve con un’attività tra 10-30%, c) i cosiddetti portatori “carriers”- eterozigoti - con un’attività tra 40-70%. In conclusione la possibilità di misurare l’attività del canale CFTR in HNECs fornisce un importante contributo alla diagnosi di FC, mediante individuazione di un “cut-off diagnostico”, ed anche alla previsione della gravità fenotipica della malattia; quindi quanto rilevabile dalla misura del suddetto canale permette di prospettare per il futuro la possibilità di valutare meglio i pazienti per i quali il test del sudore ha dato risultati ambigui (borderline o negativi). La metodica da noi sperimentata consente anche di monitorare i pazienti durante il trattamento farmacologico, valutando in tal modo i reali effetti delle nuove terapie.

scholarly journals Estrogen-Induced Abnormally High Cystic Fibrosis Transmembrane Conductance Regulator Expression Results in Ovarian Hyperstimulation Syndrome

2005 ◽  
Vol 19 (12) ◽  
pp. 3038-3044 ◽  
Author(s):  
Louis Chukwuemeka Ajonuma ◽  
Lai Ling Tsang ◽  
Gui Hong Zhang ◽  
Connie Hau Yan Wong ◽  
Miu Ching Lau ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
In Vitro Fertilization ◽  
Assisted Reproduction ◽  
Ovarian Hyperstimulation Syndrome ◽  
Ovarian Hyperstimulation ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Vitro Fertilization ◽  
Cystic Fibrosis Transmembrane

Abstract Ovarian hyperstimulation syndrome (OHSS) remains one of the most life-threatening and potentially fatal complications of assisted reproduction treatments, arising from excessive stimulation of the ovaries by exogenous gonadotropins administrated during in vitro fertilization procedures, which is characterized by massive fluid shift and accumulation in the peritoneal cavity and other organs, including the lungs and the reproductive tract. The pathogenesis of OHSS remains obscure, and no definitive treatments are currently available. Using RT-PCR, Western blot, and electrophysiological techniques we show that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed in many epithelia, is involved in the pathogenesis of OHSS. Upon ovarian hyperstimulation, rats develop OHSS symptoms, with up-regulated CFTR expression and enhanced CFTR channel activity, which can also be mimicked by administration of estrogen, but not progesterone, alone in ovariectomized rats. Administration of progesterone that suppresses CFTR expression or antiserum against CFTR to OHSS animals results in alleviation of the symptoms. Furthermore, ovarian hyperstimulation does not induce detectable OHSS symptoms in CFTR mutant mice. These findings confirm a critical role of CFTR in the pathogenesis of OHSS and may provide grounds for better assisted reproduction treatment strategy to reduce the risk of OHSS and improve in vitro fertilization outcome.

scholarly journals The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70

2002 ◽  
Vol 366 (3) ◽  
pp. 797-806 ◽  
Author(s):  
Carlos M. FARINHA ◽  
Paulo NOGUEIRA ◽  
Filipa MENDES ◽  
Deborah PENQUE ◽  
Margarida D. AMARAL
Keyword(s):  
Cystic Fibrosis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hsc 70 ◽  
Immature Form ◽  
Cystic Fibrosis Transmembrane

The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated intermediate in the endoplasmic reticulum (ER), probably due to misfolding that is recognized by the cellular quality control machinery involving molecular chaperones. Here, we overexpressed the heat-shock protein (Hsp) 70 chaperone in vivo and observed no changes in degradation rate of the core-glycosylated form, nor in the efficiency of its conversion into the fully glycosylated form, for either wt- or F508del-CFTR, contrary to previous in vitro studies on the affect of heat-shock cognate (Hsc) 70 on part of the first nucleotide-binding domain of CFTR. Co-transfection of Hsp70 with its co-chaperone human DnaJ homologue (Hdj)-1/Hsp40, however, stabilizes the immature form of wt-CFTR, but not of F508del-CFTR, suggesting that these chaperones act on a wt-specific conformation. As the efficiency of conversion into the fully glycosylated form is not increased under Hsp70/Hdj-1 overexpression, the lack of these two chaperones does not seem to be critical for CFTR maturation and ER retention. The effects of 4-phenylbutyrate and deoxyspergualin, described previously to interfere with Hsp70 binding, were also tested upon CFTR degradation and processing. The sole effect observed was destabilization of F508del-CFTR.

scholarly journals Renal Effects of Glibenclamide in Cystic Fibrosis Mice

2001 ◽  
Vol 12 (11) ◽  
pp. 2507-2510
Author(s):  
JONATHAN KIBBLE ◽  
ANDREW NEAL ◽  
STANLEY WHITE ◽  
ROGER GREEN ◽  
STEVE EVANS ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Distal Nephron ◽  
Transmembrane Conductance ◽  
Renal Effects ◽  
Significant Change ◽  
Cystic Fibrosis Transmembrane

Abstract. In vitro studies have shown that glibenclamide sensitivity is conferred upon Kir 1.1 K+ channels when they are co-expressed with the cystic fibrosis transmembrane conductance regulator (CFTR). In rats, glibenclamide acts as a K+-sparing diuretic by a mechanism that involves blockade of Kir 1.1 channels in the distal nephron. To test whether interaction between Kir 1.1 and CFTR is required to mediate the renal effects of glibenclamide (15 mg/kg), clearance experiments were performed comparing wild type (WT) and Cftrtm2cam ▵F508 cystic fibrosis (CF) mice. Glibenclamide treatment was associated with an equivalent diuresis in both WT and CF mice. Glibenclamide was K+-sparing in both genotypes with no significant change in urinary K+ excretion observed. That glibenclamide was an effective K+-sparing diuretic in CF animals suggests that CFTR expression is not a requirement to mediate its renal actions in mice.

scholarly journals Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation

2015 ◽  
Vol 291 (4) ◽  
pp. 2004-2017 ◽  
Author(s):  
Xiaoyan Gong ◽  
Annette Ahner ◽  
Ariel Roldan ◽  
Gergely L. Lukacs ◽  
Patrick H. Thibodeau ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ring Finger ◽  
Transmembrane Conductance Regulator ◽  
Native Conformation ◽  
Transmembrane Conductance ◽  
Sumoylation Site ◽  
Sumo Modification ◽  
Cystic Fibrosis Transmembrane

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50–60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys447, obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.

scholarly journals Screen-Printed Sensor for Low-Cost Chloride Analysis in Sweat for Rapid Diagnosis and Monitoring of Cystic Fibrosis

Biosensors ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 123 ◽  
Author(s):  
Alicia Hauke ◽  
Susanne Oertel ◽  
Leona Knoke ◽  
Vanessa Fein ◽  
Christoph Maier ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Low Cost ◽  
Fast Response ◽  
New Drugs ◽  
Remote Measurement ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sweat Chloride ◽  
Cystic Fibrosis Transmembrane ◽  
Screen Printed

Analysis of sweat chloride levels in cystic fibrosis (CF) patients is essential not only for diagnosis but also for the monitoring of therapeutic responses to new drugs, such as cystic fibrosis transmembrane conductance regulator (CFTR) modulators and potentiators. Using iontophoresis as the gold standard can cause complications like burns, is uncomfortable, and requires repetitive hospital visits, which can be particularly problematic during a pandemic, where distancing and hygiene requirements are increased; therefore, it is necessary to develop fast and simple measures for the diagnosis and monitoring of CF. A screen-printed, low-cost chloride sensor was developed to remotely monitor CF patients. Using potentiometric measurements, the performance of the sensor was tested. It showed good sensitivity and a detection limit of 2.7 × 10−5 mol/L, which covered more than the complete concentration range of interest for CF diagnosis. Due to its fast response of 30 s, it competes well with standard sensor systems. It also offers significantly reduced costs and can be used as a portable device. The analysis of real sweat samples from healthy subjects, as well as CF patients, demonstrates a proper distinction using the screen-printed sensor. This approach presents an attractive remote measurement alternative for fast, simple, and low-cost CF diagnosis and monitoring

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