Journal: Cells

Article Title: Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

doi: 10.3390/cells11244096

Figure Lengend Snippet: The complex allele [G576V;R668C] increases the activation and maturation of the CFTR channel. The functional and biochemical analysis of the [G576A;R668C] CFTR complex allele on heterologous expression systems. ( A ). Increase in CFTR activation. The bar graphs show the activity of [G576A;R668C] CFTR and, for comparison, WT-CFTR transiently expressed in CFBE41o- (upper panel) or FRT (lower panel) cells stably expressing HS-YFP. The CFTR activity was determined as a function of the YFP quenching rate following the iodide influx in cells stimulated with FSK alone (20 µM; light gray) or with FSK plus ivacaftor (1 µM; dark gray). The data are means ± SD (n = 3). The asterisks indicate statistical significance vs. the WT-CFTR protein: **, p < 0.01. ( B ). Increase in CFTR maturation. Representative Western blot images showing the electrophoretic mobility of [G576A;R668C] and, for comparison, wild-type, G576A, R668C, and F508del CFTR transiently expressed in CFBE41o- (upper panel) or FRT (lower panel) cells. The whole lysates derived from cells not expressing CFTR (null cells) are shown as controls for antibody specificity. In the case of F508del, the cells were treated for 24 h with DMSO alone (vehicle) or Elexa/Teza (3 µM/10 µM) to correct the mutant misfolding. The arrows indicate the complex-glycosylated (band C) and core-glycosylated (band B) forms of the CFTR protein. ( C ). CFTR band C densitometry of the Western blot experiments. The data are means ± SD (n = 3). The asterisks indicate statistical significance vs. WT-CFTR protein: **, p < 0.01. ( D ). CFTR half-life evaluation. Representative Western blot image showing the CFTR expression pattern of [G576A;R668C] CFTR and, for comparison, WT-CFTR transiently expressed in CFBE41o- cells at different time points (T = 0 and 24 h) following the CHX-induced block of protein synthesis. The whole lysates derived from cells not expressing CFTR (null cells) are shown as controls for antibody specificity. ( E ). The quantification of the normalized CFTR band C (left graph) following CHX treatment (T = 0 h, light gray; T = 24 h, dark gray) and band C fold-increase (right graph) over 24 h CHX treatment, obtained from experiments detailed in D, normalized with the initial value of band C. The data are means ± SD (n = 3). The asterisks indicate statistical significance vs. WT-CFTR protein: **, p < 0.01.

Article Snippet: The CFTR modulators VX-770, VX-661 and VX-809 were from TargetMol (Wellesley Hills, MA, USA, catalog ID: T2588, T2263, and T2595, respectively), while VX-445 was purchased from MedChemExpress (Monmouth Junction, NJ, USA, catalog ID: HY-111772).

Techniques: Activation Assay, Functional Assay, Expressing, Activity Assay, Stable Transfection, Western Blot, Derivative Assay, Mutagenesis, Blocking Assay

Journal: Cells

Article Title: Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

doi: 10.3390/cells11244096

Figure Lengend Snippet: Functional analysis of CFTR ultra-rare variants on heterologous expression systems identified loss-of-function alleles with reduced activity. ( A ) Functional analysis of trans CFTR alleles. ( B ) Functional analysis of cis CFTR alleles. The bar graphs show the activity of the different CFTR variants under investigation and, for comparison, WT- CFTR transiently expressed in CFBE41o- cells stably expressing HS-YFP. CFTR activity was determined as a function of the YFP quenching rate following the iodide influx in the cells stimulated with FSK alone (20 µM; light gray) or with FSK plus ivacaftor (1 µM; dark gray). The data are means ± SD (n = 3). The symbols indicate the statistical significance: # p < 0.05 vs. WT-CFTR protein; ## p < 0.01 vs. WT-CFTR protein; * p < 0.05, ** p < 0.01 vs. DMSO-treated, FSK-stimulated variant protein; §, p < 0.05, §§, p < 0.01 vs. FSK-stimulated variant protein (upon the same chronic treatment). ^ this type of variant is expected to result in little or no CFTR protein (see CFTR2 database at https://cftr2.org , accessed on 3 November 2022).

Article Snippet: The CFTR modulators VX-770, VX-661 and VX-809 were from TargetMol (Wellesley Hills, MA, USA, catalog ID: T2588, T2263, and T2595, respectively), while VX-445 was purchased from MedChemExpress (Monmouth Junction, NJ, USA, catalog ID: HY-111772).

Techniques: Functional Assay, Expressing, Activity Assay, Stable Transfection, Variant Assay

Journal: ERJ Open Research

Article Title: BOS-318 treatment enhances elexacaftor–tezacaftor–ivacaftor-mediated improvements in airway hydration and mucociliary transport

doi: 10.1183/23120541.00445-2024

Figure Lengend Snippet: Effect of long-term BOS-318 treatment in combination with ETI on ion channel function. Cystic fibrosis human bronchial epithelial cells were treated with either vehicle or BOS-318 in the presence or absence of elexacaftor–tezacaftor–ivacaftor (ETI) for 48 h. After the treatment period, baseline I eq was established, followed by the sequential addition of 10 µM amiloride, 20 µM forskolin, 20 µM CFTRinh-172 and 100 µM UTP. (a) Representative equivalent current (I eq ) recordings. (b) CFTRinh-172-sensitive I eq representing CFTR activity. (c) Amiloride-sensitive I eq , representing epithelial sodium channel (ENaC) activity. n≥5 individual inserts from three donors. Statistical analyses were performed using a Kruskal–Wallis test and Dunn's multiple comparisons test in comparison to the relevant dimethylsulfoxide control. *: p≤0.05; **: p≤0.01. CFTR: cystic fibrosis transmembrane conductance regulator.

Article Snippet: CFTR modulators (VX-661 and VX-770) were from Adooq Biosciences (Irvine, CA, USA).

Techniques: Activity Assay, Comparison, Control

Journal: ERJ Open Research

Article Title: BOS-318 treatment enhances elexacaftor–tezacaftor–ivacaftor-mediated improvements in airway hydration and mucociliary transport

doi: 10.1183/23120541.00445-2024

Figure Lengend Snippet: BOS-318 treatment enhances ETI-mediated improvements in MCT. Cystic fibrosis human bronchial epithelial cells were treated with vehicle, BOS-318, elexacaftor–tezacaftor–ivacaftor (ETI) or a combination of ETI and BOS-318, in the presence of 30 nM vasoactive intestinal peptide, for 48 h. The mucociliary transport (MCT) rate was determined by tracking fluorescently labelled microspheres on the apical surface of the cells. (a) MCT rate shown as μm·s −1 and (b) MCT rate shown as a % of triple CFTR modulator therapy rate. n=7 individual inserts from four cystic fibrosis donors. Statistical analyses were performed using a Mann–Whitney U-test. **: p≤0.01. CFTR: CF transmembrane conductance regulator; DMSO: dimethylsulfoxide.

Article Snippet: CFTR modulators (VX-661 and VX-770) were from Adooq Biosciences (Irvine, CA, USA).

Techniques: MANN-WHITNEY

Journal: Journal of Experimental Pharmacology

Article Title: Pharmacological Modulation of Ion Channels for the Treatment of Cystic Fibrosis

doi: 10.2147/JEP.S255377

Figure Lengend Snippet: Pharmacological Therapies Commonly Used in Therapeutic Regimens of Individuals with Cystic Fibrosis

Article Snippet: These molecules were demonstrated to rescue mutant CFTR carrying G551D, G178R, R334W, S549N or F508del – the latter in combination with either VX-809 or ABBV-2222 – in both cell lines and CF HBE., , Patch-clamp functional studies showed that ABBV-974 and GLPG-2451 reduce the closed time and increase the open time of CFTR channels by a similar mechanism to that of VX-770, as no additive effects were observed when these molecules were tested together., , ABBV-3067 is another potentiator developed by Abbvie that was demonstrated to rescue F508del-CFTR in combination with the corrector ABBV-2222, and this combination is currently under clinical investigation (NCT03969888).

Techniques: Binding Assay, Translocation Assay, Clinical Proteomics, Membrane, Permeability, Muscles, Activation Assay, Inhibition, Phospho-proteomics, Activity Assay, Expressing, Transmigration Assay, Recombinant, Saline

Journal: Journal of Experimental Pharmacology

Article Title: Pharmacological Modulation of Ion Channels for the Treatment of Cystic Fibrosis

doi: 10.2147/JEP.S255377

Figure Lengend Snippet: Modulation of ion channels/transporters as alternative therapies for CF. In healthy airways, CFTR, ENaC, TMEM16A and SLC26A9 are expressed at the plasma membrane (PM) of epithelial cells where they contribute to ion and water homeostasis. In CF airways, due to the absence of functional CFTR, Cl – secretion is compromised and Na + absorption is upregulated, leading to a dehydrated air surface liquid (ASL) and impaired mucociliary clearance (MCC). Expression of TMEM16A and SLC26A9 at the PM are also diminished in CF ciliated cells, although the role of TMEM16A overexpression in secretory cells and its role in mucus secretion are still controversial. Alternative therapies for CF thus include blocking ENaC, enhancing SLC26A9 expression at the PM, and modulating TMEM16A. Although for the latter is still not clear whether activators or inhibitors are beneficial, a TMEM16A potentiator in currently under clinical investigation.

Article Snippet: These molecules were demonstrated to rescue mutant CFTR carrying G551D, G178R, R334W, S549N or F508del – the latter in combination with either VX-809 or ABBV-2222 – in both cell lines and CF HBE., , Patch-clamp functional studies showed that ABBV-974 and GLPG-2451 reduce the closed time and increase the open time of CFTR channels by a similar mechanism to that of VX-770, as no additive effects were observed when these molecules were tested together., , ABBV-3067 is another potentiator developed by Abbvie that was demonstrated to rescue F508del-CFTR in combination with the corrector ABBV-2222, and this combination is currently under clinical investigation (NCT03969888).

Techniques: Clinical Proteomics, Membrane, Functional Assay, Expressing, Over Expression, Blocking Assay

Journal: Journal of Experimental Pharmacology

Article Title: Pharmacological Modulation of Ion Channels for the Treatment of Cystic Fibrosis

doi: 10.2147/JEP.S255377

Figure Lengend Snippet: TMEM16A Activators

Article Snippet: These molecules were demonstrated to rescue mutant CFTR carrying G551D, G178R, R334W, S549N or F508del – the latter in combination with either VX-809 or ABBV-2222 – in both cell lines and CF HBE., , Patch-clamp functional studies showed that ABBV-974 and GLPG-2451 reduce the closed time and increase the open time of CFTR channels by a similar mechanism to that of VX-770, as no additive effects were observed when these molecules were tested together., , ABBV-3067 is another potentiator developed by Abbvie that was demonstrated to rescue F508del-CFTR in combination with the corrector ABBV-2222, and this combination is currently under clinical investigation (NCT03969888).

Techniques: Activation Assay, Binding Assay