Journal: Frontiers in Pharmacology

Article Title: Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators

doi: 10.3389/fphar.2024.1494327

Figure Lengend Snippet: Functional evaluation of CFTR activity and rescue by combined corrector treatment on nasal epithelia derived from CF patients carrying the G85E mutation. (A) Representative traces of the effect of vehicle (DMSO), or the combinations of elexacaftor (ELX, 3 µM) with tezacaftor (TEZ, 10 µM), ARN23765 (ARN, 10 nM and 1 µM), lumacaftor (LUM, 3 µM), or ABBV-2222 (ABV, 100 nM) on G85E/G542X nasal epithelial cells (derived from donor ID: GE143) with the short-circuit current technique. During the recordings, the epithelia were sequentially treated (as indicated by downward arrows) with amiloride (10 μM; added on the apical side), CPT-cAMP (100 μM; added on both apical and basolateral sides), ivacaftor (1 μM; apical side) and the CFTR inhibitor-172 (inh-172; 20 μM; apical side). The dashed line indicates zero current level. (B) Scatter dot plot showing the summary of results obtained from experiments described in (A) . Data reported are the amplitude of the current blocked by 20 μM inh-172 (ΔIsc inh-172 ). For each experimental condition the number of biological replicates was n = 4-6. (C) Representative traces recorded with the short-circuit current technique on nasal epithelia derived from a non-CF subject (donor ID: Ctr191) sequentially treated as indicated in (A) . (D) Dose-response relationships for ARN23765 following 24 h treatment of CFBE41o− cells expressing either F508del or G85E determined with the HS-YFP assay. Each symbol is the mean ± SD of n = 3 experiments. (E) Scatter dot plot showing the summary of results obtained from experiments similar to those described in (A) following treatment with vehicle (DMSO), with ELX/TEZ (3 µM/10 µM), or ELX/ARN (3 µM/1 µM) on nasal epithelia having genotype G85E/2372del8 (donor ID: GE004), or G85E/621 + 1G>T (donor ID: GE072), or G85E/W1282X (donor ID: FI113). Data reported are the amplitude of the current blocked by 20 μM inh-172 (ΔIsc inh-172 ). For each experimental condition the number of biological replicates was n = 4-6. Symbols indicate statistical significance of treatments: ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The CFTR modulators ivacaftor, tezacaftor, and lumacaftor were purchased from TargetMol (catalog ID: T2588, T2263, and T2595, respectively; Wellesley Hills, MA, United States).

Techniques: Functional Assay, Activity Assay, Derivative Assay, Mutagenesis, Expressing

Journal: Frontiers in Pharmacology

Article Title: Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators

doi: 10.3389/fphar.2024.1494327

Figure Lengend Snippet: Functional evaluation of the effect of single or combined corrector treatment and of chronic potentiator administration on nasal epithelia derived from CF patients carrying the G85E mutation. (A) Representative traces of the effect of vehicle (DMSO), or ELX/TEZ (3 µM/10 µM), or ELX/TEZ/IVA (3 µM/10 µM/5 µM), or ELX/ARN (3 µM/1 µM), or ELX/ARN/IVA (3 µM/1 µM/5 µM) on G85E/E379X nasal epithelial cells (derived from donor ID: GE227) with the short-circuit current technique. During the recordings, the epithelia were sequentially treated (as indicated by downward arrows) with amiloride (10 μM; added on the apical side), CPT-cAMP (100 μM; added on both apical and basolateral sides), ivacaftor (1 μM; apical side) and the CFTR inhibitor-172 (inh-172; 20 μM; apical side). The dashed line indicates zero current level. (B) Scatter dot plot showing the summary of results obtained from experiments described in (A) . Data reported are the amplitude of the current blocked by 20 μM inh-172 (ΔIsc inh-172 ). For each experimental condition the number of biological replicates was n = 4-6. (C) Representative traces of the effect of vehicle (DMSO), or ELX/TEZ (3 µM/10 µM), or ELX/ARN (3 µM/1 µM), or TEZ (10 µM), or ARN (1 µM) on G85E/R75X nasal epithelial cells (donor ID: GE155) with the short-circuit current technique. During the recordings, the epithelia were sequentially treated as indicated in (A) . (D) Scatter dot plot showing the summary of results obtained from experiments described in (C) . Data reported are the amplitude of the current blocked by 20 μM inh-172 (ΔIsc inh-172 ). For each experimental condition the number of biological replicates was n = 4-6. (E) Representative Western blot images showing the electrophoretic mobility in lysates of derived from G85E patients (GE227 and GE155) and corresponding density profiles analyses. Epithelia were treated for 24 h with vehicle alone (DMSO) or ELX/TEZ (3 µM/10 µM), or ELX/ARN (3 µM/1 µM), or TEZ (10 µM), or ARN (1 µM) prior to lysis. For comparison, lysates of nasal epithelia derived from one non-CF donor (ID: Ctr032) and one F508del homozygous patient (AN237; treated with DMSO or ELX/TEZ) have been included. Symbols indicate statistical significance of treatments: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n. s., not significant.

Article Snippet: The CFTR modulators ivacaftor, tezacaftor, and lumacaftor were purchased from TargetMol (catalog ID: T2588, T2263, and T2595, respectively; Wellesley Hills, MA, United States).

Techniques: Functional Assay, Derivative Assay, Mutagenesis, Western Blot, Lysis, Comparison

Journal: Frontiers in Pharmacology

Article Title: Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators

doi: 10.3389/fphar.2024.1494327

Figure Lengend Snippet: Functional and biochemical evaluation of the effect of tezacaftor and ARN23765, as single agents or combined with elexacaftor, on G85E CFTR mutant in immortalized bronchial cells. The bar graph shows the activity of G85E CFTR transiently expressed in CFBE41o- cells stably expressing the HS-YFP. CFTR activity was determined as a function of the YFP quenching rate following iodide influx elicited by forskolin (20 μM; light gray bars) or forskolin + ivacaftor (1 μM; dark gray bars) in cells treated for 24 h with DMSO (vehicle), or with ARN or TEZ at the indicated concentrations, alone or combined with ELX (3 µM). Data from cells transiently expressing wt-CFTR and F508del- (following treatment with DMSO, or, for F508del only, with LUM (3 µM) or ELX/TEZ (3 µM/10 µM) are also shown for comparison. (B) Biochemical analysis of the G85E-CFTR expression pattern in CFBE41o- cells. The representative western blot image shows CFTR electrophoretic mobility in cell lysates following treatment for 24 h, prior to lysis, with the correctors indicated in (A) . Lysates from cells transiently expressing wt- and F508del-CFTR are also shown for comparison. Lysates of parental cells have been included as control for antibody specificity. The bar graphs show CFTR band B and band C densitometry, as well as the band C over total CFTR ratio, of the western blot experiments. Symbols indicate statistical significance of treatments: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. DMSO-treated; § p < 0.05; §§ p < 0.01 vs. ELX-treated.

Article Snippet: The CFTR modulators ivacaftor, tezacaftor, and lumacaftor were purchased from TargetMol (catalog ID: T2588, T2263, and T2595, respectively; Wellesley Hills, MA, United States).

Techniques: Functional Assay, Mutagenesis, Activity Assay, Stable Transfection, Expressing, Comparison, Western Blot, Lysis, Control

Journal: Frontiers in Pharmacology

Article Title: Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators

doi: 10.3389/fphar.2024.1494327

Figure Lengend Snippet: Functional and biochemical evaluation of the effect of lumacaftor and ABBV2222, as single agents or combined with elexacaftor, on G85E CFTR mutant in immortalized bronchial cells. (A) The bar graph shows the activity of G85E CFTR transiently expressed in CFBE41o- cells stably expressing the HS-YFP. CFTR activity was determined as a function of the YFP quenching rate following iodide influx elicited by forskolin (20 μM; light gray bars) or forskolin + ivacaftor (1 μM; dark gray bars) in cells treated for 24 h with DMSO (vehicle), or with LUM or ABV at the indicated concentration, alone or combined with ELX (3 µM). Treatment with ELX/ARN (3 µM/1 µM) was included as control. Data from cells transiently expressing wt-CFTR and F508del- (following treatment with DMSO, or, for F508del only, with LUM (3 µM) or ELX/TEZ (3 µM/10 µM) are also shown for comparison. (B) Biochemical analysis of the G85E-CFTR expression pattern in CFBE41o- cells. The representative western blot image shows CFTR electrophoretic mobility in cell lysates following treatment for 24 h, prior to lysis, with the correctors indicated in (A) . Lysates from cells transiently expressing wt-CFTR are also shown for comparison. Lysates of parental cells have been included as control for antibody specificity. The bar graphs show CFTR band B and band C densitometry, as well as the band C over total CFTR ratio, of the western blot experiments. Symbols indicate statistical significance of treatments: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. DMSO-treated; §§ p < 0.01 vs. ELX-treated.

Article Snippet: The CFTR modulators ivacaftor, tezacaftor, and lumacaftor were purchased from TargetMol (catalog ID: T2588, T2263, and T2595, respectively; Wellesley Hills, MA, United States).

Techniques: Functional Assay, Mutagenesis, Activity Assay, Stable Transfection, Expressing, Concentration Assay, Control, Comparison, Western Blot, Lysis

Journal: Frontiers in Pharmacology

Article Title: Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators

doi: 10.3389/fphar.2024.1494327

Figure Lengend Snippet: Functional and biochemical evaluation of the effect of ARN23765, as single agent or combined with the type 3 correctors 4172 and ELX, on G85E CFTR mutant in immortalized bronchial cells. (A) The bar graph shows the activity of G85E CFTR transiently expressed in CFBE41o- cells stably expressing the HS-YFP. CFTR activity was determined as a function of the YFP quenching rate following iodide influx elicited by forskolin (20 μM; light gray bars) or forskolin + ivacaftor (1 μM; dark gray bars) in cells treated for 24 h with DMSO (vehicle), or with ARN at the indicated concentrations, alone or combined with 4172 (10 µM) or with ELX (3 µM). Data from cells transiently expressing wt-CFTR and F508del- (following treatment with DMSO, or, for F508del only, with LUM (3 µM) or ELX/TEZ (3 µM/10 µM) are also shown for comparison. (B) Biochemical analysis of the G85E-CFTR expression pattern in CFBE41o- cells. The representative western blot image shows CFTR electrophoretic mobility in cell lysates following treatment for 24 h, prior to lysis, with the correctors indicated in (A) . Lysates from cells transiently expressing wt- and F508del-CFTR are also shown for comparison. Lysates from cells transiently expressing wt-CFTR and F508del- (following treatment with DMSO, or, for F508del only, with ELX/TEZ (3 µM/10 µM) are also shown for comparison. Lysates of parental cells have been included as control for antibody specificity. The bar graphs show CFTR band B and band C densitometry, as well as the band C over total CFTR ratio, of the western blot experiments. Symbols indicate statistical significance of treatments: ** p < 0.01; *** p < 0.001 vs. DMSO-treated; §§ p < 0.01 vs. ELX-treated; # p < 0.05 vs. 4172-treated.

Article Snippet: The CFTR modulators ivacaftor, tezacaftor, and lumacaftor were purchased from TargetMol (catalog ID: T2588, T2263, and T2595, respectively; Wellesley Hills, MA, United States).

Techniques: Functional Assay, Mutagenesis, Activity Assay, Stable Transfection, Expressing, Comparison, Western Blot, Lysis, Control

Journal: Frontiers in Pharmacology

Article Title: Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators

doi: 10.3389/fphar.2024.1494327

Figure Lengend Snippet: Biochemical evaluation of the effect of chronic ivacaftor on G85E-CFTR rescue by tezacaftor or ARN23765, as single agent or combined with elexacaftor, on G85E CFTR mutant in immortalized bronchial cells. Biochemical analysis of the G85E-CFTR expression pattern in CFBE41o- cells. The representative western blot image shows CFTR electrophoretic mobility in cell lysates following treatment for 24 h, prior to lysis, with vehicle (DMSO), or IVA (5 µM), or ELX/TEZ (3 µM/10 µM), or ELX/TEZ/IVA (3 µM/10 µM/5 µM), or ELX/ARN (3 µM/1 µM), or ELX/ARN/IVA (3 µM/1 µM/5 µM). Lysates from cells transiently expressing wt- and F508del-CFTR are also shown for comparison. Lysates of parental cells have been included as control for antibody specificity. The bar graphs show CFTR band B and band C densitometry, as well as the band C over total CFTR ratio, of the western blot experiments. Asterisks indicate statistical significance of treatments: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The CFTR modulators ivacaftor, tezacaftor, and lumacaftor were purchased from TargetMol (catalog ID: T2588, T2263, and T2595, respectively; Wellesley Hills, MA, United States).

Techniques: Mutagenesis, Expressing, Western Blot, Lysis, 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: 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