Vx 809

Primary human nasal epithelial cells (pHNEs) were obtained from nasal brushing as before [37 (link)]. After expansion, cells were seeded on porous membranes and cultured under air–liquid interface (ALI) conditions for 21 days following protocols by Jeffrey Beekman’s lab (Utrecht, The Netherlands, submitted to Cell Rep Med). Differentiated monolayers of pHNEs were incubated with 3 µM VX809, 5 µM VX661 (Selleckchem, TX, USA), or DMSO alone for 48 h prior to measurements in an Ussing Chamber. The pHNE monolayers were mounted on micro-Ussing chambers and analyzed under open-circuit conditions, as described previously [16 (link)]. CFTR was stimulated by the presence of forskolin (2 µM) + IBMX (100 µM), and specificity was evaluated after using CFTR inhibitor 172 (25 µM). Experiments were performed in the presence of amiloride (20 µM) to inhibit the Epithelial Sodium (Na+) Channel (ENaC) and thus avoid interference of ENaC-mediated currents. In pHNEs, the maximum CFTR activation corresponds to the sum of IBMX/Fsk and IBMX/Fsk+VX-770 responses [16 (link)]. CFTR rescue by CFTR modulators in pHNEs was assessed by comparison of the maximum activation of CFTR between control pHNE (DMSO) and treated pHNE (VX809 or VX661). The individuals were considered responders when this difference was statistically different (p-value < 0.05). Each experiment was performed at least three times.

CF-HBE and CFBE cells were treated with VX-809 (3 μM, Selleckchem, Houston, TX) for 48 h to increase Phe508del CFTR Cl^- secretion. VX-809 is a key component of Orkambi, an FDA drug approved for use in CF patients to stimulate Phe508del CFTR Cl^- secretion by CF-HBE cells. To provide biological validation for our proteomics analysis, in one set of experiments, the same number of OMVs isolated from control or Tobramycin treated P. aeruginosa were added to the apical side of CF-HBE or CFBE cells for 1.5 h before measurements of Phe508del CFTR Cl^- secretion. OMVs were quantified using FM 4–64 fluorescent dye (Invitrogen) as described previously [25 (link)]. In a second validation experiment either P. aeruginosa (PA14-wt) or PA14, in which the aprA gene was deleted (PA14-ΔaprA), was added to the apical side of CF-HBE cells at a multiplicity of infection (MOI) of 30:1 for 6 hours. Phe508del CFTR Cl^- secretion was measured as described in detail previously [9 (link)].

To determine the cytotoxicity of commercial compounds that modulate CFTR protein activity, cells were incubated with different concentrations (1 to 50 μM) of ivacaftor (VX-770, Merck) or lumacaftor (VX-809; Selleck Chemicals) and cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide] reduction assay (88 (link)).

The assay was performed as previously described (Botelho et al, 2015 (link)) CFBE cells expressing the inducible mCherry‐Flag‐CFTR reporter (WT‐ or F508del‐CFTR variants) were grown to confluence and split to 50% confluency. Twenty‐four hours later, the cells were trypsinized to antibiotic‐free medium and seeded in siRNA coated 384‐well plates (1,000 cells/well) using a Multidrop™ Combi peristaltic dispenser (Thermo Scientific 5840300). CFTR expression was induced for 48 h (24 h after seeding) by supplementing the medium with 1 μg/ml of doxycycline (Sigma 9891). At this point, VX‐809 (3 µM) or VX‐661 (5 µM) (Selleckchem (S7059)) were added to selected wells containing Neg1 siRNA as a positive control for F508del‐CFTR traffic rescue. Extracellular Flag tags were immunostained in non‐permeabilized cells 72 h after seeding. The primary anti‐Flag antibody (Sigma F‐1804, 1:500) was incubated 1 h at 4°C, the cells were fixed with PFA 3% for 20 min at 4°C, an anti‐mouse IgG antibody conjugated with Alexa 647 (Invitrogen A‐31571. 1:500) was incubated 1 h at room temperature and Hoechst 33342 (200 ng/ml, Sigma B2261) was incubated 1 h at room temperature. Four independent biological replicates were performed.

The 3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid (Lumacaftor, VX809), 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl]-cyclopropanecarboxamide (Tezacaftor, VX661), N-[2-(5-chloro-2-methoxyphenylamino)-2 0-yl]benzamide (corr4a), 4-(cyclohexyloxy)-2-{1-[4-(4-metho-xybenzenesulfonyl)piperazin-1-yl]ethyl}quinazoline (VX325) correctors were purchased from Selleck Chemicals (Munich, Germany). If not explicitly indicated in the text, all other chemicals and culture media components were provided by Sigma-Aldrich (Milan, Italy).