Vx 770

Electrophysiological analyses were performed in an Ussing Chamber (Physiologic Instruments, Reno, NV, USA) under current clamp (0 μA) conditions with intermittent current pulsing (200 ms pulses at ±5 μA). Transepithelial current (∆I_T), resistance (∆TEER), and potential difference (TEPD) values were continuously monitored. I_T values for data obtained in HNECs are shown in all figures, and the TEPD and TEER values for Figure 1, Figure 2 and Figure 3 are shown in Supplemental Figures S1–S3, respectively. Cells analyzed under symmetrical chloride were bathed in a modified Ringer’s solution (120 mM NaCl, 10 mM D-Glucose, 3.3 mM KH₂PO₄, 0.83 mM K₂HPO₄, 1.2 mM MgCl₂, 1.2 mM CaCl₂, 25 mM NaHCO₃, pH 7.4), maintained at 37 °C, and gassed with 5% CO₂/95% O₂. For cells analyzed under a chloride gradient, a solution with gluconate substituted for chloride (115 mM NaC₆H₁₁O₇, 10 mM D-Glucose, 3.3 mM KH₂PO₄, 0.83 mM K₂HPO₄, 1.2 mM MgSO₄, 5 mM CaC₆H₁₁O₇, 25 mM, and NaHCO₃, pH 7.4) was applied to the side of the transwell containing the cells (for HNECs, this is defined as the apical surface). During analysis of HNECs, the following concentrations of compounds were utilized as indicated: apical 10 μM amiloride (#J62168, Alfa Aesar, Haverhill, MA, USA); apical 1 μM VX-770 (#S1144, Selleck Chemicals, Huston, TX, USA); apical 10 μM C_act-A1 (#505985, EMD Millipore, Burlington, MA, USA), 20 μM forskolin (#11018, Cayman Chemical)/100 μM IBMX (#I5879, Sigma-Aldrich) to both chambers; apical 20 μM CFTR(inh)-172 (Cystic Fibrosis Foundation Therapeutics CFTR Compound Program). During the analysis of FRT cells, the same concentrations of drugs were utilized and applied to both chambers.

VX-445 (#S8851), VX-661 (#S7059), VX-770 (#S1144) and VX-809 (#S1565) were purchased from Selleckchem, USA. Amiloride, 3-isobutyl-1-methylxanthine (IBMX, #I5879), Forskolin (#F6886) were purchased from Sigma-Aldrich, USA. CFTRinh-172 (#HY-16671) and CCG-273441 (9j, #HY-47573) were purchased from MedChemExpress, USA. 9g (CCG-273463) was synthesized in-house, as described below.

Electrophysiological measurements were performed directly on the filter using specific Ussing chambers (P2300) and sliders (P2302T) (Physiologic Instruments, San Diego, CA, USA) and a voltage clamp EVC4000 (World Precision Instruments, Sarasota, FL, USA). For transepithelial current measurement, the colonocyte monolayers were incubated with Elexacaftor, ELEXA (VX-445, 3 μM; Med Chem Express, Monmouth Junction, NJ, USA), Lumacaftor, LUMA (VX-809; 3 μM; Selleck Chemicals LLC, Houston, TX, USA) and Tezacaftor, TEZA (VX-661, 3 μM; Selleck Chemicals LLC, Houston, TX, USA), or vehicle (dimethyl sulfoxide, DMSO, 0.1%) for 20–24 h. Monolayers were bathed in symmetrical Meyler saline solution (pH 7.4) [10 mM Hepes; 0.3 mM Na₂HPO₄; 0.4 mM NaH₂PO₄; 1.0 mM MgCl₂; 1.3 mM CaCl₂; 4.7 mM KCl; 128 mM NaCl; 20.2 mM NaHCO₃; 10 mM D-glucose] for the measurement of chloride secretion mediated by CFTR. Solutions were maintained at 37 °C, gassed with 95% O₂, 5% CO₂. The transepithelial potential difference was clamped at 0 mV with a VVC-MC8 module (Physiologic Instruments), and the resulting short-circuit current (Isc) was recorded using a PowerLab 8/35 AD-converter (AD Instruments, Bella Vista, Australia) and associated software (LabChart v8; AD Instruments, Bella Vista, Australia). First, short circuit current reduction was blocked by 100 µM Amiloride (M) stimulus that inhibits the sodium channel EnaC; then filters were tested with components that act positively on CFTR activity: 10 µM forskolin (Sigma) applied to both apical (ap) and basolateral (bl) surfaces and 0.3 µM VX-770 (Selleckchem) (ap + bl). The experiment was concluded with the addition of the CFTR inhibitor, 20µM PPQ-102 (Tocris), from the apical and basolateral sides. At the end, 20 µM ATP (ap + bl) were just used to assess filters viability.

WT and F508del-CFTR cells were grown at 37 °C for five days post-confluence submerged on 96-well black, clear bottom culture plates (Costar) in EMEM media (Wisent BioProducts) with 10% Fetal Bovine Serum (Wisent BioProducts) and 1% Penicillin/Streptomycin (Wisent BioProducts). Twenty-four hours before the assay, the F508del-CFTR HBE cells were treated with DMSO or 3 µM VX-809 in EMEM media with 10% of FBS. The cells were then loaded with blue membrane potential dye dissolved in chloride-free buffer (150 mM NMDG-gluconate, 3 mM potassium gluconate, 10 mM HEPES, pH 7.30, 300 mOsm). The plate was then read in a fluorescence plate reader (SpectraMax i3; Molecular Devices) at 37 °C (excitation: 530 nm; emission: 560 nm). CFTR was stimulated with 10 µM Forskolin (Sigma–Aldrich, St. Louis, MO, USA) and 1 µM VX-770 (Selleck Chemicals) for cells treated with VX-809 for 24 h. The assay was terminated with 10 µM CFTRinh172 (Cystic Fibrosis Foundation Therapeutics). The changes in membrane potential were normalized to the point before addition of agonist and to the DMSO control response [22 (link),35 (link)].