Tezacaftor

The molecules, 2a, 7a, and 7m, were synthetised, as previously published [37 (link),38 (link)]. The known CFTR modulators, Tezacaftor, VX661, Elexacaftor, VX445 and Ivacaftor, and VX770 were purchased from Selleck Chemicals (Munich, Germany). If not explicitly indicated in the text, all other chemicals and culture media components were provided by Merck (Milan, Italy).

CF HNECs were differentiated from ALI + 0 to 21 days in the presence or absence of 125 nM LY450139 with and without 3 µM elexacaftor and 3 µM tezacaftor (Selleckchem). Transwells were mounted in an Ussing chamber for electrophysiological short circuit current (I_sc) measurements using standard methods (34 (link)). Solutions in the serosal and mucosal baths were prepared so that a chloride gradient was established between both sides. After stable baseline current recordings were obtained, agonists were added in the following order to the apical side: amiloride (10 µM) to block sodium channel activity, IBMX and forskolin (10 µM) to stimulate CFTR through increased cAMP, ivacaftor (10 µM) to potentiate CFTR activity, and CFTR_inh-172 (20 µM) to block CFTR current. For each agonist, signals were monitored until a plateau in current was noted before adding the next agonist. The delta-I_sc in response to CFTR_inh-172 was used as the chief readout for CFTR-mediated chloride transport.

Fisher Rat Thyroid (FRT) cells stably transfected with a wild type (WT) CFTR, or CFTR carrying the cystic fibrosis mutation p.F508del, were grown at 37 °C and 5% CO₂ in modified F12 Coon’s medium with addition of 10% FBS and, 2 mM of Glutamine, 1 mg ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin and the addition of 1 mg ml⁻¹ geneticin (G418) and 0.6 mg ml⁻¹ zeocin as selection agents. For the iodide influx assay, cells stably co-transfected with the halide-sensitive yellow fluorescent protein, YFP-p.H148Q/p.I152L^{28 (link),29 (link)}, were seeded in 96-well microplates at a density of 40,000 cells/well. For intracellular pH measurements, cells were seeded on glass-bottom Petri dishes. Measurements were carried out 48 h after seeding. To evaluate the correctors p.F508del rescue on the cells expressing the mutated CFTR, cells were incubated for 18 h with 5 µM of VX809 (lumacaftor; Selleck Chemicals, Huston, TX, USA), VX661 (tezacaftor; Selleck Chemicals), Corr4a (ChemBridge Corp. San Diego, CA, USA), or 2.5 µM of VX809 with 2.5 µM of Corr4a together (final concentration 5 µM). Except when indicated, all chemicals compounds were purchased from Sigma-Aldrich (Milan, Italy).