Elexacaftor

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.

The following chemicals were used: DMSO (Sigma-Aldrich, St Louis, MO, Cat# D2650), MG-132 (Cayman Chemical, Ann Arbor, MI, Cat# 10012628), Lumacaftor (Cayman Chemical, Cat# 22196), Tezacaftor (Selleck Chemicals, Houston, TX, Cat# S7059), Elexacaftor (Selleck Chemicals, Cat# S8851), Ivacaftor (Chemscene LLC, Monmouth Junction, NJ, Cat# CS-0497), cycloheximide (CHX, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, Cat# 3720991), doxycycline (dox, FU-JIFILM Wako Pure Chemical Corporation, Cat# 049-31121), glycerol (FU-JIFILM Wako Pure Chemical Corporation, Cat# 075-00616), cyclosporin A (CLP-A, FU-JIFILM Wako Pure Chemical Corporation, Cat# 031-24931), E-4031 (Selleck Chemicals, Cat# S6627). Hit compounds from the in silico screening and the FR3 analog compounds are listed in Supplementary Table S1.