The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway
Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.
Voltage-clamp studies of primary human bronchial epithelial (HBE) cells
HBE cells were seeded onto fibronectin-coated Snapwell inserts (Corning, Tewksbury MA), and the apical medium was removed after 24 h to establish an air–liquid interface68. Transepithelial resistance was monitored using an EVOM epithelial volt-ohmmeter (World Precision Instr. Sarasota FL), and monolayers were used after 4 weeks when the resistance was 300–400 Ω cm2. HBE monolayers expressing F508del-CFTR were treated on both sides with 0.1% dimethylsulfoxide (negative control) or 10 μM test compound (except VX-809; 1 μM) in OptiMEM containing 2% (v/v) fetal bovine serum. The short-circuit current (Isc) was measured across monolayers mounted in modified Ussing chambers and was voltage clamped using a VCCMC6 multichannel current–voltage clamp (Physiologic Instruments San Diego CA.).
Apical membrane conductance was functionally isolated by permeabilizing the basolateral membrane with 200 μg/ml nystatin and imposing an apical-to-basolateral Cl− gradient. The basolateral solution contained 1.2 mM NaCl, 115 mM Na-gluconate, 25 mM NaHCO3, 1.2 mM MgCl2, 4 mM CaCl2, 2.4 mM KH2PO4, 1.24 mM K2HPO4, and 10 mM glucose (pH 7.4). The apical solution contained 115 mM NaCl, 25 mM NaHCO3, 1.2 mM MgCl2, 1.2 mM CaCl2, 2.4 mM KH2PO4, 1.24 mM K2HPO4, and 10 mM mannitol (pH 7.4). Apical glucose was replaced with mannitol to eliminate current mediated due to Na+-glucose cotransport. Successful permeabilization of the basolateral membrane under these conditions was obvious from the reversal of Isc. Solutions were maintained at 37 °C and continuously stirred by gassing with 95% O2/5% CO2. The transepithelial voltage was measured, and currents passed through agar bridge Ag/AgCl electrodes. Pulses (1 mV amplitude, 1 s duration) were delivered every 90 s to monitor resistance, and a PowerLab/8SP interface was used for data acquisition. CFTR was activated by adding 10 µM forskolin + 50 µM genistein to the apical bathing solution, and the resulting Isc was sensitive to CFTR inh-172 [10 µM]69, confirming that it was mediated by CFTR.