The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508-NBD1 and housekeeping proteins prevents ΔF508-CFTR delivery to the plasma membrane. Based on this assumption we applied structure-based virtual screening to identify new low-molecular-weight compounds that should bind to ΔF508-NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico-selected compounds induced functional expression of ΔF508-CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508-CFTR mice. The proposed compounds disrupt keratin8-ΔF508-CFTR interaction in ΔF508-CFTR HeLa cells. Structural analysis of ΔF508-NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508-CFTR trafficking defect known to date.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF-β1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB-crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB-crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad-dependent pathway. We demonstrate here that αB-crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB-crystallin-deficient mice are protected from bleomycin-induced fibrosis. Similar protection from fibrosis was observed in αB-crystallin KO mice after transient adenoviral-mediated over-expression of IL-1β or TGF-β1. We show in vitro in primary epithelial cells and fibroblasts that αB-crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF-β1-Smad pathway and the consequent activation of TGF-β1 downstream genes. αB-crystallin over-expression disrupts Smad4 mono-ubiquitination by interacting with its E3-ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB-crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono-ubiquitination and nuclear export are favoured and thus TGF-β1-Smad4 pro-fibrotic activity is inhibited. This study demonstrates that αB-crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases.

Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTR inh -172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTR inh -172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/ W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTR inh -172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTR inh -172, but not GlyH-101, induced nuclear translocation of nuclear factor-B (NF-B). CFTR inh -172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-B signaling pathway, independently of CFTR inhibition.