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Background and Purpose: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects.Experimental Approach: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques.Key Results: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR.Conclusions and Implications: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent. [ABSTRACT FROM AUTHOR]

Background and Purpose Dysfunction of the cystic fibrosis transmembrane conductance regulator ( CFTR) Cl− channel causes the genetic disease cystic fibrosis ( CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561 E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561 E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. Experimental Approach Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques. Key Results Low temperature incubation delivered a small proportion of A561 E- CFTR protein to the cell surface. Like F508del- CFTR, low temperature-rescued A561 E- CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561 E- CFTR also exhibited thermoinstability, losing function more quickly than F508del- CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561 E- CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561 E- and F508del- CFTR Cl− channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches. Conclusions and Implications Like F508del- CFTR, A561 E- CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561 E- CFTR. Transformational drug therapy for A561 E- CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability. [ABSTRACT FROM AUTHOR]