High-throughput screening yielded several classes of small molecules that corrected ΔF508-CFTR cellular misprocessing and restored plasma membrane expression and halide permeability to levels greater than those achieved by low-temperature rescue. We verified correction by electrophysiological and biochemical measurements, as well as using control (non–ΔF508-CFTR–expressing) cells and a CFTR-selective inhibitor. The micromolar potency of the correctors is orders of magnitude better than the millimolar potency reported for correction of ΔF508-CFTR misprocessing by 4-PBA (11
). The identification of small-molecule ΔF508-CFTR correctors presented a greater conceptual difficulty than that of ΔF508-CFTR potentiators or CFTR activators/inhibitors because correction of cellular misprocessing could involve multiple targets, whereas the primary target for potentiators, activators, and inhibitors is CFTR itself.
The cell line used for primary screening was chosen to be of epithelial origin (to resemble native CFTR-expressing cells), to permit rapid assessment of chloride currents in cell monolayers, and to give a robust low-temperature rescue response. Additional requirements for the cell line for high-throughput screening included rapid growth in multiwell plates, stable and bright YFP-H148Q/I152L expression, and low basal halide permeability. The YFP-H148Q/I152L fluorescent halide indicator was developed by our lab (30
) to have bright cellular expression and ultra-high iodide sensitivity. The transfected FRT cell line used here was selected after many transfected and natively expressed epithelial cell lines were screened, and well as more than 100 ΔF508-CFTR/YFP-H148Q/I152L–transfected FRT cell clones (18
). Although primary screening using natively expressed human airway cells may be preferable on theoretical grounds, practical considerations of stable cell phenotype, relatively high ΔF508-CFTR expression, and robust signal (Z′ factor > 0.5) precluded the use of available human ΔF508-CFTR cell lines.
CFTR cellular processing involves translation, folding at the ER, Golgi transport, posttranslational glycosylation, and apical plasma membrane targeting (6
). Plasma membrane CFTR is internalized by endocytosis and then recycled to the plasma membrane or targeted for lysosomal degradation (9
). ΔF508-CFTR folding is inefficient, with 99.5% of newly synthesized ΔF508-CFTR in BHK cells targeted for degradation without reaching the Golgi apparatus. Near-complete ER retention of ΔF508-CFTR was reported in other model systems as well (2
Screening of 150,000 chemically diverse compounds produced several classes of candidate correctors of defective ΔF508-CFTR cellular processing. Functional and biochemical analysis identified the class 4 bisaminomethylbithiazoles as the most promising class of compounds for further development. Corr-4a and -4c increased ΔF508-CFTR folding efficiency nearly 3-fold without affecting translational rate, which suggests that the correctors could partially overcome the posttranslational folding barrier. Based on recent structural studies, small molecules might facilitate the folding of the nucleotide binding domain 2 (NBD2) and/or transmembrane domains (7
). The simplest interpretation of the peripheral stabilizing effect is that the conformationally stabilized mutant is less susceptible to the ubiquitin-dependent peripheral quality control mechanism and lysosomal degradation than the low temperature–rescued ΔF508-CFTR (24
). The lack of effect of some ΔF508-CFTR correctors on the temperature-sensitive mutant P574H-CFTR and the mutant DRD4-M345T suggests their specificity for the ΔF508 mutation, though further studies are needed to evaluate the numerous potential off-target specificities.
Our results thus provide proof-of-principle for discovery of small-molecule correctors of ΔF508-CFTR cellular misprocessing. While some correctors, such as those of class 4, have favorable properties, including efficacy in human airway epithelia, partial correction of the gating defect, apparent ΔF508-CFTR specificity, and a mechanism involving improved ΔF508-CFTR protein folding, additional analysis and compound optimization are needed prior to the initiation of clinical studies, to include in vivo testing of compound pharmacology, toxicity, and efficacy. The effect of class 4 correctors on Cl–
secretion in human airway CF epithelial cells was small (approximately 8%) compared with that in non-CF epithelia. However, the correction conferred by class 4 compounds is comparable to that obtained by low-temperature rescue and within the range considered to be therapeutically beneficial. It has been estimated that 6–10% of normal CFTR activity might prevent or significantly reduce lung pathology in CF (32
), although this remains the subject of debate. Finally, the general paradigm of small molecule discovery of correctors of mutant protein misfolding may be applicable to a wide range of protein folding diseases such as Alzheimer disease, Parkinson disease, nephrogenic diabetes insipidus, and α1-antitrypsin deficiency (33