Small-molecule correctors of defective ΔF508-CFTR cellular processing identified by high-throughput screening
Nicoletta Pedemonte, Gergely L. Lukacs, Kai Du, Emanuela Caci, Olga Zegarra-Moran, Luis J.V. Galietta, A.S. Verkman
Nicoletta Pedemonte, Gergely L. Lukacs, Kai Du, Emanuela Caci, Olga Zegarra-Moran, Luis J.V. Galietta, A.S. Verkman
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Research Article

Small-molecule correctors of defective ΔF508-CFTR cellular processing identified by high-throughput screening

Abstract

The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (ΔF508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The ΔF508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in ΔF508-CFTR–transfected epithelial cells using a fluorescent halide indicator. Screening of 150,000 chemically diverse compounds and more than 1,500 analogs of active compounds yielded several classes of ΔF508-CFTR correctors (aminoarylthiazoles, quinazolinylaminopyrimidinones, and bisaminomethylbithiazoles) with micromolar potency that produced greater apical membrane chloride current than did low-temperature rescue. Correction was seen within 3–6 hours and persisted for more than 12 hours after washout. Functional correction was correlated with plasma membrane expression of complex-glycosylated ΔF508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved ΔF508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected ΔF508-CFTR in ΔF508/ΔF508 human bronchial epithelia but did not correct a different temperature-sensitive CFTR mutant (P574H-CFTR) or a dopamine receptor mutant. Small-molecule correctors may be useful in the treatment of CF caused by the ΔF508 mutation.

Authors

Nicoletta Pedemonte, Gergely L. Lukacs, Kai Du, Emanuela Caci, Olga Zegarra-Moran, Luis J.V. Galietta, A.S. Verkman

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Biochemical analysis of corrector mechanism of action. (A) Effect of the...
Biochemical analysis of corrector mechanism of action. (A) Effect of the indicated correctors (10 μM) on the expression pattern of ΔF508-CFTR-CtHA in BHK cells. Cells were cultured for 24 hours at 37°C with or without correctors or at 27°C. Top: CFTR was visualized using anti-HA primary and HRP-conjugated secondary antibodies. Filled arrowhead, complex-glycosylated form (band C); open arrowhead, core-glycosylated form (band B). Bottom: Quantification of bands B and C (SEM; n = 4–5). (B) Effect of the indicated correctors (10 μM) on the expression pattern of ΔF508-CFTR in FRT epithelia. Confluent monolayers were treated as described in A, and immunoblot analysis was performed with the indicated primary antibodies. (C) Correlation between cell-surface density and PKA-activated chloride current. Cell-surface density of ΔF508-CFTR was determined using the radioactive anti-HA antibody binding assay and plotted against ΔF508-CFTR apical membrane currents in parallel experiments done on FRT cells. (D) Translational rate of ΔF508-CFTR was measured in the presence of correctors (10 μM) by monitoring [35S]methionine/cysteine incorporation into CFTR during a 15-minute pulse labeling (SEM; n = 3). (E) Folding efficiency measured by pulse-chase assay. Top: The amount of newly synthesized ΔF508-CFTR was computed from radioactive incorporation during a 15-minute pulse (P). For measurement of folding efficiency, cells were pulsed for 150 minutes and than chased for 150 minutes (C). The amounts of core- (open arrowhead) and complex-glycosylated (filled arrowhead) forms were determined by phosphorimage analysis. Bottom: Maturation efficiency expressed as the percent of mature, complex-glycosylated ΔF508-CFTR relative to the newly synthesized pool, as shown in the top panel. (F) Stability of the core-glycosylated ΔF508-CFTR was measured following a 15-minute pulse labeling in the presence of the indicated correctors. Remaining radioactivity associated with CFTR was measured after 1 hour chase (SEM; n = 3). (G) Cell-surface stability of the rescued ΔF508-CFTR was measured by the anti-HA antibody binding assay before and after 2 hours chase in the presence of the indicated correctors. Data are expressed as the mean ± SEM of 3 independent experiments.