Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening

Abstract

The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (DeltaF508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The DeltaF508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in DeltaF508-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 DeltaF508-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 DeltaF508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved DeltaF508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected DeltaF508-CFTR in DeltaF508/DeltaF508 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 DeltaF508 mutation.

Figure 1

Discovery of ΔF508-CFTR correctors by high-throughput screening. (A) Screening procedure. FRT cells coexpressing ΔF508-CFTR and a halide-sensitive YFP were incubated with test compounds (10 μM) at 37°C. ΔF508-CFTR function was assayed at 18–24 hours in a plate reader according to YFP fluorescence quenching by iodide in the presence of forskolin (20 μM) plus genistein (fsk + gen) (50 μM). (B) Representative traces showing iodide influx under control conditions (37°C) or after 24 hours incubation at 27°C or with 4-PBA (4 mM) or correctors (10 μM; 37°C). (C) Chemical structures of correctors of 4 chemical classes. See Supplemental Table 1 for full listing of correctors and their activities. (D) Dose-response data for indicated correctors (SEM; n = 5). The dashed line indicates the level of activity of the low-temperature rescue used as a positive control.

Figure 2

Properties of ΔF508-CFTR correctors. (A) Maximal iodide influx (normalized to 37°C control) in ΔF508-CFTR–expressing FRT cells incubated at 37°C or 27°C (SEM; n = 5). Iodide influx increased significantly (P < 0.01) for all compounds compared with control. (B) Apical membrane chloride current measured in Ussing chambers after basolateral membrane permeabilization and in the presence of a chloride gradient (see Methods). The concentrations were: forskolin, 20 μM; genistein, 50 μM; CFTR_inh-172, 10 μM. Measurements were performed on ΔF508-CFTR–expressing FRT cells, except in the experiment represented in the lower-left panel, which was performed on FRT-null cells. (C) Time course of correction. Cells incubated for different times at 27°C or with indicated correctors or 4-PBA (4 mM) at 37°C. ΔF508-CFTR activity was assayed in the presence of foskolin/genistein. (D) Persistence of correction. Cells were treated for 24 hours with correctors (or at 27°C). ΔF508-CFTR activity was assayed at different times after washout (or return from 27°C to 37°C).

Figure 3

Increased ΔF508-CFTR sensitivity to activators after incubation with correctors. (A) Effect of forskolin (20 μM) or forskolin plus genistein (50 μM) in ΔF508-CFTR–expressing FRT cells kept at 37°C or 27°C with or without correctors. (B) Forskolin dose-response relationships. Cells stimulated with forskolin (in the absence of genistein) after incubation with correctors (at 37°C) or at 27°C. (C) Apical membrane chloride current after incubation for 24 hours at 37°C with DMSO vehicle (left curve) or corr-2b (middle curves). The curve at the far right was obtained from an experiment on cells grown at 27°C with compounds added as shown (20 μM forskolin, 20 μM corr-2b, 50 μM genistein).

Figure 4

Biochemical analysis of corrector mechanism of action. (A) Effect of the indicated correctors (10 μM) on the expression pattern of ΔF508-CFTR-C_tHA 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.

Figure 5

Specificity for corrector action on ΔF508-CFTR cellular misprocessing. (A) Apical membrane chloride current in FRT cells expressing the temperature-sensitive mutant P574H-CFTR. Experiments were carried out as described in Figure 2B. I_SC, short-circuit current. (B) CHO cells expressing Flag-tagged wild-type or mutant (M345T) dopamine receptor (DRD4) were treated for 16 hours with the indicated correctors (5 μM corr-4c and -4b; 10 μM corr-4a) or 10 μM domperidone, a dopamine antagonist. The cell-surface density of DRD4 was measured using anti-Flag primary and HRP-conjugated goat anti-mouse antibodies. Data represent the mean of 2 independent experiments, each performed in triplicate.

Figure 6

Correction of ΔF508-CFTR misprocessing in human bronchial epithelial cells. (A) Representative short-circuit current recordings on primary cultures of human bronchial epithelial cells from a ΔF508/ΔF508 subject (top 3 curves) and a non-CF subject (bottom curve). ΔF508 cells maintained at 37°C for 24 hours in the presence of DMSO vehicle (top curve) or corr-4a (third curve) or incubated at 27°C (second curve). Concentrations were: amiloride, 10 μM; forskolin, 20 μM; genistein, 50 μM; CFTR_inh-172, 10 μM. (B) Summary of CFTR_inh-172–inhibited short-circuit current (ΔI_sc) for a series of experiments as described in A (SEM; n = 12–14). *P < 0.05; **P < 0.01. (C) Short-circuit current recordings of primary cultures of human bronchial epithelial cells from a homozygous N1303K-CFTR subject taken under conditions as described in A.