Cyanoquinolines with independent corrector and potentiator activities restore ΔPhe508-cystic fibrosis transmembrane conductance regulator chloride channel function in cystic fibrosis

Abstract

The ΔPhe508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein impairs its folding, stability, and chloride channel gating. Although small molecules that separately correct defective ΔPhe508-CFTR folding/cellular processing ("correctors") or chloride channel gating ("potentiators") have been discovered and are in clinical trials, single compounds with bona fide dual corrector and potentiator activities have not been identified. Here, screening of ∼110,000 small molecules not tested previously revealed a cyanoquinoline class of compounds with independent corrector and potentiator activities (termed CoPo). Analysis of 180 CoPo analogs revealed 6 compounds with dual corrector and potentiator activities and 13 compounds with only potentiator activity. N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3-methoxybenzamide (CoPo-22), which was synthesized in six steps in 52% overall yield, had low micromolar EC(50) for ΔPhe508-CFTR corrector and potentiator activities by short-circuit current assay. Maximal corrector and potentiator activities were comparable with those conferred by the bithiazole Corr-4a and the flavone genistein, respectively. CoPo-22 also activated wild-type and G551D CFTR chloride conductance within minutes in a forskolin-dependent manner. Compounds with dual corrector and potentiator activities may be useful for single-drug treatment of cystic fibrosis caused by ΔPhe508 mutation.

Fig. 1.

Dual-acting ΔPhe508-CFTR CoPo identified by high-throughput screening. A, screening procedure. Left, corrector assay: FRT cells coexpressing human ΔPhe508-CFTR and a halide-sensing YFP were incubated with test compounds at 37°C for 24 h. ΔPhe508-CFTR function was assayed in a plate reader from YFP fluorescence quenching in response to iodide addition in the presence of forskolin (20 μM) plus genistein (50 μM). Right, potentiator assay: cells were incubated at 27°C for 24 h before assay (temperature rescue) to target ΔPhe508-CFTR to the plasma membrane. Test compounds were added for 10 min at room temperature in presence of forskolin (20 μM) before iodide addition. B, structures of novel ΔPhe508-CFTR correctors identified in the primary screen. C, representative traces showing iodide influx at different [CoPo-22] for corrector assay (left) and potentiator assay (right). D, dose-response data of CoPo-22 in corrector (left) and potentiator (right) assays (S.E.M., n = 3). Fits to single-site activation model shown.

Fig. 2.

Structure-activity relationship analysis of CoPo analogs. A, structural determinants of CoPo corrector (left) and potentiator (right) activities. (see Table 1 for activity data of CoPo analogs). B, concentration-dependence of corrector (left) and potentiator (right) activities of CoPo-01, CoPo-02, CoPo-05, and CoPo-08. C, CoPo-22 synthesis scheme.

Fig. 3.

Characterization of CoPo-22 corrector activity. A, ΔPhe508-CFTR expressing FRT cells were incubated at 37 or 27°C with or without CoPo-22 (20 μM) or Corr-4a (10 μM). Iodide influx (S.E.M., n = 4) shown in the presence of forskolin (20 μM) or forskolin (20 μM) plus genistein (50 μM). *, P < 0.01 compared with control. B, forskolin dose-response for experiments as in A, measured in the presence of genistein (50 μM). C, representative short-circuit measurements showing apical membrane chloride current after incubation for 24 h at 37°C with indicated [CoPo-22]. Incubation with Corr-4a (5 μM) shown as reference (right). Forskolin (20 μM), genistein (50 μM), and CFTR_inh-172 (10 μM) added where indicated. D, CoPo-22 concentration-dependence deduced from experiments as in C (S.E.M., n = 3–4). Fits to single-site binding model shown.

Fig. 4.

Additive corrector efficacy of CoPo-22 and Corr-4a. A, Corr-4a concentration-dependence of iodide influx (measured with 20 μM forskolin + 50 μM genistein) in the presence of indicated (submaximal) [CoPo-22] (S.E.M., n = 4). B, additivity studies showing iodide influx after incubation with maximal CoPo-22 and Corr-4a (S.E.M., n = 4, *, P < 0.01 compared with CoPo-22 or Corr-4a alone). C, immunoblot (anti-CFTR antibody) after 24-h incubation at 37°C of ΔPhe508-CFTR expressing FRT cells with Corr-4a or CoPo-22 (or DMSO vehicle). Bands B (core-glycosylated) and C (complex-glycosylated) indicated. For comparison, data shown for (untreated) FRT cells expressing wild-type CFTR.

Fig. 5.

Characterization of CoPo-22 potentiator activity. Short-circuit current measured in FRT cells expressing ΔPhe-CFTR (A) and wild-type (B), showing responses to indicated forskolin and CoPo-22 concentrations. ΔPhe508-CFTR expressing cells were incubated at 27°C for 24 h before measurement. Where indicated, genistein (50 μM) and CFTR_inh-172 (10 μM) were added. Representative of two to four sets of measurements.

Fig. 6.

CoPo-22 activity in G551D-CFTR expressing FRT cells and ΔPhe508-CFTR-expressing A459 cells. A, top, short-circuit current measured in FRT cells expressing G551D-CFTR, showing responses to indicated forskolin and CoPo-22 concentrations. Where indicated, genistein (100 μM) and CFTR_inh-172 (10 μM) were added. Representative of three sets of measurements. Bottom, plate reader assay of G551D-CFTR chloride conductance showing representative fluorescence quenching curves (inset) and deduced concentration dependence of CoPo-22 and genistein potentiator action (S.E.M., n = 4). Measurements were made in the presence of 20 μM forskolin. B, potentiator assay done in ΔPhe508-CFTR expressing A549 cells by YFP/iodide fluorescence quenching as in Fig. 1. Representative fluorescence quenching curves (top) shown with deduced CoPo-22 and genistein concentration-dependence (bottom S.E.M., n = 4). C, corrector assay done in ΔPhe508-CFTR expressing A549 cells by YFP/iodide fluorescence quenching, in which cells were incubated with vehicle or indicated correctors at 37°C (top) or 27°C (bottom) for 24 h before iodide influx measurement. Representative of three sets of measurements.