Fig 1.
Identification of novel potentiators of human CFTR.
(A) Assay protocol. FRT cells stably expressing the halide-sensitive cytoplasmic fluorescent sensor YFP-148Q/I152L and CFTR were incubated with forskolin and test compounds. The YFP fluorescence was measured in response to addition of iodide (top panel). Chemical structure of CP7q (bottom panel). (B) In WT-CFTR expressing FRT cells, the YFP fluorescence measured in single wells of 96-well plates, showing vehicle control, 10 μM forskolin and 3 μM hydroxypyrazolines in the presence of 0.1 μM forskolin. (C) In FRT cells expressing ΔF508-CFTR that has been rescued by low temperature, the YFP fluorescence measured in single wells of 96-well plates, showing vehicle control, 50 μM genistein and 3 μM hydroxypyrazolines in the presence of 10 μM forskolin. (D) Representative traces showing iodide influx via WT-CFTR in the presence of the indicated concentrations of CP7q and 0.1 μM forskolin. (E) Representative traces showing iodide influx via low temperature-rescued ΔF508-CFTR in the presence of the indicated concentrations of CP7q and 10 μM forskolin.
Table 1.
CFTR potentiation by hydroxy pyrazoline compounds.
Structure-activity relationship of CFTR potentiator is shown. EC50 values were determined by microplate reader assay.
Fig 2.
Synthesis of the Hydroxy Pyrazolines as CFTR potentiators.
(i) KOH, EtOH, stir; (ii) Chloro(methoxy)methane (MOMCl), K2CO3, Acetone, reflux; (iii) substituted benzaldehydes 2a-2c, KOH, EtOH, stir; (iv) 3N HCl, reflux; (v) 4-hydrazinobenzenesulfonamide hydrochloride (see Materials and Methods).
Table 2.
Substitution pattern of hydroxypyrazolines (7a-7r) and intermediate hydroxychalcones (3a-3r).
Fig 3.
Characterization of CP7q, a small-molecule potentiator of CFTR.
(A) Apical membrane currents measured in FRT cells expressing WT-CFTR. CFTR was potentiated by indicated concentrations of forskolin and CP7q (left and middle panel). CFTR current was inhibited by 10 μM CFTRinh-172. Summary of CP7q dose-response data (right panel) (mean ± S.E., n = 3). (B) Summary of peak currents (mean ± S.E., n = 3). (C) Whole-cell CFTR Cl- currents were recorded at a holding potential at 0 mV, and pulsing to voltages between ± 80 mV (in steps of 20 mV) in FRT cells expressing WT-CFTR (left panel). CFTR was slightly activated by 0.1 μM forskolin and potentiated by 10 μM CP7q. Current/voltage (I/V) plot of mean currents at the middle of each voltage pulse (middle panel). Summary of current density data measured at + 80 mV (mean ± S.E., n = 5, right panel). (D) Intracellular cAMP accumulation in FRT cells in response to addition of CP7q (10 μM) and forskolin (10 μM) (mean ± S.E., n = 4). (E) Effect of CP7q on cell viability 24 h after treatment as evaluated by MTS assays in HT-29 cells (mean ± S.E., n = 6). *P < 0.05, **P < 0.01.
Fig 4.
Apical membrane currents were measured in FRT cells expressing human ΔF508-CFTR rescued by low temperature (27°C) incubation for 24 hours. (A, B) CFTR was activated by addition of 20 μM forskolin and potentiated with 50 μM genistein or different concentrations of CP7q. CFTR-dependent current was inhibited by 10 μM CFTRinh-172. (C) Summary of CP7q dose-response data (mean ± S.E., n = 3). (D) Summary of peak current (mean ± S.E., n = 3). (E) Whole-cell ΔF508-CFTR Cl- currents were measured in FRT cells expressing ΔF508-CFTR rescued by low temperature (27°C) incubation for 24 hours (left panel). ΔF508-CFTR was activated by 10 μM forskolin and potentiated by 10 μM CP7q. Current/voltage (I/V) plot of mean currents at the middle of each voltage pulse (middle panel). Summary of current density data measured at + 80 mV (mean ± S.E., n = 5, right panel). **P < 0.01.
Fig 5.
CP7q potentiates G551D-CFTR in FRT cells and WT-CFTR in primary cultured human nasal epithelial cells.
(A) Apical membrane currents were measured in FRT cells expressing human G551D-CFTR. CFTR was stimulated by application of 20 μM forskolin and then 50 μM genistein (left panel), 10 μM VX-770 (middle panel) or indicated concentrations of CP7q (right panel) were applied to bath solution. CFTR-dependent current was inhibited by 10 μM CFTRinh-172. (B) Summary of CP7q-induced fold increase in G551D-CFTR current stimulated by 20 μM forskolin (mean ± S.E., n = 3). (C) Summary of 50 μM genistein, 10 μM VX-770 and 30 μM CP7q induced peak current (mean ± S.E., n = 3). (D) Short-circuit currents were measured in normal human nasal epithelial cells (left panel). CFTR was activated by 0.5 μM forskolin. CFTR was potentiated by indicated concentrations of CP7q and inhibited by 10 μM CFTRinh-172. ENaC was inhibited by 100 μM amiloride. Bar graph showing the summarized data of peak current (mean ± S.E., n = 3, right panel). **P < 0.01.
Fig 6.
CP7q increases the functional rescue of ΔF508-CFTR by VX-809 in A549 and FRT cells.
(A) ΔF508-CFTR expressing A549 cells were incubated with 5 μM VX-809 in the presence and absence of 10 μM CP7q or 5 μM VX-770 for 48 hours. The traces are showing iodide influx via the rescued ΔF508-CFTR stimulated with 10 μM forskolin and 10 μM VX-770 (mean ± S.E., n = 6). (B) Representative ΔF508-CFTR immunoblot at 48 h after treatment with 5 μM VX-809 in the presence and absence of 10 μM CP7q or 5 μM VX-770 in A549-ΔF508-CFTR cells. (C) The ratio of ΔF508-CFTR band C/B was summarized in bar graph (mean ± S.E., n = 3). (D) Representative apical membrane current traces showing effect of CP7q and VX-770 on VX-809-induced functional rescue of ΔF508-CFTR in FRT-ΔF508-CFTR cells. Well differentiated FRT cells were incubated with 5 μM VX-809 in the presence and absence of 10 μM CP7q or 5 μM VX-770 for 48 hours. ΔF508-CFTR currents were inhibited by 10 μM CFTRinh-172. (E) Bar graph showing the summarized data of peak current (mean ± S.E., n = 6). *P < 0.05.