The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway

Abstract

Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.

Figure 1

Protein trafficking and electrophysiological assays revealed correction of F508del-CFTR by glafenine. (A) A 2-dimensional chemical structure of glafenine (2,3-dihydroxypropyl 2-[(7-chloroquinolin-4-yl) amino] benzoate; hydrochloride). (B) CETSA assay showing representative immunoblots of the pellets after cell lysates were incubated for 10 min at different temperatures (33, 38, 43, 47, 52, 57 and 61 °C) in the presence of CFTR correctors. RDR1 and VX-809 are known pharmacological chaperones and were used here as positive controls. All correctors were tested at 10 μM except VX-809 (1 μM). Blots were probed with a monoclonal anti-CFTR antibody (n = 4). (C) Graph representing the band intensities for the immunoblots show in (B). (D) Immunoblot of F508del-CFTR expressed in BHK cells after 24 h of treatment with glafenine (10 μM) and with BHK cells expressing wild-type CFTR. (n = 4), (E) Relative intensity of bands B and band C in each lane in (D) as measured by ImageJ. (F) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine at 10 μM and either VX-809 at 1 µM or Trikafta both separately and together with glafenine (n = 5). The asterisk in the graph represent a value significantly (level) above the results recorded for Trikafta alone (G). Representative I_sc response traces for F508del-CFTR functional expression in well-differentiated primary human bronchial epithelial (HBE) cells determined from the increase in short-circuit current stimulated by acute addition of forskolin + genistein (ΔIsc). The basolateral membrane was permeabilized using nystatin, and an apical-to-basolateral chloride gradient was imposed by sequential addition of 10 µM forskolin, 50 µM genistein, and 10 µM CFTRinh-172 after 24 h of preincubation with 0.1% dimethylsulfoxide (vehicle), glafenine (10 µM) or VX-809 (1 μM) individually and in combination (n = 4). (H) Graph for each compound for the data attained from the Ussing chamber (G). Data in (F) and (H) are presented as the means ± SEM, n = 4, *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2

Cell-based and electrophysiological assays demonstrate the ability of glafenine derivatives to correct F508del-CFTR. (A) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine and its analogs at 10 μM. Glafenine (compound 37) is marked in green, most analogs in black and the best responding analogs in red (n = 4). (B) FMP assay (FMP) that monitors membrane depolarization induced by forskolin + genistein when cells are pretreated for 24 h with glafenine and its derivatives (10 μM) performed in BHK cells expressing F508del-CFTR (n = 4). (C) F508del-CFTR functional expression in well-differentiated CFBE41o- cell epithelial cells determined from the increase in Isc stimulated by acute addition of forskolin + genistein (ΔIsc). The basolateral membrane was permeabilized using nystatin, and an apical-to-basolateral chloride gradient was imposed. All compounds tested at 10 µM for 24 h (n = 4). (D) Immunoblot of F508del-CFTR in BHK cells after 24 h of treatment with VX-809 (1 µM) glafenine and selected derivatives (compounds 49, 53, 54, 55, 56) (10 μM) and with BHK cells expressing wild-type CFTR (n = 4). (E) Relative intensity of bands B and band C in each lane in (D). Data in panels E is presented as the means ± SEM, n = 4.

Figure 3

Demonstration of the ability of glafenine and selected derivatives to correct F508del-CFTR in primary human HBE cells. (A) F508del-CFTR functional expression in well-differentiated primary human bronchial epithelial (HBE) cells determined from the increase in short-circuit current. The basolateral membrane was permeabilized using nystatin, and an apical-to-basolateral chloride gradient was imposed. Representative I_sc responses of primary HBE cells expressing F508del-CFTR to sequential addition of 10 µM forskolin, 50 µM genistein, and 10 µM CFTRinh-172 after 24 h preincubation with 0.1% dimethylsulfoxide (vehicle), glafenine and compounds 49, 53, 54, 55 and 56 (10 µM), or VX-809 (1 μM) (n = 4). As a control Trikafta is tested with using VX = 770 (100 nM) instead of genistein. The asterisks mark glafenine derivatives (49, 56) that give a response significantly above the control level. (B,C) This is represented in two graphs, first as the change in current and second as the percentage of VX-809 response. (D) Additionally, included are the traces for each compound attained from the Ussing chamber in this experiment. Data in panels A,B and C are presented as the means ± SEM, n = 4, *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4

This study demonstrates that COX2 inhibition is required for glafenine-mediated CFTR correction and that glafenine interacts directly with COX2. (A) Cell surface CFTR in HEK cells expressing F508del-CFTR, treated with different concentrations of glafenine (0 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 10 µM, and 30 μM) in combination with siRNA knockdown, of control scrambled siRNA COX1 (filled square) COX2 (open circle), or both COX1 and COX2 (open square) for 24 h before 24-h incubation with glafenine (n = 4). (B) Q-PCR to show the effectiveness of siRNA to COX1 and COX2 both when used separately and together. (C) Immunoblots showing that the reduction in mRNA resulted in less COX1 and COX2 protein, as shown in (B). (D) Graph to show the relative intensity of bands B and band C in each lane in panel C as monitored by image J. (E) Graph to show the COX enzyme activity remaining in the cells 48 h after siRNA treatment for COX-1 and COX-2 separately and together. Also the COX enzyme activity detected upon 24 h treatment with glafenine and 3 analogs compounds 1, 8 and 49 all at 10 µM. Black bars represent total COX enzymatic activity measured from cell lysates, the white and shaded bars represent COX-1 and COX-2 enzymatic activity respectively obtained due to the inhibition of recombinant COX enzymes (n = 3). Data in (B), (D) and (E) are presented as the means ± SEM.

Figure 5

The mechanism of glafenine-mediated CFTR correction works via the arachidonic acid pathway. (A) A cartoon of the arachidonic acid pathway with specific enzyme inhibitors marked in red. (B) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine, 10 μM and Trikafta in the presence and absence of PGH2 at 1 μM (n = 4). (C) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine (10 μM), MF63 (10 μM), picotamide (1 μM), tranyl cypromine (10 μM), sorbinil (10 μM), and suramin (5 μM) (n = 4). (D) FMP assay (FMP) that monitors membrane depolarization induced by forskolin + VX-770 when cells are pretreated for 24 h with MF63 (10 μM) and Trikafta in the presence and absence of CFTR172inh (10 μM). Additionally, MF63 (10 μM) and Trikafta were added together for 24 h (n = 4). (E) Representative I_sc responses of primary HBE cells expressing F508del-CFTR functional expression in well-differentiated primary human bronchial epithelial (HBE) cells determined from the increase in short-circuit current stimulated by acute addition of forskolin + genistein (ΔIsc). The basolateral membrane was permeabilized using nystatin, and an apical-to-basolateral chloride gradient was imposed by sequential addition of 10 µM forskolin, 100 nM vx-770, and 10 µM CFTR inh-172 after 24 h of preincubation with 0.1% dimethyl sulfoxide (vehicle) and MF63 (10 µM). (F) Graph for each compound for the data attained from the Ussing chamber (E) (n = 4). Data in (B), (C), (D) and (F) are presented as the means ± SEM, n = 4, *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 6

The mechanism of glafenine-mediated CFTR correction works via the inhibition of prostaglandin E₂ receptor 4 (EP4) activation. (A) FMP assay that monitors membrane depolarization induced by forskolin + genistein when BHK cells are pretreated with glafenine (10 µM) and various concentrations of the arachidonic pathway derivative prostaglandin E2 for 24 h prior to assaying (n = 4). (B) FMP assay (FMP) of BHK cells pretreated for 24 h with MF63 (10 µM) and an agonist (AG) and antagonist (AAG) for each of the four EP receptors (agonists for EPs 1 to 4 are ONO-D1-OO4, butaprost, sulprostone and CAY10598, respectively; for the antagonists, they are SC-51089, PF0044189948, L798106 and ONO-AE3-208, respectively) (10 μM) performed in BHK cells expressing F508del-CFTR (n = 4). (C) FMP assay in cells pretreated for 24 h with MF63 (10 µM) and a range of concentrations (10 µM to 100 pM) of the EP4 agonist CAY10598. The vertex corrector VX-809 (3 µM) was used as a positive control, and the EP2 agonist butaprost (10 µM) was used in combination with MF63 to show that the effects seen with the EP4 agonist were specific to EP4 (n = 4). (D) FMP assay in HEK cells expressing F508del-CFTR, treated with different concentrations of MF63 (0 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 10 µM, and 30 μM) alone (filled circle) or in combination with siRNA knockdown, of control scrambled siRNA (filled rhombus) or siRNA to EP4 (filled square) for 24 h before 24-h incubation with MF63 (n = 4). (E) Q-PCR to show the effectiveness of siRNA to EP4.

Figure 7

Primary HBE cells revealed that glafenine, compound 49 and MF63 are potent correctors of class 2 CFTR mutations. (A) FMP assay (FMP) that monitors membrane depolarization induced by forskolin + genistein when cells are pretreated for 24 h with glafenine, its derivative, compound 49 and MF63 (all at 10 μM) and separately with VX-809 (1 µM) and Trikafta, performed in Fischer rat thyroid (FRT) expressing a selection of class 2 CFTR mutations (F508del-, G85E, and N1303K), (n = 3). (B) G85E-CFTR functional expression in well-differentiated primary human bronchial epithelial (HBE) cells determined from the increase in short-circuit current stimulated by acute addition of forskolin + VX-770 (ΔI_sc), (NB It should be noted that the cells of this patient were heterozygotic in which one allele expressed G85E-CFTR and the other expressed the class 1 type mutation 621-1GT-CFTR). Representative I_sc responses of primary HBE cells expressing G85E-CFTR to sequential addition of 10 µM forskolin, 100 nM VX-770, and 10 µM CFTRinh-172 after 24 h preincubation with 0.1% dimethyl sulfoxide (vehicle), glafenine (10 µM) or VX-809 (1 μM), compound 49 (10 μM) and MF63 (10 μM), and Trikafta (N = 3). (C) Graphical representation of the Ussing chamber experiment outlined in (B). (D) Class 1 type 621-1GT-CFTR functional expression in well-differentiated primary human bronchial epithelial (HBE) cells determined from the increase in short-circuit current stimulated by acute addition of forskolin + genistein (ΔIsc). Representative I_sc responses of primary HBE cells expressing 621-1GT-CFTR to sequential addition of 10 µM forskolin, 50 µM genistein, and 10 µM CFTRinh-172 after 24 h preincubation with 0.1% dimethylsulfoxide (vehicle), VX-809 (1 μM), glafenine, compound 49, and MF63 (all at 10 µM), (n = 3). Data in A and D are presented as the means ± SEM, n = 4, *p < 0.05, **p < 0.01 and ***p < 0.001.