Synthetic aminoglycosides efficiently suppress cystic fibrosis transmembrane conductance regulator nonsense mutations and are enhanced by ivacaftor

Abstract

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

Figure 1.

Chemical structures of synthetic aminoglycosides.

Figure 2.

Readthrough assays in immortalized cystic fibrosis (CF) bronchial epithelial (CFBE) cells transiently transfected with readthrough reporters. (A) The diagram of dual-luciferase (Luc) reporters. Four CF transmembrane conductance regulator (CFTR) premature termination codons (PTCs; bold), together with their natural context, are shown. The corresponding WT codons are GGA for G542, CGA for R553, CGA for R1162, and TGG for W1282. pA, Poly(A) tail; P_CMV, CMV promoter. (B–E) CFBE41o⁻ cells are transfected with one of the reporters; 3 hours later, various doses (3, 10, 30, and 100 μM) of five drugs (gentamicin, NB54, NB74, NB84, and NB124) were added into medium. Luciferase assays were performed after 24 hours. The numbers of readthrough fold for the four PTCs are shown. Readthrough fold is determined as the ratio of readthrough percent after treatment to that of untreated, whereas readthrough percent is the ratio of Firefly number to Renilla number normalized to wild-type (WT). Gent, gentamicin. The data are shown as means ± SD of at least six replicates. *Significance relative to untreated; ^†significance relative to 100 μM gentamicin; * or ^†P < 0.05, **P < 0.01, ***P < 0.001, **** or ^††P < 0.0001.

Figure 3.

Improved CFTR function and expression in CFTR-G542X–expressing Fischer rat thyroid (FRT) monolayers. (A) Representative tracings of the change in the transepithelial conductance (Δ Conductance, Δ transepithelial conductance [Gt]) of FRT cell monolayers (n = 9) expressing CFTR-G542X treated with synthetic aminoglycosides and gentamicin (250 μg/ml, unless indicated otherwise). CFTR activity was measured as the change from baseline conductance after the addition of forskolin (10 μM); CFTR-_Inh172 (10 μM) was used to block CFTR-mediated Gt for confirmation. (B and C) Mean change in Gt induced by forskolin (B) and CFTR_Inh-172 (C) for experiments shown in (A). *P < 0.05, ***P < 0.001, ****P < 0.0001 versus vehicle or gentamicin (n = 9 per condition, performed in three replicate experiments). (D) Representative Western blot demonstrating presence of CFTR C-band (closed arrowhead) and B-band (open arrowhead) after treatment with synthetic aminoglycosides (n = 3 monolayers/lane) in comparison to vehicle and gentamicin control.

Figure 4.

Restoration of CFTR function in CF primary human bronchial epithelial (HBE) cells derived from a G542X/F508del donor. The forskolin (20 μM) -stimulated short-circuit current (I_sc) of HBE cells treated with one of the five drugs (250 μg/ml) or vehicle control for 48 hours is shown and plotted in comparison to the relative percent values obtained in a panel of non-CF HBE cells tested under the exact same conditions. The data are shown as means ± SEM of at least four replicates. **P < 0.01.

Figure 5.

c-AMP–activated transepithelial currents in ileum sections from mice treated with gentamicin or NB124. Representative tracings of (A) untreated Cftr^+/+ hCFTR-G542X mice (WT control), (B) untreated Cftr^−/− hCFTR-G542X (negative control), (C) gentamicin-treated Cftr^−/− hCFTR-G542X (60 mg/kg), and (D) NB124-treated Cftr^−/− hCFTR-G542X (30 mg/kg). (E) Scatter plot of all data from Cftr^−/− hCFTR-G542X mice (untreated, gentamicin treated, and NB124 treated). The mean is shown as a bar. **P < 0.01, ***P < 0.001, ****P < 0.0001. (F–H) Human CFTR immunofluorescence of excised intestine from mice treated with gentamicin or NB124. The tissue was cut 5-μM thick and incubated with CFTR-NBD1 antiserum and AlexaFluor488-labeled secondary antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Representative images are shown from (F) untreated Cftr^−/− hCFTR-G542X (negative control), (G) gentamicin-treated Cftr^−/− hCFTR-G542X (60 mg/kg), and (H) NB124-treated Cftr^−/− hCFTR-G542X (30 mg/kg). Fsk, forskolin; Gly, glybenclamide; IBMX, 3-isobutyl-1-methylxanthine.

Figure 6.

Toxicity study in cochlear hair cells in vitro. Loss of cochlear hair cells was measured 72 hours after treating with gentamicin, NB124, or NB84. The data are shown as means ± SD of at least three replicates per concentration. IC₅₀, inhibitory concentration at which 50% of cells were lost.

Figure 7.

Ivacaftor further stimulates cAMP-dependent CFTR function in CFBE41o⁻ monolayers after translational readthrough. (A and B) CFBE41o⁻ cells stably expressing CFTR-R1162X (A) or W1282X (B) were incubated with synthetic aminoglycosides (250 μg/ml) for 48 hours, followed by I_sc measurements in Ussing chambers. Cells were sequentially stimulated with forskolin (100 nM) then ivacaftor (VX-770; 10 μM). Comparisons of total stimulated current, ivacaftor-stimulated current, and forskolin-stimulated current are each designated as compared with control (asterisk noted top to bottom, respectively). The data shown are means ± SEM of at least six replicates per condition. *P < 0.05, **P < 0.01, ***P < 0.001.