Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems

Abstract

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after treatment with the selected molecules in cells expressing nonsense-CFTR-mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

Keywords: genetic disorder; nonsense mutation; oxadiazoles; premature termination codon; translational readthrough inducing drugs.

Figure 1

Codons language and most frequent misreadings for premature termination codon (PTC)’s readthrough.

Figure 2

Illustrations of cystic fibrosis transmembrane conductance regulator (CFTR) nonsense mutations and their effect. (A) CFTR mRNA sequence of the stop codon G542X and W1282X (in red the PTC UGA codons). (B) CFTR localization and effect of the nonsense mutation on the CFTR gene, impairing cell homeostasis and causing the accumulation of mucus and the following infection.

Figure 3

Different structures of proposed translational readthrough-inducing drugs (TRIDs).

Figure 4

1,2,4-oxadiazoles selected structures.

Scheme 1

Synthesis of NV930.

Scheme 2

Synthesis of NV914.

Figure 5

The newly synthesized molecules show readthrough activity in the FLuc cell model system. Histogram shows luciferase (FLuc) activity (RLU) after 24 h of exposition to PTC124, NV848, NV930, and NV914 (all at the concentration of 12 μM) in HeLa FLuc-opal transfected cells. Data were analyzed by GraphPad Prism 6 software and expressed as mean values ± standard error of the mean (SEM; n = 3). Symbol (*) represents the statistical significance of data regarding PTC124, NV848, NV914, and NV930 versus Untr: ***, p < 0.001 (ANOVA with Dunnett’s posthoc test).

Figure 6

Immunofluorescence analysis of the full-length CFTR protein in wild-type (CFTR-WT) and CFTR^G542X Fisher rat thyroid (FRT) cells, untreated (Untr) or treated with 12 μM PTC124, NV848, NV930, or NV914 for 24 h. CFTR protein was revealed by a specific antibody (ab570-green). Nuclei (blue) were DAPI (4′,6-diamidino-2-phenylindole)-stained and the cellular membrane was stained by wheat germ agglutinin (WGA)-Alexa 594 (red).

Figure 7

Immunofluorescence analysis of the full-length CFTR protein in wild-type (CFTR-WT) and CFTR^W1282X FRT cells, untreated (Untr) or treated with 12 μM PTC124, NV848, NV930, or NV914 for 24 h. CFTR protein was revealed by a specific antibody (ab570-green). Nuclei (blue) were DAPI-stained and the cellular membrane was stained by WGA-Alexa 594 (red).

Figure 8

Western blot analysis of the CFTR protein in wild-type (WT) CFTR, and CFTR^G542X (A) and CFTR^W1282X (B) FRT cells, untreated (Untr) or treated with 12 μM PTC124 (positive control), NV848, NV930, or NV914 for 24 h; β-tubulin was used as control for the protein loading. The graphs show the quantification of the gel bands by ImageJ software. The experiment was performed in triplicate and data were normalized by β-tubulin expression, analyzed by GraphPad Prism 6 software. Data are expressed as mean values ± standard error of the mean (SEM). Symbols (* and §) represent the statistical significance of NV848, NV914, and NV930 versus Untr (*) and versus PTC124 (§). two symbols, p < 0.01; three symbols, p < 0.001 (Student’s t-test and ANOVA with Dunnett’s posthoc test).

Figure 9

CFTR functionality in FRT cells measured by the yellow fluorescent protein (YFP) assay (pink: cells, green: YFP protein expression, blue: multiwell), (A) in YFP-CFTR^WT FRT, and (B) CFTR^G542X and CFTR^W1282X FRT cells, untreated or treated with 12 μM PTC124, NV848, NV914, or NV930 for 24 h (C). Cells were prestimulated with 20 μM forskolin for 20 min. The experiment was performed in triplicate. Images were taken before (t⁰) and after (t^30s) the addition of NaI. Percentages of YFP-positive cells before (− dark green bars) and after NaI addition (+ light green bars); data were analyzed by GraphPad (n = 3) and expressed as mean values ± standard error of the mean (SEM) (D). Data were analyzed comparing treated FRT cells versus untreated FRT cells by GraphPad Prism 6 software (n = 3). ***, p < 0.001 (ANOVA with Dunnett’s posthoc test).