American Chemical Society, Molecular Pharmaceutics, 3(11), p. 653-664, 2014
DOI: 10.1021/mp400230s
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The presence in the messenger RNA (mRNA) of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is Cystic Fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF trans-membrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTCs readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the Green Fluorescent Protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation, showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the Molecular Dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.