The presence in the 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 transmembrane 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 PTC 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.
|Numero di pagine||12|
|Stato di pubblicazione||Published - 2014|
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Pharmaceutical Science
- Drug Discovery
Melfi, R., Spinello, A., Pibiri, I., Palumbo Piccionello, A., Lentini, L., Barone, G., Di Leonardo, A., Pace, A., Pace, A., Barone, G., & Di Leonardo, A. (2014). Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay. Molecular Pharmaceutics, 11, 653-664.