Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.

Abstract

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.

• Authors: Laura Lentini, Raffaella Melfi, Aldo Di Leonardo, Angelo Spinello, Giampaolo Barone, Andrea Pace, Antonio Palumbo Piccionello, Ivana Pibiri
• Journal: Molecular pharmaceutics (Mol Pharm) Vol. 11 Issue 3 Pg. 653-64 (Mar 03 2014) ISSN: 1543-8392 [Electronic] United States
• PMID: 24483936 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• CFTR protein, human
• Codon, Nonsense
• Codon, Terminator
• Oxadiazoles
• RNA, Messenger
• Cystic Fibrosis Transmembrane Conductance Regulator
• Green Fluorescent Proteins
• ataluren

Topics

• Blotting, Western
• Cells, Cultured
• Codon, Nonsense (genetics, metabolism)
• Codon, Terminator (genetics, metabolism)
• Cystic Fibrosis Transmembrane Conductance Regulator (chemistry, genetics, metabolism)
• Green Fluorescent Proteins (genetics, metabolism)
• HeLa Cells
• Humans
• Molecular Dynamics Simulation
• Mutagenesis, Site-Directed
• Mutation (genetics)
• Nucleic Acid Conformation
• Oxadiazoles (chemistry, metabolism)
• Protein Conformation
• RNA, Messenger (chemistry, genetics)
• Real-Time Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction