Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay

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RESEARCH PRODUCT

Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay

Laura Lentini Ivana Pibiri Andrea Pace Aldo Di Leonardo Giampaolo Barone Raffaella Melfi Angelo Spinello Antonio Palumbo-piccionello

subject

Duchenne muscular distrophy (DMD)Protein Conformation Nonsense mutation Blotting Western Green Fluorescent Proteins Pharmaceutical Science Cystic Fibrosis Transmembrane Conductance Regulator Settore BIO/11 - Biologia Molecolare Biology Molecular Dynamics Simulation medicine.disease_cause Real-Time Polymerase Chain Reaction premature termination codons (PTC)Article Green fluorescent protein chemistry.chemical_compound Drug Discovery medicine Coding region Humans RNA Messenger molecular dynamics (MD)Gene Cells Cultured Genetics nonsense mutation readthrough Messenger RNA Mutation Oxadiazoles Reverse Transcriptase Polymerase Chain Reaction green fluorescent protein (GFP)ataluren Settore CHIM/06 - Chimica Organica Stop codon Ataluren Settore BIO/18 - Genetica chemistry Codon Nonsense Settore CHIM/03 - Chimica Generale E Inorganica Mutation Codon Terminator Mutagenesis Site-Directed Molecular Medicine Nucleic Acid Conformation cystic fibrosis (CF)oxadiazole HeLa Cells

description

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.

year	journal	country	edition	language
2014-02-04

10.1021/mp400230s http://hdl.handle.net/10447/88508