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

Mol Pharm. 2014 Mar 3;11(3):653-64. doi: 10.1021/mp400230s. Epub 2014 Feb 7.

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.

Keywords: Duchenne muscular distrophy (DMD); ataluren; cystic fibrosis (CF); green fluorescent protein (GFP); molecular dynamics (MD); nonsense mutation readthrough; oxadiazoles; premature termination codons (PTC).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

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

Grant support

National Institutes of Health, United States