Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.
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.
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università degli Studi di Palermo , Viale delle Scienze Ed. 17, 90128 Palermo, Italy.
Laura
L
Lentini
Raffaella
R
Melfi
Aldo
A
Di Leonardo
Angelo
A
Spinello
Giampaolo
G
Barone
Andrea
A
Pace
Antonio
A
Palumbo Piccionello
Ivana
I
Pibiri
D015153:Blotting, Western; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics / Q000378:metabolism*; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010069:Oxadiazoles / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
C505032:CFTR protein, human; D018389:Codon, Nonsense; D018388:Codon, Terminator; D010069:Oxadiazoles; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; C515878:ataluren
Duchenne muscular distrophy (DMD); ataluren; cystic fibrosis (CF); green fluorescent protein (GFP); molecular dynamics (MD); nonsense mutation readthrough; oxadiazoles; premature termination codons (PTC)