PTC124 DERIVATIVES AS A NOVEL APPROACH TO IMPROVE THE READTHROUGH OF PREMATURE AMBER AND OCHRE STOP CODONS

Nucleotide changes within an exon may alter the trinucleotide normally encoding a particular amino acid, such that a new \u201cstop\u201d signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to the lack of production of a normal full-length protein. Such premature termination codon (PTC) mutations occur in an estimated 10% to 15% of many genetically based disorders (1). Pathological nonsense mutations resulting in TAG (40.4%), TGA (38.5%), and TAA (21.1%) occur in different proportions to naturally occurring stop codons (2). Several genetic disorders are characterized by opal (TGA; Cystic fibrosis, Duchenne/Becker muscular dystrophy), amber (TAG; \uf062-thalassemia, emphysema, cystic fibrosis) and ochre mutations (TAA; APC gastric cancer, Haemophilia B, Hypothyroidism) (3). Messenger RNA containing a nonsense mutation is often degraded rapidly through the process of nonsense-mutation-mediated decay (NMD) resulting in the lack of the protein (4). A recent approach to directly overcome the deleterious effects caused by nonsense mutations is represented by readthrough strategies which take advantage of the known properties of aminoglycosides that can suppress stop codons (5). Several aminoglycosides (gentamicin, amikacin, hygromycin, etc.) can suppress the accurate identification of translation termination codons in cultured eukaryotic cells. Unfortunately, aminoglycoside action lacks specificity resulting in readthrough of many correctly positioned stop codons. Consequently, long- term use of aminoglycosides may originate toxic aggregates or dominant negative readthrough products (6). By an high throughput screening it was identified the PTC124 (Ataluren), a small molecule that has been suggested to allow PTCs readthrough (7). However, despite the results obtained on opal mutation it was shown that it has a lower activity against ochre and amber nonsense mutations (7). In the attempt to identify molecules with an activity against ochre and amber nonsense mutations, we designed and synthesized new PTC124 derivatives to be tested in human cultured cells to see if they have higher and wider activity towards PTCs than PTC124. To this aim we generated a reporter vector with non-sense mutation by introducing in the pBOS-H2BGFP plasmid a TAG codon (amber) and TAA codon (ochre) by site-directed mutagenesis. PCR and sequencing analyses confirmed the presence of the stop codons in the plasmids that were transfected in HeLa cells to explore the ability of the derivatives to promote the translational read-through. Immunofluorescence analyses showed that one of the analyzed derivatives was able to restore GFP fluorescence in HeLa H2BGFP-amber cells, as indicated by GFP-localization in the nuclei of treated cells (figure 1). This positive response also confirmed the correct functioning of the model system which will allow us to perform the screening of a greater number of molecules with read-through action