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[automatically translated] Specific objectives: The nonsense mutations (mutations STOP), a common genetic defect in individuals with cystic fibrosis (CF), causing the synthesis of CFTR protein truncated and non-functioning that are associated with a more severe phenotype of the CF (McKone EF. et al., Chest 2006). The aim of our study was to draw derivatives dell'Ataluren (PTC124), a 'small molecule' that has been attributed readthrough activities, and evaluate its activities on three different experimental model systems containing premature STOP codons (UGA , UAG, UAA). Materials and methods: 24 were synthesized molecules derived from PTC124 and analyzed by spectroscopic techniques to assess their molecular structure and their degree of purity. To assess the ability readthrough, was used (1) a reporter vector (FLuc190) in which is present the UGA mutation in the coding portion Fluc and (2) the PBOS-H2BGFP plasmid in which, by means of site directed mutagenesis, they were alternately introduced into the portion encoding the GFP protein the three different types of stop codons (UGA, UAG, UAA). The plasmid DNA was purified and isolated from the bacteria by 'Colony PCR', subjected to selective-PCR and sequenced. HeLa cells were then transfected with vectors containing the STOP mutation also were also used IB3.1 cell (FC) to evaluate the re-expression of the CFTR protein. Both cell types were treated with different derivatives of PTC124 whose activity was verified by direct observation by fluorescence microscopy, enzymatic assays and indirect immunofluorescence. Results: The functional screening which makes use of the vector in which is cloned the gene of the luciferase contains the stop mutation UGA (opal), on the PTC124 derivative 24 has allowed the identification of three molecules (derived: # 1, # 3 and # 5) showing a readthrough activity higher than PTC124. That answer is also evident in the 'recoding' H2BGFP expression of protein in HeLa cells expressing the reporter vector PBOS-H2BGFP-opal, whose presence has been highlighted in vivo / in vitro and using indirect immunofluorescence analysis. As for the other two stop codons, UAG, UAA (amber and ocher), our results demonstrate that one of the molecules (# 3) had given a good result on the mutation UGA (opal) seems to also have a good effect in the recovery of H2BGFP protein in HeLa cells H2BGFP-amber (UAG). On the contrary in HeLa cells H2BGFP-ocher (UAA), none of the molecules tested gave a positive result. Experiments conducted on IB3.1 cells (F508 / W1282X) showed at 24 hours after treatment with daughter # 5 the increase in the amount of CFTR protein probably attributable to readthrough of nonsense mutation. This result is even more evident after 10 days of continued treatment in which the derivative # 5 was added every 24 hours. Conclusions: The research identified three molecules, among the 24 we designed and synthesized, able to induce readthrough of premature stop mutations, in particular the mutation UGA (opal). Furthermore, contrary to the aminoglycosides, the treatments with the derivatives of PTC124 not toxic at the cellular level, the fact that index such molecules may find better application in substitution therapy with aminoglycoside antibiotics.
Original languageItalian
Number of pages1
Publication statusPublished - 2013

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