X CONVENTION OF INVESTIGATORS IN CYSTIC FIBROSIS.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, despite the results obtained "in vitro" and "in vivo" as well the clinical trials done with PTC124, some caveats exist. In particular Ataluren showed a lower activity against UAA and UAG than UGA nonsense mutations and also there is no general consensus about its mechanism of action. Hypothesis and Objectives Our proposal is aimed to design and synthesize new small molecules, structurally related to PTC124 and to establish a new cell based approach to evaluate their ability to promote translational read-through of PTCs present in a reporter plasmid generated by us and transfected in human cells. Preliminary results Twelve derivatives were designed and synthesized and tested in HeLa cells transfected with a plasmid harbouring an in-frame PTC (TGA). To this aim we generate a reporter vector with non-sense mutation by introducing in the pBOS-H2BGFP plasmid a TGA codon (opal) by site-directed mutagenesis. PCR and sequencing analyses confirmed the presence of the TGA codon in the plasmid that was transfected in HeLa cells to explore the ability of the derivatives to promote the translational read-through. Treatments with PTC124 of HeLa transfected cells confirmed the correct functioning of the model system. Moreover, HeLa cells transfected with a reporter vector encoding the mutated FLuc (opal mutation) when treated with the derivatives showed a positive response. Spin-off for research and clinical purpose Since there is not a general consensus on the mechanism of action of the PTC124 (protein stabilization or PTCs read-through) development of molecules capable of promoting the read-through of all PTCs without adverse effects, as Ataluren is suggested to do for the TGA codon, would be helpful as therapeutic strategy for other nonsense mutations found in CF