PTC124 derivatives as a novel approach to improve the readthrough of premature stop codons in the CFTR gene.

The presence of Premature Stop Codons (PTCs) in mRNA results in protein truncation that is responsible for inherited (genetic) diseases. Approximately 10% (worldwide) of patients affected by cystic fibrosis (CF) have nonsense mutations (UAA, UAG or UGA) in the CF trans-membrane regulator (CFTR) gene. CFTR mutations in the two genes (alleles) of a patient can be different, with one mutation being delta-F508 and the other a nonsense mutation. Pharmacological approaches aimed to rescue protein function have been proposed to directly overcome nonsense mutations. PTC124 (Ataluren) a small molecule that mimic the activity of aminoglycosides has been suggested to allow PTCs readthrough (Welch EM et al. Nature. 2007 May 3;447(7140):87-91.). However, despite the results obtained from "in vitro" and "in vivo" experiments as well the advanced clinical trials done with Ataluren, some caveats exist. In fact Ataluren has a lower activity against UAA and UAG than UGA nonsense mutations and also there is no general consensus about its mechanism of action. We think that is very important to develop drugs capable of promoting better PTCs read-through found in CF patients. Our project is aimed to design and synthesize new small molecules possessing wider activity towards PTCs than Ataluren. To this end Ataluren derivatives with the geometrical requirements to match the hydrogen bonding of the PTCs present in the mRNA will be synthesized. To evaluate these small molecules we will use human cultured cells engineerized with plasmids harboring PTCs in the H2BGFP, FLuc and RLuc reporter genes (Auld DS et al. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3585-90.). Ability of the new molecules to promote PTCs read-through will be assessed by detecting fluorescence of the H2BGFP reporter gene by microscopy and by luminometer, and at molecular level by immunoblotting and Real time RT-PCR. Validation will consist in assessing CFTR status showed by IB3-1epithelial cells after treatment with the newly synthesized small molecules. We expect to obtain newly synthesized compounds displaying, in preclinical settings, better bioavailability and better activity than Ataluren to restore expression of the full-length proteins. The project deals with innovative research and its results could open up new avenues for the understanding of the mechanism of action of these small molecules as well for the development of new drugs for PTCs caused pathology