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

Progetto: Research project


The presence in mRNA of premature stop codons (PTCs) results in protein truncation responsible for inherited (genetic) diseases. A well-known example is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF trans-membrane regulator (CFTR) gene. 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. However, despite the results obtained from "in vitro" and "in vivo" experiments as well the advanced clinical trials done with PTC124, some caveats exist. In fact PTC124 has a lower activity against ochre and amber non sense mutations. There is no general consensus about its mechanism of action and very high doses have to be administered to reach working blood concentrations. The project is aimed to design and synthesize new molecules, alternatives or complementary to PTC124, able to promote the PTCs readthrough and with better bioavailability. Heterocycles possessing the geometrical requirements to match the hydrogen bonding of the nonsense stop codon in the mRNA will be synthesized. Ability of the new molecules to promote the readthrough will be tested in cells transfected with pBOS-H2BGFP plasmid harboring PTCs and in CF epithelial cells (IB3-1). Cells will be treated with PTC124, as a control, and with different PTC124 derivatives. The effect of these compounds will be monitored by fluorescence microscopy and at molecular level by immunoblotting and Real time PCR.First year: design and synthesis of 24 new PTC124 derivatives; additional reporter vector mutagenesis, cell transfection and treatment with new synthesized molecules.Second year: synthesis of selected derivatives with improved bioavailability as suggested by biological data; treatments on transfected cells with reporter vector and their validation on IB3-1 cells.

Layman's description

-Design and synthesis of new molecules, structurally related to PTC124, with higher bioavailability and wider activity towards PTCs than PTC124.-Evaluation of their ability to promote readthrough of PTCs in human cells engineerized with plasmids harboring PTCs, and in IB3-1 CF cells.The synthesis of PTC124 derivatives will be realized by already reported synthetic protocols, the improvement of the bioavailability will be realized by introducing on the carboxylic function glycosidic units or aminoacidic residues, or charged/polar moieties. All the new synthesized compounds will be analyzed by spectroscopic techniques such as 1H-NMR, 13C-NMR, UV, IR, GC-MS, to assess their molecular structure and their purity grade.Site directed mutagenesis with the QuickChange kit (Statagene, CA, USA) will be performed using PfuTurbo DNA polymerase. The bacterial transformation will be performed as described by Sambrook (1989).Colonies will be screened first by "colony PCR", with primers internal to the GFP sequence. Positive clones will be further screened by selective PCR using primers with differing 3’ termini matching to the potential mutation site, in order to allow selective amplification of the corresponding wt and mutant target. Plasmid DNA will be purified from bacteria with NucleoSpin Plasmid miniprep kit (Macherey-Nagel) for sequencing.Cells will be transfected with wild type and mutant contructs, using lipofectamine 2000. To overcome problems due to drug response variability, already reported for different cell lines, the contructs will be transfected in SW480, HCT116, cell lines. Stably transfected cells will be selected by antibiotic (blasticidin) resistance and charaterized by PCR to verify the integrity of the reporter gene. Cells will be then treated with the PTC124 (as a control) and with PTC124 derivatives at different doses to promote the PTCs readthrough. Cells will be analyzed, by fluorescence microscopy and by Real time RT-PCR using specific primers designed to amplifiy mutated or wild type GFP. The presence of the truncated and full-length fusion protein will be revealed by western blotting against human H2B or GFP on protein extracts from treated and non-treated cells, by using antibodies which will allow to discriminate the endogenous protein (about 14 kds) and the full-lenght or truncated GFP fusion protein. The CF cell line IB3-1 will be treated with the new molecules and the expression of CFTR gene will be evaluated by Real time RT-PCR using specific primers and Western blot analysis with two different antibodies specific for the amino-terminus and carboxyl-terminus of the protein.
Data di inizio/fine effettiva1/1/11 → …