Optimization of a new lead promoting the readthrough of the nonsense mutations for CFTR rescue in human CF cells

Optimization of a new lead promoting the readthrough of the nonsense mutations for CFTR rescue in human CF cells Laura Lentini, Raffaella Melfi, Sara Baldassano, Marco Tutone, Aldo Di Leonardo, Andrea Pace, Ivana Pibiri Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo Background and rationale Cystic Fibrosis patients with nonsense mutations in the CFTR gene have a more severe form of the disease. Nonsense mutations represent about 10% of the mutations that affect the CFTR gene and they are frequently associated to the classical F508 mutation (1). A potential treatment for this genetic alteration is to promote the translational readthrough of premature termination codons (PTCs) by Translational Read-Through-Inducing Drugs (TRIDs) (2-4). Hypothesis and objectives Our objective is to evaluate the functionality of the CFTR channel after treatment with a new molecule that we individuated in a precedent FFC project, and the activity of new lead molecules in cells stably expressing a nonsense-CFTR-mRNA (ns CFTR) in CF cellular model systems. We want also to study the supramolecular interactions among TRIDs, CFTR mRNA and the ribosomal A-site to identify the biological target and the mechanism of action. Essential methods QSAR, carried out on the basis of our preliminary results, will allow to achieve lead optimization and synthesize then a small library of analogs to be tested and compared to the Lead. We will mutagenize the CFTR cDNA by introducing the most diffuse nonsense mutations. Subsequently, FRT cells engineered with the vector expressing mutagenized nsCFTR, and nonsense-CF-human broncoepithelial cells will be grown in the air-liquid culture system to reproduce in vitro the epithelial organization. CFTR expression after treatments with our molecule will be evaluated by biomolecular techniques. CFTR activity will be revealed by specific CFTR-functionality assays. Finally, in vitro-in vivo (Zebrafish model) analyses of the safety profile for the set of synthesized molecules will complete the study. Preliminary results We screened the activity of several molecules synthetized by us in a precedent FFC project, identifying some molecules that showed high readthrough activity associated to the expression of the CFTR protein in ns CF immortalized cells. Expected final results and their significance We are confident that our findings will provide the validation of molecules with readthrough activity for the recovery of the CFTR function. Moreover, our pre-clinical study will assess the presence of toxic effects caused by the molecules in vivo. References 1. Sermet-Gaudelus I, Boeck KD, Casimir GJ, Vermeulen F, Leal T, Mogenet A, Roussel D, Fritsch J, Hanssens L, Hirawat S, Miller NL, Constantine S, Reha A, Ajayi T, Elfring GL, Miller LL. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis., Am J RespirCrit Care Med. 2010 Nov 15;182(10):1262-72. 2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I. Towards a rationale for the PTC124 (Ataluren) promoted read-through of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol. Pharm. 2014 11, 653-664. 3. Pibiri I, Lentini L, Melfi R, Gallucci G, Pace A, Spinello A, Barone G, Di Leonardo A. Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives European Journal of Medicinal Chemistry 06/2015; 101. 4. Nagel-Wolfrum K, Möller F, Penner I, Baasov T4, Wolfrum U. Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs), BioDrugs. 2016 Apr;30(2):49-74.