Novel Readthrough compounds for nonsense mutations in Cystic Fibrosis. Bridging the translational gap – STOP-CF

Premature stop codons (PTC), UAA, UAG and UGA, are responsible for 12% of genetic diseases. PTCs readthrough strategies aim to restore the synthesis of a complete and functional protein. Cystic fibrosis, which may be due to CFTR mutations linked to PTCs, is a good model to evaluate these strategies.
Thanks to the very high resolution determination of the structure of the eukaryotic 80S ribosome, we have identified a new compound, named URN-1, effective on the 3 PTCs, and demonstrating a restoration of CFTR activity superior to that obtained with the compound currently being tested in the clinic (ELX-02).
This project aims to identify compounds with higher readthrough activity than URN-1 through 4 main objectives.
(1) search for more powerful compounds using a unique structure-function approach based on the co-crystallization of the molecule to be tested with the translation module (80s ribosome/messenger RNA/transfer RNA/elongation factor eEF2). The most interesting compounds will undergo a first selection step in a heterologous expression system expressing the main CFTR mutations linked to PTCs.
(2) evaluation of the bioactivity of the best URNs compounds on the readthrough of CFTR mutations linked to PTCs in respiratory cell lines and then in primary respiratory and intestinal cultures of patients. This will be based on change in transcript levels, expression and activity of the CFTR protein. The combination with CFTR modulators and inhibitors of the NMD system will maximize the functional restoration of CFTR. This step will determine the most effective and least toxic compound.
(3) characterization of the mechanism of action of the best compound by evaluating its impact on translational reprogramming, in particular on the readthrough of terminal stop codons, and by identifying the amino acids whose incorporation it promotes.
(4) characterization of the effect of the best compound on CFTR readthrough using the mouse model carrying the G542X CFTR mutation. The restoration of CFTR activity will be evaluated in vivo in the nasal mucosa and ex vivo in the intestinal mucosa after intranasal topical administration or short- and long-term systemic administration.

The originality of the project is based on the identification of a completely new family of readthrough inducers of the 3 PTCs and their optimization by drug design on the 80s ribosome structure. If this approach proves effective, it will be a major breakthrough, as there is currently no cure for most hereditary diseases caused by PTCs and some cancers.