Abstract
C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.
Highlights
C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation
Aiming to gain further insights into the mechanism of action of this corrector and optimize its efficacy, we performed a 3 step study (i) we mapped the putative binding site on the 3D structures of both isolated Nucleotide Binding Domain 1 (NBD1) and the whole Membrane Spanning Domains (MSDs):NBDs assembly; (ii) we designed and synthetized novel derivatives to improve correction efficacy; (iii) we investigated the effect of the combination of the most potent derivative with VX-809
The synthesis of the designed extended c407 analog G1 was carried out according to a strategy similar to the one we developed for the synthesis of C1–C11 compounds (Fig. 4E, Supplementary information and Supplementary Fig. 7)
Summary
Recent years have unraveled amazing progress in development of CFTR correctors. little mechanistic insights are available to rationalize their mode of action. It has been shown that robust correction of F508del-CFTR defects requires a cooperative rescue involving distinct structural defects, including stabilization of NBD1, as well as the secondary effects of the mutation (NBD1–MSD1 or − MSD2 interfaces; NBD2 folding and NBD1–NBD2 interface)[4,6,16,28] This was recently confirmed by clinical studies showing that a triple combination therapy based on the association of VX-770 with 2 correctors, was able to very significantly improve the respiratory status of patients in association with CFTR restoration to more than 80% of the n ormal[14,15]. We postulate that this observation could have important consequences for therapeutic strategies aimed at modulating the dynamics of this interface and addressing corrections of mutations affecting this interface
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