Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel expressed at the apical membrane of epithelial cells. High-throughput screens (HTS) identified several novel molecules potentially targeting the underlying CFTR defect but only for some patients: potentiator VX-770 (Ivacaftor/Kalydeco), for subjects bearing G551D and other gating mutations, the combination corrector/potentiator VX-809 (Lumacaftor)/VX-770 (Orkambi) for F508del-homozygous patients and another similar combination VX-661 (Tezacaftor)/VX-770 is under approval. The main objective of this PhD work was to study new compounds that correct the basic CF defect, by rescuing CFTR protein traffic and function, focusing both on individual responses of CF patients with different CFTR mutations to these new drugs, and their mechanism of action. Chapter 1 focusses on the measurement of functional responses on human bronchial epithelial cells (HBE’s) derived from CF lung explants bearing different CFTR mutations to VX-809 namely: A561E, N1303K, G542X and Y1092X. Our data showed a positive response of A561E/A561E to VX-809 and F508del/Y1092X but not F508del/G542X. In Chapter 2, we evaluated the efficacy of CFTR modulators (correctors/potentiators) in physiologically relevant tissues, namely rectal biopsies, intestinal organoids, (HBE’s) and human nasal epithelial cells (HNE’s), from CF patients with rare CFTR mutations. Data obtained here showed that neither R560S nor H1079P-could not be rescued by any of the CFTR modulators, but 3849+10kbC>T and R334W and c.120del23-CFTR were rescued by VX-770 alone or with VX-809. In Chapter 3 we evaluated the efficacy of two novel CFTR correctors (B9, E12) in primary HBE cells, and three novel compounds E-act mimics (C2, C5, and C7) as enhancers of alternative Cl- channel TMEM16A in human intestinal organoids. In Chapter 4 (final) we assessed the effect of CFTR modulators and their possible additivity with F508del-CFTR genetic revertants 4RK, R1070W, and G550E to understand the mechanism of action of small molecule correctors and another variant diacidic ER exit code DD/AA in CFBE mCherry cells expressing these varinats by Ussing chamber analysis with or without CFTR modulators. Our data show that C18 and VX-661 and low temperature (But not VX-809) rescued DD/AA to the cell surface and genetic revertants restore the channel function without any CFTR modulator. Altogether, results from this work bring new insights into how the CFTR genotype may influence CFTR function and response to CFTR modulators and how each patient should be assessed individually for the responsiveness to the CFTR modulators towards personalized therapeutics
