Design of new molecules as a therapeutic strategy for Cystic Fibrosis

The approval of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator therapy by global health agencies over the past few decades has fundamentally changed the course of patients affected by Cystic Fibrosis (CF). CFTR is a membrane protein that functions as a chloride channel in epithelial cells. Mutations in the CFTR gene lead to absent or impaired chloride transport across the apical membranes, primarily in the respiratory and glandular epithelia. This defect results in the extracellular accumulation of thick, sticky mucus, leading to chronic conditions such as sinusitis, bronchitis, pneumonia, and asthma, often refractory to standard therapy, as well as nasal polyposis, digital clubbing, and bronchiectasis. CFTR modulators are categorized, based on their mechanism of action, as correctors, which enhance CFTR folding and trafficking to the cell surface, and potentiators, which improve ion transport through CFTR channels already present on the apical membrane. The four currently approved modulators are the potentiator Ivacaftor (VX770 or IVA) and the correctors Lumacaftor (VX809 or LUM), Tezacaftor (VX661 or TEX), and Elexacaftor (VX445 or ELX). Combinations of these modulators, such as the triple combination Ivacaftor/Tezacaftor/Elexacaftor (Kaftrio®), represent the best therapeutic option available for patients with sensitive CFTR mutations, including the most common one, the phenylalanine 508 deletion (F508del-CFTR). However, current therapies do not guarantee complete CFTR functional recovery, highlighting the need for developing new molecules to fill these therapeutic gaps. This thesis reports on the development and study of novel molecules featuring an arylthiazole scaffold for their potential to restore CFTR functionality. The goal was to develop compounds that are more active than those already clinically approved, both when administered alone and in combination, aiming for a synergistic effect due to complementary mechanisms of action between them and already approved modulators. Functional channel activity assays using the Yellow Fluorescent Protein (YFP) reporter in Fisher Rat Thyroid (FRT) and CFBE41o- cells stably expressing F508del-CFTR demonstrated a significant additive/synergistic effect when the novel VX809-hybrids 2a and 3a were combined with VX445. Furthermore, eight other newly synthesized compounds (4a, 5a, 1b, 2b, 3b, 7b, and 1c), when tested individually, significantly increased F508del-CFTR activity compared to the vehicle treatment in CFBE bronchial epithelial cells. Notably, the combinations 2b + 5a and 7b + 4a resulted in 2 approximately 20% and 29% greater F508del-CFTR rescue, respectively, than the clinically used VX809 + VX445 combination. To confirm this efficacy, a functional measurement of CFTR activity using the Fluorescence Microplate Polarimetry (FMP) assay was performed in Human Nasal Epithelial (HNE) primary cells from four CF patients homozygous for the F508del-CFTR mutation. The results confirmed that the novel corrector combinations (2b + 5a and 7b + 4a) significantly increased FSK-activated and VX770-potentiated F508del-CFTR function compared to the rescue levels mediated by either the VX809 + VX445 or VX661 + VX445 combinations in HNE cells. Finally, recognizing the emerging role of molecular chaperone modulators as a strategy to enhance F508del-CFTR rescue, combining them with already approved modulators, a small library of Hsc70/Hsp70 modulators was developed based on the scaffold of MKT-077, a known allosteric Hsp70 inhibitor. Among the new library, three MKT-077 analogues, i.e. DL79, DL90, and AP161, showed an inhibitory effect on human recombinant Hsp70 ATPase activity. Significantly, DL79 demonstrated a higher ability than MKT-077 to enhance the corrective effect of VX809 on F508del-CFTR in CFBE41o- cells at a remarkably low concentration. The combination of DL79 with other clinically approved correctors revealed a significant synergistic interaction with VX661 and with the VX661/VX445 combination, though no meaningful effect was observed in combination with VX445 alone. Results reported in this thesis identified and characterized novel CFTR modulators and molecular chaperone enhancers that demonstrate superior efficacy, particularly in combination, compared to current standard-of-care therapies for F508del-CFTR. In summary, these results provide strong preclinical evidence supporting the further development of these novel arylthiazole correctors and the Hsp70 modulators as potential next-generation therapeutic agents for CF patients with the F508del-CFTR mutation.