Restoration of Cellular Homeostasis by Vx-445 Under ER Stress Conditions: Insights from Biochemical and Proteomic Assays _ Expression Proteomics_2

Endoplasmic reticulum (ER) stress is a patho-physiological condition that plays a central role in the development of various human diseases. Multiple intracellular and extracellular factors can impair protein folding, leading to protein misfolding diseases (PMDs). Cystic fibrosis (CF) is a notable example, whose pharmacological management relies on combinations of modulators—such as the corrector Vx-445, that targets the defect in the cystic fibrosis transmembrane conductance regulator (CFTR). In vitro binding assays so far are unable to demonstrate a direct interaction between the corrector and CFTR in the cellular environment, leaving its precise mechanism of action unresolved. Thus, this study aims to investigate the role of Vx-445 in neuronal cells, following the induction of ER stress with Thapsigargin, a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), by a multi-disciplinary approach involving biochemical assays, label-free and functional proteomics. Our findings revealed that Vx-445 may participate in the regulation of oxidative stress, as evidenced by its ability to reduce intracellular ROS levels. Additionally, it influences calcium homeostasis, as it was able to restore this system to its basal state and it seems to play a role in modulating apoptosis, as indicated by its capacity to lower cytochrome c release. These results were supported by both expression proteomics and DARTS (Drug Affinity Responsive Target Stability) analyses, aimed at identifying the main cellular pathways restored by Vx-445 and potential protein interaction partners. Overall, the findings could improve our understanding of the cellular targets of CFTR modulators and offer new opportunities for drug repurposing strategies.
This dataset comprises Thapsigargin raw data of the expression proteomics experiments.