Lysosomal accumulation of Masitinib alters autophagy via pH-dependent trapping.

Masitinib is an oral tyrosine kinase inhibitor developed initially for the treatment of mastocytosis. Preclinical studies suggest that masitinib can modulate mast cell activity and neuroinflammation, making it a potential therapeutic candidate for neurodegenerative diseases. However, clinical outcomes remain inconclusive, which may be linked to a lack of understanding of its molecular mechanism of action and potential off-target effects. Using a panel of cancer cell lines, we found that masitinib suppresses mTORC1 activity while simultaneously inducing AKT phosphorylation. Interestingly, thermal proteome profiling analysis revealed that lysosomal proteins were the most thermally affected by masitinib. Using large unilamellar vesicle systems that mimic the lipid composition and physicochemical environment of lysosomal membranes, we observed that masitinib preferentially accumulates in acidic vesicle membranes, consistent with a pH-dependent trapping mechanism. Furthermore, cellular assays demonstrated that masitinib disrupts autophagy by impairing lysosomal acidification. Together, these findings elucidate the lysosomal accumulation of masitinib, providing a molecular basis for its mechanism of action.