Lysosomal accumulation of masitinib alters autophagy via pH-dependent trapping
Abdulrahman El Sayed1, Monika Kluzek2, Remigiusz Serwa3
1Laboratory of Lipids and Chronobiology, International Institute of Molecular Mechanisms and Machines (IMol), Polish Academy of Sciences, 00-783, Warsaw, Poland.
Abstract:
Small-molecule kinase inhibitors often exhibit complex cellular behaviors that cannot be explained solely by target inhibition. Masitinib is a clinically investigated tyrosine kinase inhibitor with reported anti-inflammatory and neuroprotective effects, yet its intracellular mechanism of action remains poorly defined. Here, we show that masitinib undergoes pH-dependent lysosomal sequestration that dominates its cellular activity. Across multiple cell lines, masitinib suppresses mTORC1 signaling while paradoxically inducing AKT phosphorylation through a VPS34 and rapamycin-sensitive pathway independent of class I PI3K. Thermal proteome profiling identifies lysosomal proteins as the primary off-target signature of masitinib. Using defined membrane model systems that recapitulate lysosomal lipid composition and acidity, we demonstrate that masitinib preferentially accumulates and intercalates into acidic, negatively charged membranes. This lysosomal accumulation impairs lysosomal acidification and disrupts autophagic flux, providing a mechanistic link between the physicochemical properties of masitinib and its downstream signaling effects. Together, our findings highlight lysosomal sequestration as a key determinant of kinase inhibitor behavior and underlie the importance of subcellular drug distribution in modulating cellular responses.
Insights
Masitinib, a kinase inhibitor, accumulates in lysosomes, affecting cell signaling and autophagy. This lysosomal sequestration explains its complex cellular effects beyond target inhibition.
Area of Science:
- Cellular Biology
- Pharmacology
- Biochemistry
Background:
- Small-molecule kinase inhibitors display complex cellular activities not solely explained by target inhibition.
- Masitinib, a tyrosine kinase inhibitor, has known anti-inflammatory and neuroprotective effects, but its intracellular mechanism is unclear.
Purpose of the Study:
- To elucidate the intracellular mechanism of action of masitinib.
- To investigate the role of lysosomal sequestration in masitinib's cellular effects.
- To link masitinib's physicochemical properties to its downstream signaling.
Main Methods:
- Cellular assays across multiple cell lines.
- Thermal proteome profiling.
- In vitro membrane model systems mimicking lysosomal conditions.
Main Results:
- Masitinib exhibits pH-dependent lysosomal sequestration, dominating its cellular activity.
- Masitinib suppresses mTORC1 signaling and induces AKT phosphorylation via a VPS34-dependent pathway.
- Lysosomal protein interactions were identified as masitinib's primary off-target signature.
- Masitinib accumulates in acidic membranes, impairs lysosomal acidification, and disrupts autophagic flux.
Conclusions:
- Lysosomal sequestration is a critical determinant of masitinib's cellular behavior and signaling outcomes.
- Subcellular drug distribution significantly modulates cellular responses to kinase inhibitors.
- Understanding drug sequestration is vital for predicting and optimizing kinase inhibitor efficacy.
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