Modification-free and Site-specific Global Profiling of Cinnamaldehyde Targets in Living Cells

The Modification-free Site-specific Identification of Cinnamaldehyde Targeting (MOSCAT) strategy has been developed to enable the global profiling of cinnamaldehyde (CA) targets in living cells at a site-specific level. Compared to classic probe-based approaches, the MOSCAT method bypasses the use of CA derivatives, allowing for the identification of genuine in vivo target proteins. Using the MOSCAT method, 632 CA-modification sites across 480 proteins were identified, with 70% of these sites also modified by other post-translational modifications, particularly N-nitrosylation and N-sulfenylation, consistent with CA’s anti-inflammatory effects. Pathways enriched in this study are associated with neurodegeneration and microbial infections, suggesting CA’s potential as a multi-target-directed ligand in polypharmacology. Furthermore, 60% of the proteins targeted by CA have yet to be explored in DrugBank, providing new prospects for potent therapeutic ligands. This study also reveals that the CA-targeted conserved site C93 on GPX4, a crucial member of the GPX family involved in ferroptosis, may serve as a potential allosteric target for covalent warheads.