Chemical proteomics reveals sinomenine’s anti-inflammatory mechanism through serum protein covalent modification

BACKGROUND: Covalent protein modification by drugs or their reactive metabolites has emerged as an important mechanism underlying pharmacological activity. However, its contribution to the therapeutic effects of compounds derived from traditional Chinese medicine remains insufficiently characterized. Sinomenine (SIN), an active alkaloid from Sinomenium acutum, has long been utilized in managing inflammatory disorders, yet the molecular basis of its efficacy is not fully elucidated. METHODS: A discovery-driven chemical proteomics approach was deployed to investigate the role of covalent protein modification in the action of SIN. Metabolite profiling and in vitro reactivity assays were initially conducted to evaluate the modification potential of SIN and its metabolites. Subsequently, in vivo serum proteomics analysis in rat was performed to map the covalent modification landscape. Enriched biological pathways were identified through bioinformatics analyses, and functional validation was executed using western blotting and enzymatic activity assays. RESULTS: Results demonstrated that SIN, along with its oxygenated and demethylated metabolites, could covalently modify cysteine and lysine residues on proteins via three distinct modification patterns. Serum proteomics analysis identified seven proteins modified by SIN in vivo. Pathway enrichment analysis revealed that these target proteins were predominantly involved in coagulation and complement pathways. Functional validation demonstrated that SIN treatment significantly suppressed the activation of the coagulation cascade, the kallikrein-kinin system (KKS), and the complement cascade. CONCLUSIONS: This study provides evidence that covalent protein modification may contribute to the pharmacological effects of SIN by modulating coagulation and complement pathways, which are implicated in inflammatory regulation. The findings offer new mechanistic insights into SIN's action and underscore the utility of covalent proteomics as an effective strategy for uncovering the molecular mechanisms of bioactive compounds derived from traditional Chinese medicine.