Abstract

A two-step bio-orthogonal strategy was developed to capture the targets of SKN • Six SKN-derived probes were rationally designed and applied for target identification • SQSTM1/P62 was identified as potential target of SKN by ABPP • SQSTM1/P62 mediates SKN-driven ATM degradation and chemosensitization Background Shikonin (SKN), a dihydroxy-1,4-naphthoquinone extracted from the roots of Lithospermum spp, exhibited diverse pharmacological activities, including potent antitumor effects. Our previous work demonstrated that SKN enhanced chemosensitivity by suppressing activation of the DNA damage response (DDR) pathway through autophagy-dependent degradation of the ATM kinase; however, its direct molecular targets remained unidentified. Purpose This study aimed to identify the cellular targets of SKN and elucidate the molecular mechanisms underlying its chemosentizing activity. Methods To identify SKN-associated cellular targets, we employed activity-based protein profiling (ABPP) combined with functional validation. SKN-derived probes were developed to enable chemoproteomic target identification using LC-MS/MS followed by bioinformatic analysis. Interactions between SKN and candidate proteins were validated using pull-down assays coupled with western blotting, cellular thermal shift assay (CETSA), and co-immunoprecipitation. Functional roles of identified targets were further examined using RNA interference (RNAi) and overexpression approaches. Results Six SKN-derived probes were developed with minimal modifications to support chemoproteomic target identification while maintaining the native activity of shikonin. Chemoproteomic profiling identified SQSTM1/P62, an autophagy cargo receptor, as a critical mediator of SKN-induced chemosensitization. Functional validation revealed that modulation of SQSTM1/P62 markedly affected SKN-induced ATM degradation and the associated enhancement of chemosensitivity. Conclusion This study reveals a previously unrecognized molecular