Photoaffinity Labeling Strategy Reveals Tetraspanin CD9 as a Transient Target of Anticancer Yaku’amide B

Innovative chemical strategies are needed to understand the molecular targets of natural products, especially when they engage with multiple targets via transient interactions. Yaku'amide B (<b>1</b>), an anticancer natural product, inhibits mitochondrial F_oF₁-ATP synthase with nanomolar affinity; however, this mechanism alone does not explain its unique activity. Here, we employed photoaffinity labeling (PAL), a powerful, chemical strategy, to discover that tetraspanin CD9 is a transient cell surface target of <b>1</b> in cancer cells. Comprehensive analyses revealed that <b>1</b> binds to the cytoplasmic N-terminal region of CD9 with micromolar affinity, which accelerates its cellular entry, inducing CD9 depletion via lysosomal proteolysis. Both <b>1</b> and siCD9 inhibit cancer cell migration, suggesting that the <b>1</b>-CD9 interaction plays a role in the mechanism of action of <b>1</b>. Thus, <b>1</b> represents a novel natural product that binds to and promotes the degradation of CD9. This study underscores the versatility of applying the PAL strategy for mechanistic studies and highlights <b>1</b> as a promising molecular platform for targeting CD9-mediated cellular pathways.