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 ( 1 ), an anticancer natural product, inhibits mitochondrial F o F 1 -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 1 in cancer cells. Comprehensive analyses revealed that 1 binds to the cytoplasmic N-terminal region of CD9 with micromolar affinity, which accelerates its cellular entry, inducing CD9 depletion via lysosomal proteolysis. Both 1 and siCD9 inhibit cancer cell migration, suggesting that the 1 –CD9 interaction plays a role in the mechanism of action of 1 . Thus, 1 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 1 as a promising molecular platform for targeting CD9-mediated cellular pathways.