Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

The ipomoeassin family of natural resin glycosides is underexplored chemical space with potent antitumor activity revealed in the NCI-60 cell lines screen; however, its mode of action has so far remained unexplored. In this manuscript, we report our chemical proteomics and subsequent biology studies that transform our collective knowledge of the ipomoeassin glycolipids from Organic Synthesis and Medicinal Chemistry to biological mechanism and provide a step change in our understanding of its action at a cellular level. Hence, we created an ipomoeassin F-based biotin affinity probe and used it in live cells to isolate the ER membrane protein Sec61α as its presumptive molecular target. A direct interaction between Sec61α and ipomoeassin F was confirmed by cell imaging, pulldown from purified ER membranes and competition studies using a photo-crosslinking analogue of the cyclodepsipeptide cotransin, a known Sec61α inhibitor. Crucially, we then showed that ipomoeassin F binding has a profound effect on Sec61 function, using both in vitro and in vivo assays for protein translocation and protein secretion respectively. Although structurally quite distinct, the potency of ipomoeassin F is comparable to that of mycolactone, a recently identified and intensely studied inhibitor of Sec61. The ~1,000 fold increase in the ipomoeassin F resistance of two cell lines expressing mutant forms of Sec61α strongly supports our conclusion that the effect of the compound on Sec61α is the primary basis for its potent cytotoxicity. However, we also provide evidence that ipomoeassin F is mechanistically distinct from known Sec61α inhibitors, suggesting that it is a novel structural class that may offer new opportunities to explore the Sec61 protein translocation complex as a therapeutic target for drug discovery.