Structure-based design of Sec61 translocon targeting prodrugs minimize off-target toxicity

Coibamide A (CbA) is a cyclic depsipeptide that inhibits the function of the Sec61 translocon and exhibits significant antitumor activity. However, its broad Sec61 inhibition results in non-selective cytotoxicity, limiting therapeutic applications. To elucidate the molecular mechanism of CbA-mediated Sec61 blockade and enable rational prodrug design, we determined the cryo-EM structure of human Sec61 bound to CbA at 3.1 Å resolution. The structure reveals that CbA adopts a distinctive lasso-like conformation and occupies the lateral gate of Sec61, a binding site shared with other Sec61 inhibitors, while forming a particularly more expansive set of interactions with the lateral gate. Guided by these structural insights, we conducted structure-activity relationship studies and developed prodrug strategies that modulate CbA's antitumor activity through the controlled perturbation of intramolecular and protein hydrogen bonding interactions. Together, these results establish a structure-guided strategy for prodrug design of CbA and demonstrate the general applicability of backbone-caging to enhance the tolerability of Sec61 inhibitors.