Abstract
Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.
Highlights
Natural products are a rich source of bioactive and specific chemical probes and serve as starting points for development of new therapeutics once their mechanism of action and cellular targets have been identified.[1,2] Coibamide A (CbA)[3] is an N-methyl-stabilized lariat depsipeptide (Figure 1) isolated from a Caldora species[4] of marine cyanobacterium collected in Panama
Obtaining sufficient quantities of CbA for detailed mechanism of action studies from field-collected material is highly challenging,[3] and we first set out to establish a total synthesis for this N-methylated peptidic macrocycle (Figure 1A) using a modification of a previously reported method[8]
We initially constructed the middle part of CbA (MeThr5−MeIle[7]: fragment 2) on (2-Cl)Trt resin by standard Fmoc-solid phase peptide synthesis, and conjugated the N-terminal four residues (Me2Val1−MeLeu[4]: fragment 1)
Summary
Natural products are a rich source of bioactive and specific chemical probes and serve as starting points for development of new therapeutics once their mechanism of action and cellular targets have been identified.[1,2] Coibamide A (CbA)[3] is an N-methyl-stabilized lariat depsipeptide (Figure 1) isolated from a Caldora species[4] of marine cyanobacterium collected in Panama. CbA inhibits expression of the integral membrane receptor, vascular endothelial growth factor receptor 2 (VEGFR-2), and its secreted ligand vascular endothelial growth factor A (VEGF-A). It induces mTOR-independent autophagy in a manner similar to apratoxin A (AprA), a previously characterized inhibitor of protein import into the early secretory pathway,[5] despite yielding different cytotoxic profiles against cell lines of the NCI-60 tumor cell line panel.[3,11] Protein secretion is a complicated multistep process[12] that begins when nascent secretory proteins are synthesized in the cytosol. Small molecule probes with a defined mechanism have allowed dissection of the basic function of the secretory pathway[13] and provided new insights into the mechanism of protein transport into the endoplasmic reticulum.[14−17] Such
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