Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Guanghui Zong,Mugunthan Govindarajan,Peristera Roboti,Mark J Henderson,Katherine Corfield,Jason M Rohde,Ludivine Baron,Sarah O’Keefe,Xianwei Sun,Stephen High,Jianhong Zhou,Nicolas Blanchard,James Inglese,Dale Tranter,Caroline Demangel,Zhijian Hu,Michael J Iannotti,Ville O Paavilainen,Belinda S Hall ,Rachel E Simmonds ,Yong Du ,Anja Paatero ,Wei Shi

doi:10.1021/jacs.8b13506

Abstract

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

Highlights

Because of significant activity loss after reducing one of the two double bonds in the α,βunsaturated esters to a single bond,[18] we hypothesized that the cinnamate and/or the tiglate (Figure 1) may enable irreversible binding between ipomoeassin F and its biological target(s)
We conclude that the pulldown of Sec61α with probe 7 represents a successful biotin affinity enrichment of a reversible ligand−membrane protein complex formed in live cells, most likely reflecting a slow disassociation rate of ipomoeassin F from Sec61α
The binding of ipomoeassin F to cellular Sec61α is supported by the endoplasmic reticulum (ER) staining seen with a fluorescent version of the compound, the highly selective pulldown of Sec61α from ER-derived microsomes, and the ability of ipomoeassin F to compete with cotransin for Sec61α binding

Summary

■ INTRODUCTION

Natural products have significantly contributed to the development of drugs for human disorders,[1] most notably as Received: December 31, 2018 Published: May 6, 2019. Our cytotoxicity data show that probe 7 closely resembles ipomoeassin F, and we reasoned that if a stable covalent complex was formed between 7 and Sec61α, the later addition of ipomoeassin F would not significantly decrease the signal of Sec61α in an affinity pulldown When this experiment was carried out, we observed a time-dependent decrease in the probe 7-dependent recovery of Sec61α in the presence of excess ipomoeassin F, such that after 60 min, the signal was substantially reduced (Figure 13A and B, cf lanes 2 and 4; see Supporting Information, Figure S10, for complete gel images). On the basis of these results, we conclude that the binding of ipomoeassin F to Sec61α is strong yet apparently reversible, suggesting that it does not involve a stable covalent interaction

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES