De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

Chen, Kai-En; Guo, Qian; Cui, Yi; Kendall, Amy K.; Hill, Timothy A.; Hall, Ryan J.; Sacharz, Joanna; Norwood, Suzanne J.; Xie, Boyang; Leneva, Natalya; Yang, Zhe; Ghai, Rajesh; Stroud, David A.; Fairlie, David; Suga, Hiroaki; Jackson, Lauren P.; Teasdale, Rohan D.; Passioura, Toby; Collins, Brett M.

The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.