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De Novo Macrocyclic Peptides for Inhibiting, Stabilising and Probing the Function of the Retromer Endosomal Trafficking Complex

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De Novo Macrocyclic Peptides for Inhibiting, Stabilising and Probing the Function of the Retromer Endosomal Trafficking Complex bioRxiv preprint doi: https://doi.org/10.1101/2020.12.03.410779; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex Kai-En Chen1, Qian Guo1, Yi Cui2, Amy K. Kendall3,4, Timothy A. Hill5, Ryan J. Hall1, Joanna Sacharz6, Suzanne J. Norwood1, Boyang Xie3,4, Natalya Leneva1,6, Zhe Yang2, Rajesh Ghai1,7, David A. Stroud6, David Fairlie5, Hiroaki Suga8, Lauren P. Jackson3,4, Rohan D. Teasdale2, Toby Passioura8,9, Brett M. Collins1* 1. The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Queensland, 4072, Australia. 2. The University of Queensland, School of Biomedical Sciences, St Lucia, Queensland, 4072, Australia. 3. Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA. 4. Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA. 5. ARC Centre of Excellence for Innovations in Peptide and Protein Science, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. 6. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, 3052 Victoria, Australia. 7. Current address: Cambridge Institute for Medical Research, Cambridge, CB2 0XY, UK. 8. Current address: CSL Ltd, Parkville, Victoria, 3052, Australia. 9. Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan. 10. Sydney Analytical, School of Life and Environmental Sciences and School of Chemistry, The University of Sydney, Camperdown, New South Wales 2050, Australia. *Corresponding author: Brett M. Collins, Tel: +61-7-3346-2043, [email protected] Running title: Cyclic peptides targeting Retromer 24/2/20 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.03.410779; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 ABSTRACT (150 words) 2 The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and 3 signalling. Mutations in Retromer cause late-onset Parkinson’s disease, while viral and bacterial 4 pathogens can hijack the complex during cellular infection. To modulate and probe its function we have 5 created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. 6 Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing 7 seQuence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction 8 with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the 9 Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, 10 suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to 11 probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools 12 for studying Retromer function. 24/2/20 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.03.410779; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 13 INTRODUCTION 14 Endosomal compartments serve as central hubs for transmembrane protein and lipid sorting, and as platforms 15 for cell signalling. Transmembrane protein cargos that arrive in the endosomal network via endocytosis or 16 anterograde trafficking are routed either for lysosomal degradation or recycling to other compartments 17 including the trans-Golgi network (TGN) and the cell surface. Endosomal trafficking thus plays a pivotal role 1-3 18 in maintaining cellular homeostasis and is controlled by a number of essential protein machineries . 19 The evolutionarily conserved Retromer complex is a 150 kDa heterotrimer composed of Vps35, Vps29 4 20 and Vps26; with two paralogues Vps26A or Vps26B in vertebrates . Retromer is a master regulator of 2, 5- 21 endosomal dynamics responsible for cargo sorting and recycling within tubulovesicular transport carriers 9 22 , and in higher eukaryotes cooperates with an array of cargo adaptors and accessory proteins to allow 2, 3, 10 23 membrane recruitment, cargo sorting and trafficking to occur . Known accessory or regulatory proteins 24 include the small GTPase Rab7, the Rab7 GTPase activating protein (GAP) TBC1 domain family member 5 25 (TBC1D5), VPS9-ankyrin-repeat protein (VARP/Ankrd27), and Fam21, a subunit of the WASP and scar 26 homology (WASH) complex. The known cargo adaptors are derived from the sorting nexin (SNX) protein 27 family and include SNX3 and SNX27. SNX3-Retromer mediated trafficking is primarily thought to mediate 11, 12 28 the trafficking of cargos from endosomes to the TGN , whereas SNX27-Retromer is important for retrieval 13-15 29 of endocytosed cargos from endosomes back to the cell surface . 30 Retromer mutation or dysregulation in humans leads to defective endosomal and lysosomal function 16-30 31 implicated in neurodegenerative disorders including Parkinson’s disease (PD) , Alzheimer’s disease (AD) 31-37 38 32 and amyotrophic lateral sclerosis (ALS) . Notably, generalised dysregulation of endosomal, lysosomal 33 and autophagic organelles is a common hallmark of neurodegenerative disorders including familial and 20, 39-44 34 sporadic AD, PD, ALS and hereditary spastic paraplegia (HSP) . Retromer dysfunction impacts 20, 45 35 endosomal and lysosomal homeostasis in neurons and microglia in multiple ways ; involving deficits in 19, 23, 27 36 regulatory protein interactions such as WASH and Leucine-rich repeat kinase 2 (LRRK2) , mis-sorting 18, 26, 28, 46-51 21, 29, 52, 53 37 of specific endosomal cargos , defects in mitochondrial function and mitophagy , and other 23, 24, 32, 34, 37, 54-57 38 widespread deficiencies in lysosomal and autophagic degradation of toxic material . 39 Retromer is also a prominent target hijacked by intracellular pathogens to facilitate their transport and 40 replication. This includes viruses such as SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis C 58-70 41 virus (HCV), and human papilloma virus (HPV) , and intracellular bacteria such as Coxiella burnetii and 71-77 42 Legionella pneumophila . Mechanistic studies have shown that the secreted effector protein RidL from L. 43 pneumophila binds directly to Vps29, competing with endogenous regulators TBC1D5 and VARP, inhibiting 44 Retromer mediated cargo transport, and supporting the growth of L. pneumophila in intracellular endosome- 71, 73, 76, 77 45 derived vacuoles . Similarly, the minor capsid protein L2 from human Papilloma virus (HPV) is 46 thought to hijack Retromer-SNX3-mediated endosomal transport by mimicking sequence motifs found in 47 endogenous cargoes such as the cation-independent mannose-6-phosphate receptor (CI-MPR) and divalent 63, 66, 78, 79 48 metal transporter 1-II (DMT1-II) . 24/2/20 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.03.410779; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 Given the importance of Retromer in endosomal trafficking, neurodegenerative disease, and cellular 50 infection, there is significant interest in developing molecular approaches to either inhibit or enhance Retromer 51 activity. Inhibition of Retromer may provide a novel avenue for targeting infectious pathogens; with peptide- 52 based inhibitors of Retromer, derived from the HPV L2 protein, able to reduce infection by HPV by slowing 66, 78, 79 53 the normal retrograde transport of incoming virions . Conversely, because of its neuroprotective role, it 54 has been proposed that a Retromer-binding ‘molecular chaperone’ could be used to enhance Retromer function 80-83 55 in diseases including AD and PD , with the goal of boosting normal endosome-dependent clearance 56 pathways to reduce accumulation of toxic aggregates of proteins such as amyloid b (Ab), tau and a-synuclein. 57 Previously a small molecule called R55 (a thiophene thiourea derivative, also called TPT-260) was identified, 58 which can bind with modest affinity to Retromer at the interface between Vps35 and Vps29 and stabilise its 84 59 structure . This molecule has since been shown to have activity in stabilising Retromer in cells, and as 60 predicted can enhance the transport of essential receptors and reduce the accumulation of toxic material 54, 84-90 61 including Ab and a-synuclein in cell, fly, and mouse models . Recently a derivative of R55 was found 38 62 to improve Retromer stability and lysosomal dysfunction in a model of ALS . Nonetheless, the low potency 63 and specificity of these compounds mean that other molecules are actively being sought. 64 We have adopted a novel screening strategy, referred to as the RaPID (Random nonstandard Peptides 65 Integrated Discovery) system, to identify a series of eight de novo macrocyclic peptides with high affinity and 66 specificity for Retromer, possessing either inhibitory or stabilising activities. These peptides bind to Retromer
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