De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains

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A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.

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Fig. 1: RaPID selection of K48Ubn binding cyclic peptides.
Fig. 2: Cyclic peptides bind to residues at the hydrophobic Ub–Ub interface in K48-linked di- and tetra-Ub.
Fig. 3: Cyclic peptides inhibit the DUB cleavage of K48-linked di/tetra-Ub.
Fig. 4: Ub4ix cyclic peptide inhibits 26S proteasomal activity in vitro.
Fig. 5: Uptake of Ub4ix by living cells and effect on ubiquitination.
Fig. 6: Ub4ix reduces cell viability and induces apoptosis.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.


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A.B. holds the Jordan and Irene Tark Academic Chair. Ha.S. is supported at the Technion by a Technion-Guangdong Fellowship. This work was supported by the Japan Agency for Medical Research and Development, Basic Science and Platform Technology Programme for Innovative Biological Medicine (JP18am0301001) to Hi.S., and by NIH grant GM065334 to D.F. J.M.R. was supported by Grants-in-aid for JSPS Fellows (P13766) and a joint ANR-JST grant (ANR-14-JITC-2014-003 and JST-SICORP). The authors thank A. Majumdar for help with triple-resonance NMR experiments. A.C. is supported by the Dr Miriam and Sheldon Adelson Medical Research Foundation (AMRF), the Israel Science Foundation (ISF), the German–Israeli Foundation for Research and Development (GIF) and a Professorship funded by the Israel Cancer Research Fund (ICRF).

Author information

J.M.R. utilized the cyclic peptide discovery RaPID system, carried out SPR assays and data analysis, and co-wrote the paper. M.N. assisted in the chemical synthesis of cyclic peptides, carried out in vitro and cellular assays and co-wrote the paper. I.L. carried out the confocal microscopy assay and assisted with cellular studies. S.M.B. and B.L. synthesized isotope-labelled Ub chains, conducted the NMR studies and assisted with writing the paper. S.M.M. assisted with chemical synthesis of the ubiquitin chains. G.B.V. assisted in the synthesis of cyclic peptides. Ha.S. prepared the δ-mercaptolysine used in the ubiquitin chain synthesis. B.B. assisted with the design of the in vitro proteasomal degradation assay. D.F. designed and supervised the NMR studies, carried out data analysis and assisted with writing the manuscript and the Supplementary Information. Y.H. performed additional SPR studies against K11- and K63-linked Ub chains. A.C. assisted in the design of the confocal microscopy assay and in vitro and cellular studies. Hi.S. supervised the RaPID study and assisted in the writing of the paper. A.B. designed and supervised the entire project and the writing of the paper.

Correspondence to Hiroaki Suga or Ashraf Brik.

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Supplementary Figs. 1–20. Supplementary methods. Supplementary Table 1. Supplementary references 1–14

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Nawatha, M., Rogers, J.M., Bonn, S.M. et al. De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains. Nat. Chem. 11, 644–652 (2019) doi:10.1038/s41557-019-0278-x

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