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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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).
The authors declare no competing interests.
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Supplementary Figs. 1–20. Supplementary methods. Supplementary Table 1. Supplementary references 1–14