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

Abstract

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.

References

  1. 1.

    Glickman, M. H. & Ciechanover, A. The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    CAS  Article  Google Scholar 

  2. 2.

    Hersko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  Google Scholar 

  3. 3.

    Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    CAS  Article  Google Scholar 

  4. 4.

    Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    CAS  Article  Google Scholar 

  5. 5.

    Gopinath, P., Ohayon, S., Nawatha, M. & Brik, A. Chemical and semisynthetic approaches to study and target deubiquitinases. Chem. Soc. Rev. 45, 4171–4198 (2016).

    CAS  Article  Google Scholar 

  6. 6.

    Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep. 9, 536–542 (2008).

    CAS  Article  Google Scholar 

  7. 7.

    Finley, D. Recognition and processing of ubiquitin–protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).

    CAS  Article  Google Scholar 

  8. 8.

    Reyes-Turcu, F. E. & Wilkinson, K. D. Polyubiquitin binding and disassembly by deubiquitinating enzymes. Chem. Rev. 109, 1495–1508 (2009).

    CAS  Article  Google Scholar 

  9. 9.

    Huang, X. & Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res. 26, 484–498 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Pickart, C. M. & VanDemark, A. P. Opening doors into the proteasome. Nat. Struct. Biol. 7, 999–1001 (2000).

    CAS  Article  Google Scholar 

  11. 11.

    Adams, J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 5, 417–421 (2004).

    CAS  Article  Google Scholar 

  12. 12.

    Goldberg, A. L. Development of proteasome inhibitors as research tools and cancer drugs. J. Cell Biol. 199, 583–588 (2012).

    CAS  Article  Google Scholar 

  13. 13.

    Groll, M., Berkers, C. R., Ploegh, H. L. & Ovaa, H. Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure 14, 451–456 (2006).

    CAS  Article  Google Scholar 

  14. 14.

    Richardson, P. G. et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N. Engl. J. Med. 352, 2487–2498 (2005).

    CAS  Article  Google Scholar 

  15. 15.

    Deshaies, R. J. Proteotoxic crisis, the ubiquitin–proteasome system, and cancer therapy. BMC Biol. 12, 94 (2014).

    Article  Google Scholar 

  16. 16.

    Lee, D. H. & Goldberg, A. L. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 8, 397–403 (1998).

    CAS  Article  Google Scholar 

  17. 17.

    Harrigan, J. A., Jacq, X., Martin, N. M. & Jackson, S. P. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat. Rev. Drug Discov. 17, 57–77 (2018).

    CAS  Article  Google Scholar 

  18. 18.

    Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117–120 (2004).

    CAS  Article  Google Scholar 

  19. 19.

    Nakasone, M. A. et al. Structural basis for the inhibitory effects of ubistatins in the ubiquitin–proteasome pathway. Structure 25, 1839–1855 (2017).

    CAS  Article  Google Scholar 

  20. 20.

    Ye, Y. et al. Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266–270 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Castañeda, C. A. et al. Linkage-specific conformational ensembles of non-canonical polyubiquitin chains. Phys. Chem. Chem. Phys. 18, 5771–5788 (2016).

    Article  Google Scholar 

  22. 22.

    Jongkees, Sa. K., Hipolito, C. J., Rogers, J. M. & Suga, H. Model foldamers: applications and structures of stable macrocyclic peptides identified using in vitro selection. New J. Chem. 39, 3197–3207 (2015).

    CAS  Article  Google Scholar 

  23. 23.

    Zorzi, A., Deyle, K. & Heinis, C. Cyclic peptide therapeutics: past, present and future. Curr. Opin. Chem. Biol. 38, 24–29 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    Goto, Y. et al. Reprogramming the initiation event in translation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol. 3, 120–129 (2008).

    CAS  Article  Google Scholar 

  26. 26.

    Yamagishi, Y. et al. Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem. Biol. 18, 1562–1570 (2011).

    CAS  Article  Google Scholar 

  27. 27.

    Jongkees, S. A. K. et al. Rapid discovery of potent and selective glycosidase-inhibiting de novo peptides. Cell. Chem. Biol. 24, 381–390 (2017).

    CAS  Google Scholar 

  28. 28.

    Mali, S. M., Singh, S. K., Eid, E. & Brik, A. Ubiquitin signaling: chemistry comes to the rescue. J. Am. Chem. Soc. 139, 4971–4986 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Orlowski, R. Z. The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ. 6, 303 (1999).

    CAS  Article  Google Scholar 

  30. 30.

    Maki, C. G., Huibregtse, J. M. & Howley, P. M. In vivo ubiquitination and proteasome-mediated degradation of p53. Cancer Res. 56, 2649–2654 (1996).

    CAS  PubMed  Google Scholar 

  31. 31.

    Li, B. & Dou, Q. P. Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc. Natl Acad. Sci. USA 97, 3850–3855 (2000).

    CAS  Article  Google Scholar 

  32. 32.

    Lloyd, R. V. et al. P27Kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am. J. Pathol. 154, 313–323 (1999).

    CAS  Article  Google Scholar 

  33. 33.

    Vaziri, S. A. J. et al. Inhibition of proteasome activity by bortezomib in renal cancer cells is p53 dependent and VHL independent. Anticancer Res. 29, 2961–2969 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Ajish Kumar, K. S., Haj-Yahya, M., Olschewski, D., Lashuel, H. A. & Brik, A. Highly efficient and chemoselective peptide ubiquitylation. Angew. Chem. Int. Ed. 48, 8090–8094 (2009).

    CAS  Article  Google Scholar 

  35. 35.

    Bavikar, S. N. et al. Chemical synthesis of ubiquitinated peptides with varying lengths and types of ubiquitin chains to explore the activity of deubiquitinases. Angew. Chem. Int. Ed. 51, 758–763 (2012).

    CAS  Article  Google Scholar 

  36. 36.

    Eddins, M. J., Varadan, R., Fushman, D., Pickart, C. M. & Wolberger, C. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH. J. Mol. Biol. 367, 204–211 (2007).

    CAS  Article  Google Scholar 

  37. 37.

    Varadan, R., Assfalg, M., Raasi, S., Pickart, C. & Fushman, D. Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. Mol. Cell 18, 687–698 (2005).

    CAS  Article  Google Scholar 

  38. 38.

    Mevissen, T. E. T. et al. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 154, 169–184 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Renatus, M. et al. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2. Structure 14, 1293–1302 (2006).

    CAS  Article  Google Scholar 

  40. 40.

    Stanley, M. & Virdee, S. Chemical ubiquitination for decrypting a cellular code. Biochem. J. 473, 1297–1314 (2016).

    CAS  Article  Google Scholar 

  41. 41.

    Bremm, A., Freund, S. M. V. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010).

    CAS  Article  Google Scholar 

  42. 42.

    Pagano, M. et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682–685 (1995).

    CAS  Article  Google Scholar 

  43. 43.

    Devine, T. & Dai, M.-S. Targeting the ubiquitin-mediated proteasome degradation of p53 for cancer therapy. Curr. Pharm. Des. 19, 3248–3262 (2013).

    CAS  Article  Google Scholar 

  44. 44.

    Raasi, S., Varadan, R., Fushman, D. & Pickart, C. M. Diverse polyubiquitin interaction properties of ubiquitin-associated domains. Nat. Struct. Mol. Biol. 12, 708–714 (2005).

    CAS  Article  Google Scholar 

  45. 45.

    Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

    CAS  Article  Google Scholar 

  46. 46.

    Pye, C. R. et al. Nonclassical size dependence of permeation defines bounds for passive adsorption of large drug molecules. J. Med. Chem. 60, 1665–1672 (2017).

    CAS  Article  Google Scholar 

  47. 47.

    Lü, S. & Wang, J. The resistance mechanisms of proteasome inhibitor bortezomib. Biomark. Res. 1, 13 (2013).

    Article  Google Scholar 

  48. 48.

    Roscoe, B. P., Thayer, K. M., Zeldovich, K. B., Fushman, D. & Bolon, D. N. A. Analyses of the effects of all ubiquitin point mutants on yeast growth rate. J. Mol. Biol. 425, 1363–1377 (2013).

    CAS  Article  Google Scholar 

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Acknowledgements

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).

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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.

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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). https://doi.org/10.1038/s41557-019-0278-x

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