Skip to main content
SpringerLink
Log in

In Vitro Selection of Thioether-Closed Macrocyclic Peptide Ligands by Means of the RaPID System

  • Protocol
  • First Online:
Peptide Macrocycles

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2371))

Abstract

The Random nonstandard Peptides Integrated Discovery (RaPID) system enables efficient screening of macrocyclic peptides with high affinities against target molecules. Random peptide libraries are prepared by in vitro translation using the Flexible In vitro Translation (FIT) system, which allows for incorporation of diverse nonproteinogenic amino acids into peptides by genetic code reprogramming. By introducing an N-chloroacetyl amino acid at the N-terminus and a Cys at the downstream, macrocyclic peptide libraries can be readily generated via posttranslational thioether formation. Here, we describe how to prepare a thioether-closed macrocyclic peptide library, and its application to the RaPID screening.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 149.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Driggers EM, Hale SP, Lee J, Terrett NK (2008) The exploration of macrocycles for drug discovery--an underexploited structural class. Nat Rev Drug Discov 7(7):608–624

    Article  CAS  Google Scholar 

  2. Beck JG, Chatterjee J, Laufer B, Kiran MU, Frank AO, Neubauer S, Ovadia O, Greenberg S, Gilon C, Hoffman A, Kessler H (2012) Intestinal permeability of cyclic peptides: common key backbone motifs identified. J Am Chem Soc 134(29):12125–12133

    Article  CAS  Google Scholar 

  3. Takahashi N, Hayano T, Suzuki M (1989) Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin. Nature 337(6206):473–475

    Article  CAS  Google Scholar 

  4. Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid FX (1989) Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337(6206):476–478

    Article  CAS  Google Scholar 

  5. Perlin DS (2011) Current perspectives on echinocandin class drugs. Future Microbiol 6(4):441–457

    Article  CAS  Google Scholar 

  6. Yamagishi Y, Shoji I, Miyagawa S, Kawakami T, Katoh T, Goto Y, Suga H (2011) 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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Goto Y, Ohta A, Sako Y, Yamagishi Y, Murakami H, Suga H (2008) Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem Biol 3(2):120–129

    Article  CAS  Google Scholar 

  9. Goto Y, Iwasaki K, Torikai K, Murakami H, Suga H (2009) Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides. Chem Commun (Camb) 23:3419–3421

    Article  Google Scholar 

  10. Yamagishi Y, Ashigai H, Goto Y, Murakami H, Suga H (2009) Ribosomal synthesis of cyclic peptides with a fluorogenic oxidative coupling reaction. Chembiochem 10(9):1469–1472

    Article  CAS  Google Scholar 

  11. Sako Y, Goto Y, Murakami H, Suga H (2008) Ribosomal synthesis of peptidase-resistant peptides closed by a nonreducible inter-side-chain bond. ACS Chem Biol 3(4):241–249

    Article  CAS  Google Scholar 

  12. Sako Y, Morimoto J, Murakami H, Suga H (2008) Ribosomal synthesis of bicyclic peptides via two orthogonal inter-side-chain reactions. J Am Chem Soc 130(23):7232–7234

    Article  CAS  Google Scholar 

  13. Takatsuji R, Shinbara K, Katoh T, Goto Y, Passioura T, Yajima R, Komatsu Y, Suga H (2019) Ribosomal synthesis of backbone-cyclic peptides compatible with in vitro display. J Am Chem Soc 141(6):2279–2287

    Article  CAS  Google Scholar 

  14. Morimoto J, Hayashi Y, Suga H (2012) Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew Chem Int Ed Engl 51:3423–3427

    Article  CAS  Google Scholar 

  15. Passioura T, Liu W, Dunkelmann D, Higuchi T, Suga H (2018) Display selection of exotic macrocyclic peptides expressed under a radically reprogrammed 23 amino acid genetic code. J Am Chem Soc 140(37):11551–11555

    Article  CAS  Google Scholar 

  16. Katoh T, Suga H (2018) Ribosomal incorporation of consecutive β-amino acids. J Am Chem Soc 140(38):12159–12167

    Article  CAS  Google Scholar 

  17. Katoh T, Sengoku T, Hirata K, Ogata K, Suga H (2020) Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing cyclic β-amino acids. Nat Chem 12(11):1081–1088

    Article  CAS  Google Scholar 

  18. Katoh T, Suga H (2020) Ribosomal elongation of cyclic γ-amino acids using a reprogrammed genetic code. J Am Chem Soc 142(11):4965–4969

    Article  CAS  Google Scholar 

  19. Kawakami T, Murakami H, Suga H (2008) Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem Biol 15:32–42

    Article  CAS  Google Scholar 

  20. Katoh T, Tajima K, Suga H (2017) Consecutive elongation of D-amino acids in translation. Cell Chem Biol 24:1–9

    Article  Google Scholar 

  21. Katoh T, Iwane Y, Suga H (2017) Logical engineering of D-arm and T-stem of tRNA that enhances D-amino acid incorporation. Nucleic Acids Res 45(22):12601–12610

    Article  CAS  Google Scholar 

  22. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H (1997) In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett 414(2):405–408

    Article  CAS  Google Scholar 

  23. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94(23):12297–12302

    Article  CAS  Google Scholar 

  24. Hayashi Y, Morimoto J, Suga H (2012) In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem Biol 7(3):607–613

    Article  CAS  Google Scholar 

  25. Matsunaga Y, Bashiruddin NK, Kitago Y, Takagi J, Suga H (2016) Allosteric inhibition of a semaphorin 4D receptor plexin B1 by a high-affinity macrocyclic peptide. Cell Chem Biol 23(11):1341–1350

    Article  CAS  Google Scholar 

  26. Murakami H, Ohta A, Ashigai H, Suga H (2006) A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 3(5):357–359

    Article  CAS  Google Scholar 

  27. Wang QS, Unrau PJ (2002) Purification of histidine-tagged T4 RNA ligase from E. coli. BioTechniques 33(6):1256–1260

    Article  CAS  Google Scholar 

  28. Pluthero FG (1993) Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res 21(20):4850–4851

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was partially supported by KAKENHI (JP20H05618 to H.S.; JP16H06444 to H.S. and Y.G.; JP17H04762, JP18H04382, JP19K22243, and JP20H02866 to Y.G.; JP18H02080 and JP18K19389 to T.K.) from the Japan Society for the Promotion of Science (JSPS), Japan Agency for Medical Research and Development (AMED), Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) under JP19am0101090 to H.S.

Author information

Author notes

  1. Takayuki Katoh and Yuki Goto contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Takayuki Katoh, Yuki Goto & Hiroaki Suga

Authors
  1. Takayuki Katoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuki Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroaki Suga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroaki Suga .

Editor information

Editors and Affiliations

  1. DEVCOM Army Research Lab, Adelphi, MD, USA

    Matthew B. Coppock

  2. DEVCOM Army Research Lab, Adelphi, MD, USA

    Alexander J. Winton

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Katoh, T., Goto, Y., Suga, H. (2022). In Vitro Selection of Thioether-Closed Macrocyclic Peptide Ligands by Means of the RaPID System. In: Coppock, M.B., Winton, A.J. (eds) Peptide Macrocycles. Methods in Molecular Biology, vol 2371. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1689-5_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1689-5_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1688-8

  • Online ISBN: 978-1-0716-1689-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation