Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Identification of highly selective covalent inhibitors by phage display

Nature Biotechnology volume 39, pages 490–498 (2021)Cite this article

Subjects

Abstract

Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic ‘warhead’ to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The phage display approach to screen for selective covalent inhibitors.
Fig. 2: Confirmation of the selectivity and reactivity of the cyclization reactions and establishment of washing conditions for the removal of non-covalently bound phage.
Fig. 3: Identification and optimization of covalent cyclic peptide inhibitors of TEV protease and FphF hydrolase.
Fig. 4: TEV13 and FphF16 are potent and specific covalent inhibitors.
Fig. 5: The fluorescent cpABP Cy5–TEV13 specifically labels TEV protease in complex proteomic samples.
Fig. 6: MD simulations to predict the interactions between TEV13 and TEV protease.

Similar content being viewed by others

Data availability

All data presented in this manuscript are available from the corresponding author upon reasonable request. The TEV protease-expressing plasmid sequence is available at GenBank with accession number MN480436. Characterization data for cyclization linker and probes are available in the Supplementary Information. Source data are provided with this paper.

References

  1. Singh, J., Petter, R. C., Baillie, T. A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10, 307–317 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Ghosh, A. K., Samanta, I., Mondal, A. & Liu, W. R. Covalent inhibition in drug discovery. ChemMedChem 14, 889–906 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cravatt, B. F., Wright, A. T. & Kozarich, J. W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Sanman, L. E. & Bogyo, M. Activity-based profiling of proteases. Annu. Rev. Biochem. 83, 249–273 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Cohen, M. S., Zhang, C., Shokat, K. M. & Taunton, J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 308, 1318–1321 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985).

    Article  CAS  PubMed  Google Scholar 

  7. Matthews, D. J. & Wells, J. A. Substrate phage: selection of protease substrates by monovalent phage display. Science 260, 1113–1117 (1993).

    Article  CAS  PubMed  Google Scholar 

  8. Heinis, C., Rutherford, T., Freund, S. & Winter, G. Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Nat. Chem. Biol. 5, 502–507 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Chen, S., Bertoldo, D., Angelini, A., Pojer, F. & Heinis, C. Peptide ligands stabilized by small molecules. Angew. Chem. Int. Ed. Engl. 53, 1602–1606 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Jafari, M. R. et al. Discovery of light-responsive ligands through screening of a light-responsive genetically encoded library. ACS Chem. Biol. 9, 443–450 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Ng, S. & Derda, R. Phage-displayed macrocyclic glycopeptide libraries. Org. Biomol. Chem. 14, 5539–5545 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. Cardote, T. A. & Ciulli, A. Cyclic and macrocyclic peptides as chemical tools to recognise protein surfaces and probe protein–protein interactions. ChemMedChem 11, 787–794 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Cesaratto, F., Burrone, O. R. & Petris, G. Tobacco etch virus protease: a shortcut across biotechnologies. J. Biotechnol. 231, 239–249 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Lentz, C. S. et al. Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP). Nat. Chem. Biol. 14, 609–617 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Assem, N., Ferreira, D. J., Wolan, D. W. & Dawson, P. E. Acetone-linked peptides: a convergent approach for peptide macrocyclization and labeling. Angew. Chem. Int. Ed. Engl. 54, 8665–8668 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Larsen, D. et al. Exceptionally rapid oxime and hydrazone formation promoted by catalytic amine buffers with low toxicity. Chem. Sci. 9, 5252–5259 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Palmer, J. T., Rasnick, D., Klaus, J. L. & Bromme, D. Vinyl sulfones as mechanism-based cysteine protease inhibitors. J. Med. Chem. 38, 3193–3196 (1995).

    Article  CAS  PubMed  Google Scholar 

  18. Oleksyszyn, J., Boduszek, B., Kam, C. M. & Powers, J. C. Novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases. J. Med. Chem. 37, 226–231 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Timmerman, P., Beld, J., Puijk, W. C. & Meloen, R. H. Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces. Chembiochem 6, 821–824 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Kather, I., Bippes, C. A. & Schmid, F. X. A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution. J. Mol. Biol. 354, 666–678 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Chen, S., Morales-Sanfrutos, J., Angelini, A., Cutting, B. & Heinis, C. Structurally diverse cyclisation linkers impose different backbone conformations in bicyclic peptides. Chembiochem 13, 1032–1038 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Hornsby, M. et al. A high through-put platform for recombinant antibodies to folded proteins. Mol. Cell. Proteom. 14, 2833–2847 (2015).

    Article  CAS  Google Scholar 

  23. Raran-Kurussi, S., Cherry, S., Zhang, D. & Waugh, D. S. Removal of affinity tags with TEV protease. Methods Mol. Biol. 1586, 221–230 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Rebollo, I. R., Angelini, A. & Heinis, C. Phage display libraries of differently sized bicyclic peptides. MedChemComm 4, 145–150 (2013).

    Article  CAS  Google Scholar 

  25. Nazif, T. & Bogyo, M. Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors. Proc. Natl Acad. Sci. USA 98, 2967–2972 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Albrow, V. E. et al. Development of small molecule inhibitors and probes of human SUMO deconjugating proteases. Chem. Biol. 18, 722–732 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hemelaar, J. et al. Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol. Cell Biol. 24, 84–95 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Choe, Y. et al. Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J. Biol. Chem. 281, 12824–12832 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Bromme, D., Klaus, J. L., Okamoto, K., Rasnick, D. & Palmer, J. T. Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L. Biochem. J. 315, 85–89 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sanman, L. E., van der Linden, W. A., Verdoes, M. & Bogyo, M. Bifunctional probes of cathepsin protease activity and pH reveal alterations in endolysosomal pH during bacterial infection. Cell Chem. Biol. 23, 793–804 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Verdoes, M. et al. Improved quenched fluorescent probe for imaging of cysteine cathepsin activity. J. Am. Chem. Soc. 135, 14726–14730 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Salomon-Ferrer, R., Case, D. A. & Walker, R. C. An overview of the Amber biomolecular simulation package. Wiley Interdiscip. Rev. Comput. Mol. Sci. 3, 198–210 (2013).

    Article  CAS  Google Scholar 

  33. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, J., Wang, W., Kollman, P. A. & Case, D. A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graph. Model. 25, 247–260 (2006).

    Article  PubMed  Google Scholar 

  35. Phan, J. et al. Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277, 50564–50572 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Chen, L., Keller, L. J., Cordasco, E., Bogyo, M. & Lentz, C. S. Fluorescent triazole urea activity-based probes for the single-cell phenotypic characterization of Staphylococcus aureus. Angew. Chem. Int. Ed. Engl. 58, 5643–5647 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hacker, S. M. et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat. Chem. 9, 1181–1190 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kapust, R. B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Shen, A. et al. Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS ONE 4, e8119 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Fellner, M. et al. Structural basis for the inhibitor and substrate specificity of the unique Fph serine hydrolases of Staphylococcus aureus. ACS Infect. Dis. 6, 2771–2782 (2020).

    Article  CAS  PubMed  Google Scholar 

  42. Bellotto, S., Chen, S., Rentero Rebollo, I., Wegner, H. A. & Heinis, C. Phage selection of photoswitchable peptide ligands. J. Am. Chem. Soc. 136, 5880–5883 (2014).

    Article  CAS  PubMed  Google Scholar 

  43. Chen, S. et al. Improving binding affinity and stability of peptide ligands by substituting glycines with d-amino acids. Chembiochem 14, 1316–1322 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Mikolajczyk, J. et al. Small ubiquitin-related modifier (SUMO)-specific proteases: profiling the specificities and activities of human SENPs. J. Biol. Chem. 282, 26217–26224 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Li, H. et al. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature 530, 233–236 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Chen, S. et al. Bicyclic peptide ligands pulled out of cysteine-rich peptide libraries. J. Am. Chem. Soc. 135, 6562–6569 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Lentz, C. S. et al. Design of selective substrates and activity-based probes for hydrolase important for pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect. Dis. 2, 807–815 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, S. et al. Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides. Nat. Chem. 6, 1009–1016 (2014).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University for providing technical assistance with mass spectrometry. We also thank D. Waugh (National Cancer Institute) for providing the TEV expression construct, pDZ2087, and C. Heinis (EPFL) for providing the phage library. This work was supported by Swiss National Science Foundation Postdoc Mobility fellowship P2ELP3_155323 P300PB_164725 (to S.C.) and by funding from National Institutes of Health grants R01 EB026285 and R01 EB026285 02S1 (to M.B.).

Author information

Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Shiyu Chen, Scott Lovell, Sumin Lee & Matthew Bogyo

  2. Biochemistry Department, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand

    Matthias Fellner & Peter D. Mace

  3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Matthew Bogyo

Authors
  1. Shiyu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Scott Lovell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sumin Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthias Fellner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter D. Mace
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matthew Bogyo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.B. and S.C. conceived the project and designed the experiments. S.C., S. Lovell, S. Lee and M.F. performed the experiments and analyzed the data. M.B. and S.C. wrote the manuscript with input from all authors. M.B. and P.D.M. obtained funding for the work.

Corresponding author

Correspondence to Matthew Bogyo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Strategy for the synthesis of DCA-VS, derivatizing a vinyl sulfone cysteine reactive warhead with 1,3-Dichloroacetone.

The cysteine protease reactive vinyl sulfone warhead was efficiently conjugated to the 1,2-dichloroacetone linker through oxime-ketone reaction.

Extended Data Fig. 2 Strategy for the synthesis of DCA-DPP, derivatizing a diphenylphosphonate serine reactive warhead with 1,3-Dichloroacetone.

a, Acetic Anhydride, paraformaldehyde, triphenyl phosphite, acetic acid, 5 h, 120 °C, 46 % b, i. HBr in acetic acid ii. (Boc-aminooxy)acetic acid, DCC, DIPEA, DMF, RT, O/N, 67 % c, i. TFA in DCM, RT, 1 h ii. Dichloroacetone, DMF, RT, O/N, 72 %.

Extended Data Fig. 3 Strategy for synthesis of the Fmoc-Gln-vinyl sulfone warhead.

The carboxyl side chain of P1 glutamine was deprotected and reacted with chlorotrityl resin. The derived resin can be used for directly synthesizing linear ABPs bearing a glutamine-VS motif at the C-terminus.

Supplementary information

Supplementary Information

Supplementary Figs. 1–14.

Reporting Summary

Supplementary Video 1

Molecular dynamic simulation of TEV13 complex with TEV protease.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 5

Unprocessed gels.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, S., Lovell, S., Lee, S. et al. Identification of highly selective covalent inhibitors by phage display. Nat Biotechnol 39, 490–498 (2021). https://doi.org/10.1038/s41587-020-0733-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-020-0733-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research