Phage-encoded combinatorial chemical libraries based on bicyclic peptides


Here we describe a phage strategy for the selection of ligands based on bicyclic or linear peptides attached covalently to an organic core. We designed peptide repertoires with three reactive cysteine residues, each spaced apart by several random amino acid residues, and we fused the repertoires to the phage gene-3-protein. Conjugation with tris-(bromomethyl)benzene via the reactive cysteines generated repertoires of peptide conjugates with two peptide loops anchored to a mesitylene core. Iterative affinity selections yielded several enzyme inhibitors; after further mutagenesis and selection, we were able to chemically synthesize a lead inhibitor (PK15; Ki = 1.5 nM) specific to human plasma kallikrein that efficiently interrupted the intrinsic coagulation pathway in human plasma tested ex vivo. This approach offers a powerful means of generating and selecting bicyclic macrocycles (or if cleaved, linear derivatives thereof) as ligands poised at the interface of small-molecule drugs and biologics.

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Figure 1: Generation of phage-encoded combinatorial chemical libraries and an isolated molecule.
Figure 2: Conjugation of peptide fusions with TBMB.
Figure 3: Sequences of selected conjugates.
Figure 4: Affinity maturation of human plasma kallikrein inhibitors.
Figure 5: Inhibition of human plasma kallikrein by conjugates and NMR solution structure of conjugate PK15.


  1. 1

    Bleicher, K.H., Bohm, H.J., Muller, K. & Alanine, A.I. Hit and lead generation: beyond high-throughput screening. Nat. Rev. Drug Discov. 2, 369–378 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Hüser, J. High-Throughput Screening in Drug Discovery Vol. 35 (Wiley-VCH, Weinheim, Germany, 2006).

    Google Scholar 

  3. 3

    Huse, W.D. et al. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275–1281 (1989).

    CAS  Article  Google Scholar 

  4. 4

    Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T. & Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544–546 (1989).

    CAS  Article  Google Scholar 

  5. 5

    McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

    CAS  Article  Google Scholar 

  6. 6

    Scott, J.K. & Smith, G.P. Searching for peptide ligands with an epitope library. Science 249, 386–390 (1990).

    CAS  Article  Google Scholar 

  7. 7

    Marks, J.D., Hoogenboom, H.R., Griffiths, A.D. & Winter, G. Molecular evolution of proteins on filamentous phage. Mimicking the strategy of the immune system. J. Biol. Chem. 267, 16007–16010 (1992).

    CAS  PubMed  Google Scholar 

  8. 8

    Lipovsek, D. & Pluckthun, A. In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51–67 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Ulrich, H. DNA and RNA aptamers as modulators of protein function. Med. Chem. 1, 199–208 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Barbas, C.F, III. Synthetic human antibodies. Nat. Med. 1, 837–839 (1995).

    CAS  Article  Google Scholar 

  11. 11

    Holliger, P. & Hudson, P.J. Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Lerner, R.A. Manufacturing immunity to disease in a test tube: the magic bullet realized. Angew. Chem. Int. Edn Engl. 45, 8106–8125 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Nygren, P.A. & Skerra, A. Binding proteins from alternative scaffolds. J. Immunol. Methods 290, 3–28 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Doyon, J.B., Snyder, T.M. & Liu, D.R. Highly sensitive in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity. J. Am. Chem. Soc. 125, 12372–12373 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Melkko, S., Scheuermann, J., Dumelin, C.E. & Neri, D. Encoded self-assembling chemical libraries. Nat. Biotechnol. 22, 568–574 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Mannocci, L. et al. High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries. Proc. Natl. Acad. Sci. USA 105, 17670–17675 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Woiwode, T.F. et al. Synthetic compound libraries displayed on the surface of encoded bacteriophage. Chem. Biol. 10, 847–858 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Brenner, S. & Lerner, R.A. Encoded combinatorial chemistry. Proc. Natl. Acad. Sci. USA 89, 5381–5383 (1992).

    CAS  Article  Google Scholar 

  19. 19

    Gartner, Z.J. et al. DNA-templated organic synthesis and selection of a library of macrocycles. Science 305, 1601–1605 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Halpin, D.R. & Harbury, P.B. DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution. PLoS Biol. 2, E174 (2004).

    Article  Google Scholar 

  21. 21

    Tse, B.N., Snyder, T.M., Shen, Y. & Liu, D.R. Translation of DNA into a library of 13,000 synthetic small-molecule macrocycles suitable for in vitro selection. J. Am. Chem. Soc. 130, 15611–15626 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Kemp, D.S. & McNamara, P.E. Conformationally restricted cyclic nonapeptides derived from L-cysteine and LL-3-amino-2-piperidino-6-carboxylic acid (LL-acp), a potent b-turn-inducing dipeptide analogue. J. Org. Chem. 50, 5834–5838 (1985).

    CAS  Article  Google Scholar 

  23. 23

    Timmerman, P., Beld, J., Meloen, R.H. & Puijk, W.C. Method for selecting a candidate drug compound. WO patent 2004077062 (2004).

  24. 24

    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).

    CAS  Article  Google Scholar 

  25. 25

    Timmerman, P., Puijk, W.C., Slootstra, J.W., Van Dijk, E. & Meloen, R.H. Binding compounds, immunogenic compounds and peptidomimetics. WO patent 2006078161 (2006).

  26. 26

    Jespers, L.S.A., Winter, G.P., Bonnert, T.P. & Simon, T.M. SBP members with a chemical moiety covalently bound within the binding site. WO patent 9501438 (1995).

  27. 27

    Jespers, L., Bonnert, T.P. & Winter, G. Selection of optical biosensors from chemisynthetic antibody libraries. Protein Eng. Des. Sel. 17, 709–713 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Wessjohann, L.A., Ruijter, E., Garcia-Rivera, D. & Brandt, W. What can a chemist learn from nature's macrocycles?–a brief, conceptual view. Mol. Divers. 9, 171–186 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Driggers, E.M., Hale, S.P., Lee, J. & Terrett, N.K. The exploration of macrocycles for drug discovery–an underexploited structural class. Nat. Rev. Drug Discov. 7, 608–624 (2008).

    CAS  Article  Google Scholar 

  30. 30

    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).

    CAS  Article  Google Scholar 

  31. 31

    Cheng, Y. & Prusoff, W.H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).

    CAS  Article  Google Scholar 

  32. 32

    Abbenante, G. & Fairlie, D.P. Protease inhibitors in the clinic. Med. Chem. 1, 71–104 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Turk, B. Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov. 5, 785–799 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Millward, S.W., Fiacco, S., Austin, R.J. & Roberts, R.W. Design of cyclic peptides that bind protein surfaces with antibody-like affinity. ACS Chem. Biol. 2, 625–634 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Litovchick, A. & Szostak, J.W. Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display. Proc. Natl. Acad. Sci. USA 105, 15293–15298 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Cremlyn, R.J. An Introduction to Organosulfur Chemistry 1st edn. (Wiley, Chichester, UK, 1996).

    Google Scholar 

  37. 37

    Wood, S.P. et al. Crystal structure analysis of deamino-oxytocin: conformational flexibility and receptor binding. Science 232, 633–636 (1986).

    CAS  Article  Google Scholar 

  38. 38

    Bhaskaran, R., Chuang, L.C. & Yu, C. Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies. Biopolymers 32, 1599–1608 (1992).

    CAS  Article  Google Scholar 

  39. 39

    Pohl, E. et al. Structure of octreotide, a somatostatin analogue. Acta Crystallogr. D Biol. Crystallogr. 51, 48–59 (1995).

    CAS  Article  Google Scholar 

  40. 40

    Melacini, G., Zhu, Q. & Goodman, M. Multiconformational NMR analysis of sandostatin (octreotide): equilibrium between beta-sheet and partially helical structures. Biochemistry 36, 1233–1241 (1997).

    CAS  Article  Google Scholar 

  41. 41

    Jackson, D.Y. et al. A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues. Science 266, 243–247 (1994).

    CAS  Article  Google Scholar 

  42. 42

    Sandman, K.E. & Noren, C.J. The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide. Nucleic Acids Res. 28, 755–761 (2000).

    CAS  Article  Google Scholar 

  43. 43

    Sandman, K.E., Benner, J.S. & Noren, C.J. Phage display of selenopeptides. J. Am. Chem. Soc. 122, 960–961 (2000).

    CAS  Article  Google Scholar 

  44. 44

    Dennis, M.S. et al. Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature 404, 465–470 (2000).

    CAS  Article  Google Scholar 

  45. 45

    Huang, L. et al. Novel peptide inhibitors of angiotensin-converting enzyme 2. J. Biol. Chem. 278, 15532–15540 (2003).

    CAS  Article  Google Scholar 

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We thank P. Jones (Laboratory of Molecular Biology, Cambridge, UK) for expert laboratory advice, L. Judd (Centre for Protein Engineering, Cambridge, UK) for media preparation, A. Jaulent (Centre for Protein Engineering, Cambridge, UK) for peptide purification, and F. Begum (Laboratory of Molecular Biology, Cambridge, UK) and S.-Y. Peak-Chew (Laboratory of Molecular Biology, Cambridge, UK) for mass spectrometric analysis. We also thank I. Kather and F.X. Schmid from the University of Bayreuth for the engineered phage with disulfide-free gene-3-protein. C.H. was supported by the Swiss National Science Foundation (SNSF) and the Novartis Foundation (formerly Ciba-Geigy Jubilee Foundation).

Author information




C.H. and G.W. conceived the experiments, analyzed the data and wrote the article; C.H. performed the experiments; T.R. and S.F. solved the NMR structure.

Corresponding authors

Correspondence to Christian Heinis or Greg Winter.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 325 kb)

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

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