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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.


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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).

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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.

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Correspondence to Christian Heinis or Greg Winter.

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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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