Bicyclic Peptide Inhibitor Reveals Large Contact Interface with a Protease Target

From a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (<i>K</i>_i = 53 nM) inhibitor of human urokinase-type plasminogen activator (uPA) and crystallized the complex. This revealed an extended structure of the peptide with both peptide loops engaging the target to form a large interaction surface of 701 Ų with multiple hydrogen bonds and complementary charge interactions, explaining the high affinity and specificity of the inhibitor. The interface resembles that between two proteins and suggests that these constrained peptides have the potential to act as small protein mimics

Bicyclic Peptide Ligands Pulled out of Cysteine-Rich Peptide Libraries

Bicyclic peptide ligands were found to have good binding affinity and target specificity. However, the method applied to generate bicyclic ligands based on phage-peptide alkylation is technically complex and limits its application to specialized laboratories. Herein, we report a method that involves a simpler and more robust procedure that additionally allows screening of structurally more diverse bicyclic peptide libraries. In brief, phage-encoded combinatorial peptide libraries of the format X_<i>m</i>CX_<i>n</i>CX_<i>o</i>CX_<i>p</i> are oxidized to connect two pairs of cysteines (C). This allows the generation of 3 × (<i>m</i> + <i>n</i> + <i>o</i> + <i>p</i>) different peptide topologies because the fourth cysteine can appear in any of the (<i>m</i> + <i>n</i> + <i>o</i> + <i>p</i>) randomized amino acid positions (X). Panning of such libraries enriched strongly peptides with four cysteines and yielded tight binders to protein targets. X-ray structure analysis revealed an important structural role of the disulfide bridges. In summary, the presented approach offers facile access to bicyclic peptide ligands with good binding affinities