Improving the binding affinity and/or stability of peptide ligands often
requires testing of large numbers of variants to identify beneficial mutations.
Herein we propose a type of mutation that promises a high success rate. In a
bicyclic peptide inhibitor of the cancer-related protease urokinase-type
plasminogen activator (uPA), we observed a glycine residue that has a positive
ϕ dihedral angle when bound to the target. We hypothesized that replacing it
with a D-amino acid, which favors positive ϕ angles, could enhance the binding
affinity and/or proteolytic resistance. Mutation of this specific glycine to
D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold)
and stability (fourfold). X-ray-structure analysis of the inhibitors in complex
with uPA showed that the peptide backbone conformation was conserved. Analysis
of known cyclic peptide ligands showed that glycine is one of the most frequent
amino acids, and that glycines with positive ϕ angles are found in many
protein-bound peptides. These results suggest that the glycine-to-D-amino acid
mutagenesis strategy could be broadly applied.
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