Protease-resistant peptide ligands from a knottin scaffold library

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Knottin peptides offer inherent stability, making them promising scaffolds for therapeutic and diagnostic agents.
• The cyclic peptide kalata B1 exhibits remarkable protease resistance.
• Developing stable peptide ligands is crucial for in vivo applications.

Purpose of the Study:

• To create a large knottin library using the kalata B1 scaffold.
• To identify peptide ligands specific for human thrombin's active site.
• To evaluate the stability and affinity of the developed ligands.

Main Methods:

• Construction of a knottin library (>10^9 variants) by randomizing seven amino acids in the kalata B1 scaffold.
• Display of the library on the surface of E. coli.
• Screening using fluorescence-activated cell sorting (FACS) for thrombin binders.
• Characterization of thrombin binders' affinity, dissociation rates, and enzymatic inhibition.

Main Results:

• Identification of high-affinity (nanomolar) thrombin binders.
• Demonstration of retained protease resistance (80% intact after 2h incubation with trypsin/chymotrypsin).
• Achieved 20-fold enhanced affinity compared to disulfide-constrained epitopes.

Conclusions:

• The kalata B1 scaffold can generate high-affinity protein ligands with excellent protease resistance.
• This strategy is effective for developing stable peptide ligands for in vivo applications.
• Knottin-based inhibitors maintain the parent scaffold's stability properties.