(331d) Protease-Resistant Peptide Ligands From a Stable Kalata B1 Scaffold Library

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents.  Given its remarkable stability to gastrointestinal digestive proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli.  A library exceeding 10⁹ variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for two distinct targets: human thrombin and human neuropilin-1.  Thrombin is an enzyme involved in the formation of blood clots and thrombin inhibitors are useful for treating numerous conditions.  Neuropilin-1 is a coreceptor for VEGF and ligands binding to this protein could be used to improve both cancer detection and treatment.

     Refolded thrombin binders were extensively characterized and exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity.  Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties.  In addition, an identified knottin-based thrombin inhibitor mediated 20-fold enhanced affinity, when compared to the same seven residue binding epitope constrained by a single disulfide bond.  We are currently engineering the scaffold to improve the folding of kalata B1 peptides produced in bacteria in addition to affinity maturing the first-generation ligands.  Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield ligands that retain the protease resistance associated with the parent peptide.  More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.