Design of a Cyclotide Antagonist of Neuropilin‑1 and -2 That Potently Inhibits Endothelial Cell Migration

• Published in: ACS chemical biology Vol. 8; no. 6; pp. 1147 - 1154
• Main Authors: Getz, Jennifer A, Cheneval, Olivier, Craik, David J, Daugherty, Patrick S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21-06-2013
• Subjects: Amino Acid Sequence; Animals; Cell Movement - drug effects; Endothelial Cells - cytology; Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells; Humans; Mice; Models, Molecular; Molecular Sequence Data; Neuropilin-1 - antagonists & inhibitors; Neuropilin-1 - metabolism; Neuropilin-2 - antagonists & inhibitors; Neuropilin-2 - metabolism; Peptides - chemistry; Peptides - pharmacology
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb4000585

Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using bacterial display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40–60 nM and slow dissociation rate constants (∼1 × 10–3 s–1). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 ≈ 100 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties.