Design of a Cyclotide Antagonist of Neuropilin‑1
and -2 That Potently Inhibits Endothelial Cell Migration
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Abstract
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<sup>–3</sup> s<sup>–1</sup>). 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 <i>in vitro</i> (EC<sub>50</sub> ≈
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