Neuropilin-1 (Nrp1) is an essential cell surface receptor for vascular endothelial growth factor-A (VEGF-A) and together these proteins control developmental and pathological angiogenesis. VEGF-A signaling via Nrp1 plays a critical role in tumorigenesis and is implicated in cancer growth and metastasis. Therefore, blocking VEGF-A binding to Nrp1 represents an important therapeutic target. A number of distinct ligand-mimetic peptides that compete with VEGF-A for Nrp1 have been reported. Biochemical and structural approaches have demonstrated that all Nrp1 ligands require a C-terminal arginine for binding, a sequence feature conserved in all known peptide inhibitors. However, it is unknown whether peptide sequence variation at other positions can modulate inhibitory potency. We hypothesized that sequence variation upstream of the C-terminal arginine could enhance Nrp1 binding and therefore improve the anti-angiogenic efficacy of these inhibitory molecules. We studied the Nrp1 binding and inhibitory potency of a peptide derived from the endogenous Nrp1 inhibitor Sema3F. Amino acids with unique physiochemical properties were substituted into this sequence and these peptides were tested for their ability to inhibit VEGF-A binding to Nrp1. These substitutions resulted in peptide potencies that varied over a 6-fold range. Isothermal titration calorimetry (ITC) analysis reveals the thermodynamic basis for the observed differences in Nrp1 binding.  The physiological significance of these findings was explored in the context of a peptide library that mimicked the natural sequence variation present for Nrp1 ligands. Surprisingly, this library of peptides varied in potency over a 26-fold range providing insight into endogenous ligand binding potency and specificity. All together these data define the sequence dependence of inhibitory peptides of Nrp1, demonstrating that residues upstream of the C-terminal arginine critically modulate peptide inhibitory potency.Supported by NIH Grant GM094155.

C-terminal sequence variation regulates neuropilin ligand binding and inhibition

246th National Meeting (2013)

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