Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor Exon 8 for NP-1 binding and role of NP-1 in KDR signaling

Neuropilin- 1 ( NP- 1) is a receptor for vascular endothelial growth factor- A(165)( VEGF- A(165)) in endothelial cells. To define the role of NP- 1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP- 1 based on the NP- 1 binding site located in the exon 7- and 8- encoded VEGF- A165 domain. The bicyclic peptide, EG3287, potently ( K-i 1.2 mu M) and effectively (> 95% inhibition at 100 mu M) inhibited VEGF- A(165) binding to porcine aortic endothelial cells expressing NP- 1 ( PAE/ NP- 1) and breast carcinoma cells expressing only NP- 1 receptors for VEGF- A, but had no effect on binding to PAE/ KDR or PAE/ Flt- 1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF- A(165). The C terminus encoded by exon 8 and the three- dimensional structure were both critical for EG3287 inhibition of NP- 1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF- A(165) binding to KDR receptors, it inhibited cross- linking of VEGF- A(165) to KDR in human umbilical vein endothelial cells co- expressing NP- 1, and inhibited stimulation of KDR and PLC- gamma tyrosine phosphorylation, activation of ERKs1/ 2 and prostanoid production. These findings characterize the first specific antagonist of VEGF- A(165) binding to NP- 1 and demonstrate that NP- 1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C- terminal exon 8 domain in VEGF- A(165) binding to NP-1.