title: 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.
abstract: Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) 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 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 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-A165. 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-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 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-A165 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-A165 binding to NP-1.
authors.affiliation: Centre for Cardiovascular Biology and Medicine, Department of Medicine, University College London, London WC1E 6JJ, United Kingdom; Ark Therapeutics Limited, The Rayne Institute, University College London, London WC1E 6JJ, United Kingdom.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Jia
authors.affiliation: Centre for Cardiovascular Biology and Medicine, Department of Medicine, University College London, London WC1E 6JJ, United Kingdom; Ark Therapeutics Limited, The Rayne Institute, University College London, London WC1E 6JJ, United Kingdom.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Bagherzadeh
authors.affiliation: NCE Discovery Ltd., Cambridge CB4 0PA, United Kingdom.
authors.forename: Basil
authors.initials: B
authors.lastname: Hartzoulakis
authors.affiliation: NCE Discovery Ltd., Cambridge CB4 0PA, United Kingdom.
authors.forename: Ashley
authors.initials: A
authors.lastname: Jarvis
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London WC1E GBT United Kingdom.
authors.forename: Marianne
authors.initials: M
authors.lastname: Löhr
authors.affiliation: NCE Discovery Ltd., Cambridge CB4 0PA, United Kingdom.
authors.forename: Shaheda
authors.initials: S
authors.lastname: Shaikh
authors.affiliation: NCE Discovery Ltd., Cambridge CB4 0PA, United Kingdom.
authors.forename: Rehan
authors.initials: R
authors.lastname: Aqil
authors.affiliation: Centre for Cardiovascular Biology and Medicine, Department of Medicine, University College London, London WC1E 6JJ, United Kingdom; Ark Therapeutics Limited, The Rayne Institute, University College London, London WC1E 6JJ, United Kingdom.
authors.forename: Lili
authors.initials: L
authors.lastname: Cheng
authors.affiliation: Centre for Cardiovascular Biology and Medicine, Department of Medicine, University College London, London WC1E 6JJ, United Kingdom; Ark Therapeutics Limited, The Rayne Institute, University College London, London WC1E 6JJ, United Kingdom.
authors.forename: Michelle
authors.initials: M
authors.lastname: Tickner
authors.affiliation: Bloomsbury Centre for Structure Biology, Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Diego
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Bloomsbury Centre for Structure Biology, Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Harris
authors.affiliation: Bloomsbury Centre for Structure Biology, Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Driscoll
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London WC1E GBT United Kingdom.
authors.forename: David L
authors.initials: DL
authors.lastname: Selwood
authors.affiliation: Centre for Cardiovascular Biology and Medicine, Department of Medicine, University College London, London WC1E 6JJ, United Kingdom. Electronic address: i.zachary@ucl.ac.uk.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Zachary
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D039942:Neuropilin-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D042461:Vascular Endothelial Growth Factor A; C511613:vascular endothelial growth factor A (138-165); D039942:Neuropilin-1; D040301:Vascular Endothelial Growth Factor Receptor-2