Lili Cheng

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-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 (Ki 1.2μm) and effectively (>95% inhibition at 100 μm) 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-γ 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.

Small Molecule Inhibitors of the Neuropilin-1 Vascular Endothelial Growth Factor A (VEGF-A) Interaction

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1−ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

Neuropilin-1 antagonism in human carcinoma cells inhibits migration and enhances chemosensitivity

Background:Neuropilin-1 (NRP1) is a non-tyrosine kinase receptor for vascular endothelial growth factor (VEGF) recently implicated in tumour functions.Methods:In this study we used a specific antagonist of VEGF binding to the NRP1 b1 domain, EG3287, to investigate the functional roles of NRP1 in human carcinoma cell lines, non-small-cell lung A549, kidney ACHN, and prostate DU145 cells expressing NRP1, and the underlying mechanisms involved.Results:EG3287 potently displaced the specific binding of VEGF to NRP1 in carcinoma cell lines and significantly inhibited the migration of A549 and ACHN cells. Neuropilin-1 downregulation by siRNA also decreased cell migration. EG3287 reduced the adhesion of A549 and ACHN cells to extracellular matrix (ECM), and enhanced the anti-adhesive effects of a β1-integrin function-blocking antibody. EG3287 increased the cytotoxic effects of the chemotherapeutic agents 5-FU, paclitaxel, or cisplatin on A549 and DU145 cells, through inhibition of integrin-dependent cell interaction with the ECM.Conclusions:These findings indicate that NRP1 is important for tumour cell migration and adhesion, and that NRP1 antagonism enhances chemosensitivity, at least in part, by interfering with integrin-dependent survival pathways. A major implication of this study is that therapeutic strategies targeting NRP1 in tumour cells may be particularly useful in combination with other drugs for combating tumour survival, growth, and metastatic spread independently of an antiangiogenic effect of blocking NRP1.

Chondroitin sulphate-modified neuropilin 1 is expressed in human tumour cells and modulates 3D invasion in the U87MG human glioblastoma cell line through a p130Cas-mediated pathway.

Neuropilin 1 (NRP1), a non‐tyrosine kinase receptor for vascular endothelial growth factor and class 3 Semaphorins, is highly expressed in many human tumour cell lines, but its function is poorly understood. Here, we describe the expression of a new chondroitin sulphate‐modified NRP1 (NRP1‐CS) in human tumour cell lines. Expression of a non‐modifiable NRP1 mutant (S612A) in U87MG human glioma cells results in enhanced invasion in three dimensions (3D), whereas wild‐type NRP1 has no effect. Furthermore, the S612A NRP1 cells show a significant increase in p130Cas tyrosine phosphorylation compared with control and wild‐type NRP1 cells. Silencing of p130Cas in S612A NRP1 cells resulted in a loss of increased invasive phenotype. Interestingly, p130Cas silencing does not inhibit basal 3D invasion, but leads to a mesenchymal to amoeboid transition. Biopsies from both low‐ and high‐grade human gliomas show strong expression of NRP1, and little expression of NRP1‐CS. Our data establish distinct roles for NRP1 and NRP1‐CS in modulating a new NRP1‐p130Cas signalling pathway contributing to glioblastoma cell invasion in 3D.

N-terminal modification of VEGF-A C terminus-derived peptides delineates structural features involved in neuropilin-1 binding and functional activity.

The interaction between VEGF‐A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7‐ and 8‐derived bicyclic peptides with N‐terminal lipophilic groups and found a simple N‐octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF‐A binding to neuropilin‐1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF‐promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.

Production of Soluble Human Vascular Endothelial Growth Factor VEGF-A165-Heparin Binding Domain in Escherichia coli

We report a method for production of soluble heparin binding domain (HBD) of human vascular endothelial growth factor VEGF-A165. Recombinant VEGF-A165-HBD that contains four disulphide bridges was expressed in specialised E. coli SHuffle cells and its activity has been confirmed through interactions with neuropilin and heparin. The ability to produce significant quantities of a soluble active form of VEGF-A165-HBD will enable further studies addressing the role of VEGF-A in essential processes such as angiogenesis, vasculogenesis and vascular permeability.