N‐Terminal Modification of VEGF‐A C Terminus‐Derived Peptides Delineates Structural Features Involved in Neuropilin‐1 Binding and Functional Activity

Abstract

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.

Immunoglobulin Fc-fused, neuropilin-1-specific peptide shows efficient tumor tissue penetration and inhibits tumor growth via anti-angiogenesis

“…It was previously reported that CendR peptides bind to the VEGF-binding site of NRP1 and NRP2 without receptor specificity [13,14,20,21]. In this study, we successfully generated an Fc-fused peptide, Fc-TPP11, that binds to the VEGF binding site of NRP1 with approximately 1,000-fold higher selectivity (K D = ~ 1.7 nM) than the closely related NRP2 (K D = ~ 1.6 μM).…”

Structural studies of neuropilin‐2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket

“…However, in both structures, effects of crystal packing on positioning of the VEGF‐A 165 components of the complexes could not be completely excluded. Our previous isothermal titration calorimetry (ITC) measurements of the in vitro interaction between NRP1, NRP2 and EG00086 , and the crystal structures of the relevant complexes, do not clarify the molecular basis for the differences in biological activity. Considering that qualitatively the same set of key molecular interactions have been identified in all instances, it is possible that additional factors such as the energy of the additional hydrogen bonds within the NRP1/ligand and NRP2/ligand complexes, entropic changes due to side chains or solvent rearrangement within the ligand binding sites, or structural differences and post‐translational modifications elsewhere on the proteins contribute to the perceived differences in the ligand affinities of NRP1 versus NRP2.…”

“…A) bound to the b1 domain of NRP2. Peptide EG00086, corresponding to the C‐terminus of VEGF‐A 165 (residues 138–165), its binding affinity to NRP1 and NRP2, and its potency to act as inhibitor of VEGF‐promoted cellular adhesion have been previously described . The peptide corresponds to the C‐terminal half (one sub‐domain) of the HBD of VEGF‐A 165 .…”

Architecture and hydration of the arginine‐binding site of neuropilin‐1

“…It is possible that other ligands containing a C‐terminal arginine will also bind in a similar fashion . Although protein–protein interactions have been considered challenging targets in drug discovery , a number of small molecules and peptides have been identified as inhibitors of the VEGF‐A 165 –NRP1 interaction . In some cases, it has been demonstrated that the inhibitors act through direct binding to the b1 domain of NRP1.…”