Peptides derived from vascular endothelial growth factor B show potent binding to neuropilin-1 Short Title: VEGFB peptides bind to neuropilin-1.

Vascular endothelial growth factors (VEGF) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. There is growing evidence that the VEGF-B isoform is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP-1). However the details of the interaction with NRP-1 and its functional importance have not been elucidated. We employed surface plasmon resonance technology and X-ray crystallography to characterize the direct binding between VEGF-B and the b1 domain of NRP-1, and developed VEGF-B – C-terminus derived peptides to be used as chemical tools for studying VEGF-B – NRP-1 related pathways. Results were compared with the corresponding VEGF-A derived peptides. Peptide lipidation was used as a means to stabilise the peptides. The binding of VEGF-B to NRP1-b1 was inhibited by a well characterized small molecule targeting the NRP1 arginine binding site. A crystal structure of a peptide with NRP-1 demonstrated that VEGF-B peptides bind at the canonical C-terminal Arginine binding site. VEGF-B – derived peptides containing a C-terminal arginine show potent binding to NRP1-b1 and inhibition of VEGF-A165 functional activity in HUVEC cells. Peptide lipidation increased binding residence time and improved plasma stability while maintaining binding affinity. The VEGF-B C-terminus imparts higher affinity for NRP-1 than the corresponding VEGF-A165 region. This binding is mediated through the canonical arginine site and the tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events.