Characterization of Konjac Ceramide (kCer) Binding to Sema3A Receptor Nrp1.

Konjac ceramide (kCer) can be prepared by a chemoenzymatic method as previously published (Usuki, S.; Tamura, N.; Sakai, S.; Tamura, T.; Mukai, K.; Igarashi, Y. Biochem. Biophys. Rep. 5, 160-167 (2016)). Thus prepared kCer showed an activation effect on Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerizaion, resulting in opposing NGF-induced neurite outgrowth. In the present study, we have shown that kCer is a potential Sema3A-like ligand that has a competitive effect on Sema3A binding to a cell surface receptor Nrp1, but animal-type ceramides have no effect on Sema3A binding to Nrp1. In addition, kCer showed a direct molecular interaction with Nrp1, but animal-type ceramides, C16Cer, C18Cer, and C24Cer show no specific bindings to Nrp1. Further, kCer showed an additive effect to activate the Sema3A signaling pathway together with low-dose Sema3A but a reversed effect to inhibit this pathway when combined with high-dose Sema3A.

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1.

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence. Here, we present a structure-activity relationship study of the systematic optimization of amino acid residues in positions 1-3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP-1.