A synthetic bivalent peptide ligand of EphB4 with potent agonistic activity.

Interaction between ephrin receptor EphB4 and its ligand EFNB2 mediates bidirectional signaling important for cancer: forward EFNB2-to-EphB4 signaling that is tumor suppressive, and reverse EphB4-to-EFNB2 signaling that promotes angiogenesis important for tumor growth and metastasis. Molecular agents targeting these forward and reverse signals of EphB4-EFNB2 interaction can be used to probe the molecular mechanisms of these complex signaling pathways and develop new anticancer therapeutics. In this study, we applied a bivalent ligand design strategy to synthesize a novel dimeric peptide based on an antagonist TNYL-RAW. The dimeric peptide possessed higher EphB4 receptor binding affinity than the monomeric TNYL-RAW peptide. Interestingly, the dimerization of TNYL-RAW peptide converted a monomeric antagonist of EphB4 to a dimeric agonist. This dimeric agonist promoted EphB4 phosphorylation, internalization and degradation, reduced cancer cell motility, and inhibited tube formation of HUVEC. To investigate the mechanism of action of this bivalent dimeric peptide, FRET experiments and molecular dynamic simulation were conducted and suggested that this bivalent ligand recognizes two EphB4 simultaneously which may promote receptor dimerization and oligomerization. This was further supported by the study of this bivalent ligand containing deletion of critical residues on one of its monomers which impaired its simultaneous binding to two EphB4 and ability to cause EphB4 dimerization and phosphorylation. These results demonstrate the value of this novel bivalent agonist ligand of EphB4 as a probe of the bidirectional signaling of EphB4-EFNB2 and lead for cancer drug development.