Design, synthesis and characterization of novel small molecular inhibitors of ephrin-B2 binding to EphB4

EphB4 is a member of the large Eph receptor tyrosine kinase family. By interacting with its preferred ligand ephrin-B2, which is also a transmembrane protein, EphB4 plays a role in a variety of physiological and pathological processes ranging from bone remodeling to cancer malignancy. EphB4-ephrin-B2 binding occurs at sites of contact between cells. Ephrin-B2 causes EphB4 clustering and increased kinase activity to generate downstream signals that affect cell behavior. Previous work identified a high-affinity antagonistic peptide that targets EphB4, named TNYL-RAW. This peptide is 15 amino acid long, has a molecular weight of ~1700 Da and binds to the ephrin-binding pocket of EphB4. Here we report the structure-based design and chemical synthesis of two novel small molecules of ~600-700 Da, which were designed starting from the small and functionally critical C-terminal portion of the TNYL-RAW peptide. These compounds inhibit ephrin-B2 binding to EphB4 at low micromolar concentrations. Additionally, although the ephrin-B2 ligand can interacts with multiple other Eph receptors besides EphB4, the two compounds retain the high selectivity of the TNYL-RAW peptide in targeting EphB4. TNYL-RAW peptide displacement experiments using the more potent of the two compounds, compound 5, suggest a competitive mode of inhibition. These EphB4 antagonistic compounds can serve as promising templates for the further development of small molecule drugs targeting EphB4.