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

Abstract

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 ~1,700 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 interact 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.

Ephrin B2/EphB4 Mediates the Actions of IGF-I Signaling in Regulating Endochondral Bone Formation

“…In the current study TNYL-RAW, a specific antagonist of ephrinB2 interactions with EphB4 (40), blocked osteoblast induced osteoclastogenesis, decreased osteoblast differentiation (as indicated by lower mRNA levels of AP) and blocked the ability of IGF-I to promote osteoblast differentiation and osteoclastogenesis in these co-cultures. Our data do not support a role for ephrin B2/EphB4 in osteoblast suppression of osteoclast differentiation as suggested by earlier studies (16); rather our results indicate the opposite—ephrin B2/EphB4 is required for osteoblasts to promote osteoclastogenesis.…”

Evaluation of novel fluorescence probes for conjugation purposes using the traceless Staudinger Ligation

“…Several Eph receptors play a major role regarding the development of cancer and metastasis and are therefore of enormous interest for optical or PET imaging [32]. Especially the EphB4 receptor is overexpressed in a variety of cancers: colon, breast, bladder, prostate or ovarian cancers [33]. The inhibition of the EphB4 receptor can lead to a reduction of cancer growth and angiogenesis [34].…”

Eph Receptors and Ephrins: Therapeutic Opportunities

“…Efforts to improve affinity have resulted in more promising but larger (>500 kDa) compounds (146–148). Small molecules are better-suited to bind the ATP-binding pocket in the Eph kinase domain (36; 39).…”