Enhancing Inhibitory Potency and Serum Stability of Peptide Inhibitors of the EphB2 Receptor

Ephrin (Eph) receptors play key regulatory roles in physiological processes, such as tissue development, cell migration, and angiogenesis. Upregulation of specific Eph receptors has been identified in many different cancers, highlighting Eph receptors as promising targets for new cancer therapeutics. Though targeting individual Eph receptors is challenging because of the high sequence homology among the 14 Eph receptors in humans, peptides have been isolated through phage display that target specific receptors (EphA2, EphA4, EphB2, EphB4). While many of these peptides have been further optimized based on the original phage display hits, the EphB2 receptor-targeting SNEW peptide has received less attention. Here we describe parallel strategies to modify SNEW, leading to improved affinity for the EphB2 receptor and greater stability in human serum while retaining SNEW's high specificity for the EphB2 receptor. Specifically, replacement of the N-terminal serine residue with cyclic α-amino acids, particularly those with saturated, six-membered rings, increased inhibitory potency against the EphB2 receptor-ephrin B2 interaction. Replacement of a central proline residue with 4,4-difluoroproline led to a significant increase in serum stability in the context of SNEW and its more potent N-terminally modified variants. SNEW variants with greater potency and serum stability offer lead candidates for targeting the EphB2 receptor in associated cancers and other diseases. Additionally, the modification approaches employed for SNEW may be extensible for N-terminal serine/threonine/cysteine substitution and/or proline substitution to improve protein targeting by other peptides.