Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor

Abstract

SYNOPSIS The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes, ranging from axon guidance and nerve regeneration to cancer malignancy. Thus, antagonists that inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic applications. Here we characterize the binding features of three antagonistic peptides (KYL, APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration calorimetry analysis demonstrates that all three peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4. Chemical shifts observed by NMR spectroscopy upon binding of the KYL peptide involve many EphA4 residues, consistent with extensive interactions and possibly receptor conformational changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM identify the residues critical for EphA4 binding. The peptides exhibit a long half-life in cell culture medium, which with their substantial binding affinity and selectivity for EphA4 makes them excellent research tools to modulate EphA4 function.

A breast cancer stem cell niche supported by juxtacrine signalling from monocytes and macrophages

“…A KYL peptide has been shown to specifically inhibit EphA4 activation 41 . Treatment of cocultures with the KYL peptide, but not the KYL-P7A control peptide 41 , blocked 90% of EphA4 phosphorylation in the HMLER90hi cells and significantly reduced the secretion of the three cytokines (Fig.…”

Blockade of EphA4 signaling ameliorates hippocampal synaptic dysfunctions in mouse models of Alzheimer’s disease

“…The present study shows that Rhy and KYL peptide (40), which binds to the ligandbinding domain of EphA4, effectively alleviated Aβ-induced synaptic dysfunction and synaptic plasticity defects in AD transgenic mouse models. This suggests that blockade of EphA4 activity can be potentially developed as a therapeutic strategy for the treatment of AD.…”

The roles and therapeutic potentials of Ephs and ephrins in lung cancer

“…Other than multi-target RTK inhibitor, Noberini et al in Pasquale EB's lab identified two isomeric 2, 5-dimethylpyrrolyl benzoic acid derivatives able to selectively inhibit ephrin binding to EphA4 and EphA2 as well as the function of these receptors in live cells [43]. In fact, this lab has discovered a series of small molecules targeting Eph receptors [44][45][46], which may serve as lead compounds for drug development, as well as targeting agents to deliver drugs or imaging agents to tumors. The antibody-based drugs targeting EphA2 have also been developed, including an antibody-drug conjugate (MEDI-547) composed of a fully human monoclonal antibody against both human and murine EphA2 (1C1) and the tubulin polymerization inhibitor monomethylauristatin F [47] and EA5 [48].…”