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

ABSTRACT: 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. In the present study 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 demonstrated 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-shift changes 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.

SUBMITTER: Lamberto I

PROVIDER: S-EPMC3677027 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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

Lamberto Ilaria I Qin Haina H Noberini Roberta R Premkumar Lakshmanane L Bourgin Caroline C Riedl Stefan J SJ Song Jianxing J Pasquale Elena B EB

The Biochemical journal 20120701 1

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. In the present study 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 demo ...[more]

PMID: 22489865

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Project description:The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, and therefore they represent a promising class of drug targets. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and likely in certain types of cancer. Here we report the first crystal structure of the EphA4 ligand-binding domain, which adopts the same jellyroll beta-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core beta-stranded regions, whereas large variations exist in the loops, particularly the D-E and J-K loops, which form the high affinity ephrin binding channel. We also used isothermal titration calorimetry, NMR spectroscopy, and computational docking to characterize the binding to EphA4 of two small molecules, 4- and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid which antagonize ephrin-induced effects in EphA4-expressing cells. We show that the two molecules bind to the EphA4 ligand-binding domain with K(d) values of 20.4 and 26.4 microm, respectively. NMR heteronuclear single quantum coherence titrations revealed that upon binding, both molecules significantly perturb EphA4 residues Ile(31)-Met(32) in the D-E loop, Gln(43) in the E beta-strand, and Ile(131)-Gly(132) in the J-K loop. Molecular docking shows that they can occupy a cavity in the high affinity ephrin binding channel of EphA4 in a similar manner, by interacting mainly with the EphA4 residues in the E strand and D-E and J-K loops. However, many of the interactions observed in Eph receptor-ephrin complexes are absent, which is consistent with the small size of the two molecules and may account for their relatively weak binding affinity. Thus, our studies provide the first published structure of the ligand-binding domain of an EphA receptor of the A subclass. Furthermore, the results demonstrate that the high affinity ephrin binding channel of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.

Project description:The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.

Dataset Information

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

Publications

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

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