Small Molecules Can Selectively Inhibit Ephrin Binding to the EphA4 and EphA2 Receptors

Abstract

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 ligandbinding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K i values of 7 and 9 M 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.

Crystal Structure and NMR Binding Reveal That Two Small Molecule Antagonists Target the High Affinity Ephrin-binding Channel of the EphA4 Receptor

“…In some structures, even two hydrogen bonds can be identified between them. This observation may explain why removal of either the carboxylic or the hydroxyl group causes a dramatic loss in the activity of the modified compounds (54).Taken together, the docking results imply that the pyrrole and benzene rings, the two methyl groups on the pyrrole ring, and the carboxylic and hydroxyl groups on the benzene ring are all critical for the binding of small molecules with a 2,5-dimethylpyrrolyl benzene scaffold to the EphA4 ligand-binding domain.…”

“…Binding Interactions Characterized by Isothermal Titration Calorimetry and Circular Dichroism-Recently, a 2,5-dimethylpyrrolyl benzoic acid derivative has been identified in a high throughput screening for inhibitors of EphA4 ligand binding (54). This small molecule and an isomeric compound were found to antagonize ephrin-induced effects in EphA4-expressing cells.…”

“…On the other hand, although the high affinity ephrin binding pocket of the Eph receptors appears to be an attractive target for design of small molecules capable of inhibiting the Eph receptor signaling by blocking ephrin binding, only now two small molecules have been identified by a high throughput screening, which are able to antagonize ephrin-induced effects in EphA4-expressing cells (see accompanying article, Ref. 54). Hence, it is of significant interest to gain structural insight into the binding interactions between the two small molecules and the EphA4 ligand-binding domain, with the ultimate goal to develop small molecule antagonists capable of inhibiting Eph-ephrin binding with high affinity and specificity.…”

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

“…Although the 12-amino acid KYL peptide was able to block the EphA4 signaling effectively, there may be huge challenges to develop this peptide as a drug candidate, e.g., bioavailability. For the identified small-molecule EphA4 inhibitors, although their effectiveness was demonstrated in in vitro or cellular assays, the in vivo effects of these compounds on inhibiting EphA4 and the bioavailability of these agents have not been reported (28,33,41). The reduction of EphA4 activation in APP/PS1 mouse brains by oral administration of Rhy (Fig.…”

Reversing Synapse Loss in Alzheimer's Disease: Rho-Guanosine Triphosphatases and Insights from Other Brain Disorders

“…In vitro use of the peptide-based inhibitor of EphA4 signaling KYL [114] was shown to block Aβ-induced synaptic dysfunction, reversing both the loss of dendritic spines in cultured hippocampal neurons and the blockage of LTP induction in hippocampal slices by Aβ oligomers [110,115]. Moreover, ICV injections (~3 weeks) of KYL was shown to be effective in blocking EphA4 signaling in the hippocampus of APP/PS-1 transgenic AD mice, resulting in the complete rescue of impaired LTP normally observed in these mice [115].…”