Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6

No ligand has hitherto been designated for the Eph receptor tyrosine kinase family member, EphB6. Here, expression of an EphB6 ligand in the pro-B leukemic cell line, Reh, is demonstrated by binding of soluble EphB6-Fc fusion protein to the Reh cells. The ligand belongs to the subgroup of membrane spanning ligands, as suggested by the fact that phosphatidylinositol-specific phospholipase C treatment did not abrogate binding of EphB6-Fc. Two transmembrane Eph receptor ligands, ephrin-B1 and ephrin-B2, were identified in Reh cells. Analysis of EphB6-Fc fusion protein binding to ephrin-B1 or ephrin-B2 transfected COS cells revealed a high-affinity saturable binding between EphB6-Fc and ephrin-B2, but not with ephrin-B1. In mice, EphB6 has previously been shown to be expressed in thymus. Here, we show expression of EphB6 in human thymus, as well as the expression of ephrin-B2 in both human and mouse thymus. We conclude that ephrin-B2 may be a physiological ligand for the EphB6 receptor.     

Multiple in vivo tyrosine phosphorylation sites in EphB receptors

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.     

PEGylation potentiates the effectiveness of an antagonistic peptide that targets the EphB4 receptor with nanomolar affinity

The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.     

Sizes of interface residues account for cross‐class binding affinity patterns in Eph receptor–ephrin families

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell–cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin‐As (Bowden et al., Structure 2009;17:1386–1397); and EphB2, which can bind ephrin‐A5 in addition to the ephrin‐Bs (Himanen et al., Nat Neurosci 2004;7:501–509). A crystal structure is available of the interacting domains of the EphA4‐ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386–1397). In this complex, the ligand‐binding domain of EphA4 adopts an EphB‐like conformation. To understand why other cross‐class EphA receptor–ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4–ephrinB1, (2) EphA4–ephrinB3, and (3) EphA2–ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity. Proteins 2014; 82:349–353. © 2013 Wiley Periodicals, Inc.     

Three-dimensional structure of the EphB2 receptor in complex with an antagonistic peptide reveals a novel mode of inhibition

The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of approximately 15 microm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-A resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities.     

Complementary expression of EphB receptors and ephrin-B ligand in the pyloric and duodenal epithelium of adult mice

Eph receptors and ephrin ligands are membrane-bound cell–cell communication molecules that regulate the spatial organisation of cells in various tissues by repulsive or adhesive signals arising from contact between EphB- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the gastric epithelium and Brunner’s glands are virtually unknown. We detected several EphB receptors and ephrin-B ligands in the pyloric and duodenal mucosa of the adult mouse by RT-PCR amplification. Immunostaining showed complementary expression patterns, with ephrin-B1 being preferentially expressed in the superficial part and EphB receptors in the deeper part of both epithelia. In the gastric pylorus, ephrin-B1 was expressed in pit cells and proliferating cells of the isthmus. In contrast, EphB2, EphB3, and EphB4 were expressed in pyloric glandular cells and proliferating cells of the isthmus. In the duodenum, ephrin-B1 was expressed in cells lining the ducts of Brunner’s glands as well as those covering villi and the upper portion of the crypts of Lieberkühn. In contrast, EphB2 and EphB3 were expressed in the gland segment of Brunner’s glands and the lower portion of the crypts and EphB4, in the crypts. In both mucosae, EphB2, EphB3, and EphB4 were found to be tyrosine phosphorylated, suggesting that EphB/ephrin-B signalling might occur preferentially in the isthmus, crypts, and duct-gland transition of Brunner’s glands, where the receptor and ligand expression overlaps. Based on these findings, we propose that EphB/ephrin-B signalling may regulate cell positioning within the pyloric and duodenal epithelium.     

Association of Dishevelled with Eph tyrosine kinase receptor and ephrin mediates cell repulsion

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, we discovered that Xenopus Dishevelled (Xdsh) forms a complex with Eph receptors and ephrin-B ligands and mediates the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affected the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induced the activation of RhoA and Rho kinase in the EphB2-overexpressed cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. We propose that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity.     

The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. Here we investigated the role of EphB receptors and their ligands during embryonic blood vessel development in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, we detected expression of EphB4 receptors in the posterior cardinal veins and their derivatives, the intersomitic veins. Vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could however not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Furthermore, we found that ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. We performed RNA injection experiments to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos resulted in intersomitic veins growing abnormally into the adjacent somitic tissue. Our findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis possibly by mediating repulsive guidance cues to migrating endothelial cells.     

The kinase-null EphB6 receptor undergoes transphosphorylation in a complex with EphB1.

Uniquely for the Eph family of receptor tyrosine kinases, the EphB6 receptor is catalytically inactive due to the alteration of several critical residues in its kinase domain. This has cast doubt upon its ability to participate in cytoplasmic signaling events. We show here that despite its lack of kinase activity, EphB6 undergoes inducible tyrosine phosphorylation upon stimulation with the Eph-B receptor subfamily ligand ephrin-B1. We also demonstrate, for the first time, evidence of cross-talk between Eph receptors. Overexpression of a catalytically active member of the Eph-B subfamily, EphB1, resulted in increased EphB6 phosphorylation. EphB1-induced EphB6 phosphorylation was ligand-dependent and required the functional catalytic activity of EphB1. EphB1 not only transphosphorylated EphB6, but together they also formed a stable hetero-complex. In addition, we identify the proto-oncogene c-Cbl as an EphB6-binding protein. Although EphB6-Cbl association appeared to be constitutive, Cbl required a functional phosphotyrosine binding domain in order to bind the receptor, whereas its RING finger motif ubiquitin-transfer domain was not necessary. Our findings demonstrate that EphB6 is an actively signaling receptor that undergoes transphosphorylation upon ligand binding and that can initiate specific cytoplasmic signaling events.     

Regulation of signaling interactions and receptor endocytosis in growing blood vessels

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRβ and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.     

Regulation of signaling interactions and receptor endocytosis in growing blood vessels

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRβ and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.     

An ephrin mimetic peptide that selectively targets the EphA2 receptor

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologic versus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolar K(D) values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

Targeting Eph receptors with peptides and small molecules: progress and challenges

The Eph receptors are a large family of receptor tyrosine kinases. Their kinase activity and downstream signaling ability are stimulated by the binding of cell surface-associated ligands, the ephrins. The ensuing signals are bidirectional because the ephrins can also transduce signals (known as reverse signals) following their interaction with Eph receptors. The ephrin-binding pocket in the extracellular N-terminal domain of the Eph receptors and the ATP-binding pocket in the intracellular kinase domain represent potential binding sites for peptides and small molecules. Indeed, a number of peptides and chemical compounds that target Eph receptors and inhibit ephrin binding or kinase activity have been identified. These molecules show promise as probes to study Eph receptor/ephrin biology, as lead compounds for drug development, and as targeting agents to deliver drugs or imaging agents to tumors. Current challenges are to find (1) small molecules that inhibit Eph receptor-ephrin interactions with high binding affinity and good lead-like properties and (2) selective kinase inhibitors that preferentially target the Eph receptor family or subsets of Eph receptors. Strategies that could also be explored include targeting additional Eph receptor interfaces and the ephrin ligands.     

Roles of Eph receptors and ephrins in segmental patterning

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have key roles in patterning and morphogenesis. Interactions between these molecules are promiscuous, but largely fall into two groups: EphA receptors bind to glycosylphosphatidyl inositol-anchored ephrin-A ligands, and EphB receptors bind to transmembrane ephrin-B proteins. Ephrin-B proteins transduce signals, such that bidirectional signalling can occur upon interaction with the Eph receptor. In many tissues, there are complementary and overlapping expression domains of interacting Eph receptors and ephrins. An important role of Eph receptors and ephrins is to mediate cell contact-dependent repulsion, and this has been implicated in the pathfinding of axons and neural crest cells, and the restriction of cell intermingling between hindbrain segments. Studies in an in vitro system show that bidirectional activation is required to prevent intermingling between cell populations, whereas unidirectional activation can restrict cell communication via gap junctions. Recent work indicates that Eph receptors can also upregulate cell adhesion, but the biochemical basis of repulsion versus adhesion responses is unclear. Eph receptors and ephrins have thus emerged as key regulators that, in parallel with cell adhesion molecules, underlie the establishment and maintenance of patterns of cellular organization.     

Looking forward to EphB signaling in synapses

Eph receptors and their ligands ephrins comprise a complex signaling system with diverse functions that span a wide range of tissues and developmental stages. The variety of Eph receptor functions stems from their ability to mediate bidirectional signaling through trans-cellular Eph/ephrin interactions. Initially thought to act by directing repulsion between cells, Ephs have also been demonstrated to induce and maintain cell adhesive responses at excitatory synapses in the central nervous system. EphB receptors are essential to the development and maintenance of dendritic spines, which accommodate the postsynaptic sites of most glutamatergic excitatory synapses in the brain. Functions of EphB receptors are not limited to control of the actin cytoskeleton in dendritic spines, as EphB receptors are also involved in the formation of functional synaptic specializations through the regulation of glutamate receptor trafficking and functions. In addition, EphB receptors have recently been linked to the pathophysiology of Alzheimer's disease and neuropathic pain, thus becoming promising targets for therapeutic interventions. In this review, we discuss recent findings on EphB receptor functions in synapses, as well as the mechanisms of bidirectional trans-synaptic ephrin-B/EphB receptor signaling that shape dendritic spines and influence post-synaptic differentiation.     

Crystal structure and NMR binding reveal that two small molecule antagonists target the high affinity ephrin-binding channel of the EphA4 receptor

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.     

Ephrin-B3 binds to a sulfated cell-surface receptor

The ephrins are a family of proteins known to bind the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinase family. In the present paper, we provide data showing that ephrin-B3 binds a sulfated cell-surface protein on HEK-293T (human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40) and HeLa cells, a binding that is nearly completely blocked by treatment of these cell lines with chlorate or heparinase, or by addition of the heavily sulfated glycosaminoglycan heparin. This indicates that heparan sulfate on these cells is essential for cell-surface binding of ephrin-B3. Heparin did not affect ephrin-B3 binding to EphB receptors expressed on transfected HEK-293T cells, indicating further that ephrin-B3 binds an alternative receptor which is a heparan sulfate proteoglycan. Site-directed mutagenesis analysis revealed that Arg178 and Lys179 are important for heparin binding of ephrin-B3 and also for ephrin-B3 binding to cells. These amino acids, when introduced in the non-heparin-binding ephrin-B1, conferred the heparin-binding property. Functional studies reveal that ephrin-B3 binding to cells induces cellular signalling and influences cell rounding and cell spreading. In conclusion, our data provide evidence for an unknown ephrin-B3-binding cell-surface proteoglycan involved in cellular signalling.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

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.     

Structure and thermodynamic characterization of the EphB4/Ephrin-B2 antagonist peptide complex reveals the determinants for receptor specificity

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 angstroms. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.