Molecular Determinants of EphA2 and EphB2 Antagonism Enable the Design of Ligands with Improved Selectivity.

With the aim of identifying novel antagonists selective for the EphA receptor family, a combined experimental and computational approach was taken to investigate the molecular basis of the recognition between a prototypical Eph-ephrin antagonist (UniPR1447) and two representative receptors of the EphA and EphB subfamilies, namely, EphA2 and EphB2 receptors. The conformational free-energy surface (FES) of the binding state of UniPR1447 within the ligand binding domain of EphA2 and EphB2, reconstructed from molecular dynamics (MD) simulations performed on the microsecond time scale, was exploited to drive the design and synthesis of a novel antagonist selective for EphA2 over the EphB2 receptor. The availability of compounds with this pharmacological profile will help discriminate the importance of these two receptors in the insurgence and progression of cancer.

EphB2 contributes to human naive B-cell activation and is regulated by miR-185.

EphB2 is an important member of the receptor tyrosine kinases. Recently, EphB2 was shown to facilitate T-cell migration and monocyte activation. However, the effects of EphB2 on B cells remain unknown. In this study, the expression of EphB2 on B cells was tested by Western blot, and the roles of EphB2 in B-cell proliferation, cytokine secretion, and immunoglobulin (Ig) production were evaluated using EphB2 siRNA interference in human B cells from healthy volunteers. Our study revealed that EphB2 was distributed on naive B cells and was up-regulated on activated B cells. Moreover, B-cell proliferation (decreased by 22%, P<0.05), TNF-α secretion (decreased by 40%, P<0.01) and IgG production (decreased by 26%, P < 0.05) were depressed concordantly with the down-regulated EphB2 expression. Subsequently, we screened microRNAs that could regulate EphB2 expression in B cells, and discovered that miR-185 directly targeted to EphB2 mRNA and suppressed its expression. Furthermore, miR-185 overexpression inhibited B-cell activation, and the inhibitor of miR-185 enhanced B-cell activation. Moreover, abatement of EphB2 through miR-185 mimics or EphB2 siRNA attenuated the activation of Src-p65 and Notch1 signaling pathways in human B cells. Our study first suggested that EphB2 was involved in human naive B cell activation through Src-p65 and Notch1 signaling pathways and could be regulated by miR-185.

Receptor tyrosine kinases in osteosarcoma: not just the usual suspects.

Despite aggressive surgical and chemotherapy protocols, survival rates for osteosarcoma patients have not improved over the last 30 years. Therefore, novel therapeutic agents are needed. Receptor tyrosine kinases have emerged as targets for the development of new cancer therapies since their activation leads to enhanced proliferation, survival, and metastasis. In fact, aberrant expression and activation of RTKs have been associated with the progression of many cancers. Studies from our lab using phosphoproteomic screening identified RTKs that are activated and thus may contribute to the signaling within metastatic human osteosarcoma cells. Functional genomic screening using siRNA was performed to distinguish which of the activated RTKs contribute to in vitro phenotypes associated with metastatic potential (motility, invasion, colony formation, and cell growth). The resulting RTK hits were then validated using independent validation experiments. From these results, we identified four RTKs (Axl, EphB2, FGFR2, and Ret) that have not been previously studied in osteosarcoma and provide targets for the development of novel therapeutics.

Enhanced EphB2-Specific Peptide Inhibitors through Stabilization of Polyproline II Helical Structure.

Ephrin (Eph) receptors are the largest family of receptor tyrosine kinases. Interactions between Eph receptors and their membrane-bound ephrin protein ligands are associated with many developmental processes as well as various cancers and neurodegenerative diseases. With significant crosstalk between different Eph receptors and ephrin ligands, there is an urgent need for high-affinity ligands that bind specifically to individual Eph receptors to interrogate and modulate their functions. Here, we describe the rational development of potent EphB2 receptor inhibitors derived from the EphB2 receptor-specific SNEW peptide. To improve inhibitory potency, we evaluated 20+ cross-linkers with the goal of spanning and stabilizing a single polyproline II helical turn observed when SNEW binds to the EphB2 receptor. Of the cross-linkers evaluated, an 11-atom cross-linker, composed of a rigid 2,7-dimethylnaphthyl moiety between two cysteine residues, was found to yield the most potent inhibitor. Analysis of the cyclized region of this peptide by NMR and molecular dynamics simulations suggests that cross-linking stabilizes the receptor-bound polyproline II helix structure observed in the receptor-peptide complex. Cross-linked SNEW variants retained binding specificity for EphB2 and showed cross-linker-dependent resistance to trypsin proteolysis. Beyond the discovery of more potent EphB2 receptor inhibitors, these studies illustrate a novel cyclization approach with potential to stabilize polyproline II helical structure in various peptides for specific targeting of the myriad protein-protein interactions (PPIs) mediated by polyproline II helices.

EphrinB2-EphB2 signaling for dendrite protection after neuronal ischemia in vivo and oxygen-glucose deprivation in vitro.

In order to rescue neuronal function, neuroprotection should be required not only for the neuron soma but also the dendrites. Here, we propose the hypothesis that ephrin-B2-EphB2 signaling may be involved in dendritic degeneration after ischemic injury. A mouse model of focal cerebral ischemia with middle cerebral artery occlusion (MCAO) method was used for EphB2 signaling test in vivo. Primary cortical neuron culture and oxygen-glucose deprivation were used to assess EphB2 signaling in vitro. siRNA and soluble ephrin-B2 ectodomain were used to block ephrin-B2-Ephb2 signaling. In the mouse model of focal cerebral ischemia and in neurons subjected to oxygen-glucose deprivation, clustering of ephrin-B2 with its receptor EphB2 was detected. Phosphorylation of EphB2 suggested activation of this signaling pathway. RNA silencing of EphB2 prevented neuronal death and preserved dendritic length. To assess therapeutic potential, we compared the soluble EphB2 ectodomain with the NMDA antagonist MK801 in neurons after oxygen-glucose deprivation. Both agents equally reduced lactate dehydrogenase release as a general marker of neurotoxicity. However, only soluble EphB2 ectodomain protected the dendrites. These findings provide a proof of concept that ephrin-B2-EphB2 signaling may represent a novel therapeutic target to protect both the neuron soma as well as dendrites against ischemic injury.

Inhibition of excessive kallikrein-8 improves neuroplasticity in Alzheimer's disease mouse model.

We recently identified excessive cerebral kallikrein-8 (KLK8) mRNA and protein levels at incipient stages of Alzheimer's disease (AD) in AD patients and TgCRND8 mice. Additionally, we showed that antibody-mediated KLK8 inhibition exerts therapeutic effects on AD along with enhancing neuroplasticity, resulting in improved spatial memory in mice. Mounting evidence further substantiates an important role of the protease KLK8 in neuroplasticity. In the present study we sought to gain new mechanistic insights in the interplay between KLK8, neuroplasticity and tau phosphorylation in the context of AD. We here demonstrate that KLK8 inhibition increased the number of hippocampal Ki-67 and doublecortin positive, proliferative neuronal progenitor cells in transgenic mice, whereas the same action in wildtypes had no effect. In line with these results, KLK8 inhibition reduced the levels of its pro-proliferative interaction partners KLK6 and protease-activated receptor 2 only in wildtypes, while the levels of its proliferation-supporting substrate neuregulin-1 and the non-complexed form of its complexing-partner phosphatidylethanolamine binding protein 1 were enhanced in both genotypes. Concomitant incubation of beta-amyloid (Aß)-producing primary neurons with KLK8 and its inhibitory antibody increased neurite complexity and soma size. KLK8 inhibition in SH-SY5Y cells or in primary neurons increased levels of the neuroplasticity-supporting KLK8 substrate ephrin receptor B2 (EPHB2) and total tau while decreasing the relative amount of phospho-tau in relation to total tau. KLK8 blockade further enhanced cell proliferation in SH-SY5Y cells. Additional co-incubation with an inhibitory anti-EPHB2 antibody decreased total tau levels and neurite complexity and increased the ratio of phospho-tau/total tau, underlining the key role of EPHB2 on this plastic change. In a reverse in vitro approach, KLK8 induction reduced EPHB2 and total tau and increased the ratio of phospho-tau/total tau, leading to impaired proliferation and neuronal differentiation. These results underline the therapeutic potential of KLK8 inhibition by counteracting plasticity deficits in AD-affected brain.

Blockade of EphB2 enhances neurogenesis in the subventricular zone and improves neurological function after cerebral cortical infarction in hypertensive rats.

EphB2/ephrinBs has been recently demonstrated to regulate cell proliferation in the neurogenic subventricular zone (SVZ). However, little is known about the role of EphB2 in adult neurogenesis following cerebral infarction. In the present study, we investigated the role of EphB2 in proliferation and differentiation of precursor cells within the SVZ, as well as the neurological function recovery after permanent middle cerebral artery occlusion (MCAO) in hypertensive rats. Bromodeoxyuridine (BrdU) was given twice per day starting from 24h after MCAO for 6-consecutive days. Recombinant EphB2-Fc or IgG-Fc was preclustered by incubation with anti-human Fcgamma and then intraventricularly administrated at 24h after MCAO. The neurological function was evaluated before operation and at 7, 14 and 21 days after MCAO respectively. The infarct size and immunoreactivities of BrdU, Nestin, DCX, GFAP and NeuN were measured at 7, 14 and 21 days after MCAO respectively. Treatment with EphB2-Fc markedly improved the neurological function recovery within 3 weeks after MCAO. In parallel, EphB2-Fc significantly increased the number of BrdU-labeled cells and led to marked increases in BrdU+/DCX+ and BrdU+/Nestin+ cells within the ipsilateral SVZ for 2 weeks after MCAO respectively (all p < 0.05). The BrdU+/NeuN+ cells in the peri-infarct area and neighboring ipsilateral striatum were significantly increased following EphB2-Fc infusion within 3 weeks after MCAO (all p < 0.05). Our data suggest that administration of exogenous clustered EphB2-Fc at 24h can enhance the endogenous neurogenesis and concomitantly improve neurological recovery after cerebral infarction.

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients' antibodies cause cross-linking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons. Our findings show that, under control conditions, NMDARs form nanosized objects and patients' antibodies increase the clustering of synaptic and extrasynaptic receptors inside the nano-objects. This clustering is subunit specific and predominantly affects GluN2B-NMDARs. Following internalization, the remaining surface NMDARs return to control clustering levels but are preferentially retained at the synapse. Monte Carlo simulations using a model in which antibodies induce NMDAR cross-linking and disruption of interactions with other proteins recapitulated these results. Finally, activation of EphB2 receptor partially antagonized the antibody-mediated disorganization of the nanoscale surface distribution of NMDARs.

EphB2 as a therapeutic antibody drug target for the treatment of colorectal cancer.

Analysis of human colorectal cancer specimens revealed overexpression of the EphB2 receptor tyrosine kinase. Monoclonal antibodies (MAbs) to extracellular sequence of EphB2 were raised and tested for activity against colorectal cancer cells. One of the MAbs, 2H9, effectively blocked the interaction of ephB2 with ephrin ligands and inhibited the resulting autophosphorylation of the receptor. However, this antibody did not affect the proliferation of cancer cells expressing ephB2. Immunocytochemical analysis revealed rapid internalization of the MAb 2H9 on binding ephB2, suggesting that target-dependent cell killing could be achieved with an antibody-drug conjugate. When MAb 2H9 was conjugated to monomethylauristatin E through a cathepsin B-cleavable linker, it specifically killed ephB2-expressing cancer cells in vitro and in vivo. Our results suggest that ephB2 is an attractive target for immunoconjugate cancer therapy.

Three stories on Eph kinase inhibitors: From in silico discovery to in vivo validation.

Several selective and potent EphB4 inhibitors have been discovered, optimized and biophysically characterized by our groups over the past years. On the outset of these discoveries high throughput docking techniques were applied. Herein, we review the optimization campaigns started from three of these hits (Xan-A1, Pyr-A1 and Qui-A1) with emphasis on their in depth in vitro and in vivo characterization, together with previously unpublished angiogenesis and fluorescence based assays.

Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 2. Identification of small-molecule inhibitors via coupling with virtual screening.

We have integrated two complementary methods, high-throughput virtual screening with a "high-content" wet screening technique based on frontal affinity chromatography with mass spectrometry detection (FAC-MS), for identification of hits against the erythropoietin-producing hepatocellular B2 (EphB2) receptor tyrosine kinase domain. Both an EphB2-directed virtual screen combining docking and scoring and a kinase-directed pharmacophore search strategy were used to identify a compound set enriched in bioactive compounds against EphB2. The coupling of virtual screening methodologies with FAC-MS is a unique hybrid approach that can be used to increase the efficacy of both hit discovery and optimization efforts in drug discovery and has successfully identified hits, in particular 19a (36% shift, IC(50) = 5.2 microM, K(d) = 3.3 microM), as inhibitors for EphB2, a potential cancer target.

Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 1. Comparison with conventional ELISA.

FAC-MS offers a convenient method for measuring the relative binding strengths of ligands in a mixture and enables a rapid ranking and identification of ligands in the mixture as potential hits against immobilized targets. Using immobilized EphB2 receptor tyrosine kinase as the target and known kinase inhibitors, the results of FAC-MS screening (% shift) have been shown to correlate with the binding constant, K(d), and with IC(50) results from the more traditional ELISA assay. Therefore, since FAC-MS can accommodate a wide variety of target proteins, its applications could play a broad role in drug discovery not only at the hit discovery stage but also during the subsequent more rigorous screening at the hit-to-lead and lead optimization stages.

Small RNAs deliver a blow to ovarian cancer.

Targeted therapeutic approaches have seen tremendous advances in the last decade, for good reason. Specifically intervening with a disease-causing gene can revert the deleterious phenotype while eliminating the toxicity often associated with broad-spectrum agents. Unfortunately, because these selective agents hit one target in a single location, acquired resistance is often high. An arguably better treatment approach includes coupling multiple targeted agents or using an agent that hits an individual target in several independent locations and/or alters multiple relevant targets in the disease-causing pathway(s), precisely the approach taken by Nishimura and colleagues in their recent report aimed at identifying a better treatment option for ovarian cancer.

An efficient bioorthogonal strategy using CuAAC click chemistry for radiofluorinations of SNEW peptides and the role of copper depletion.

The EphB2 receptor is known to be overexpressed in various types of cancer and is therefore a promising target for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of EphB2-overexpressing tumors, monitoring responses to therapy directed toward EphB2, and thus improvement in patient outcomes. We report the synthesis and evaluation of several fluorine-18-labeled peptides containing the SNEW amino acid motif, with high affinity for the EphB2 receptor, for their potential as radiotracers in the non-invasive imaging of cancer using PET. For the purposes of radiofluorination, EphB2-antagonistic SNEW peptides were varied at the C terminus by the introduction of L-cysteine, and further by alkyne- or azide-modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [(18)F]AFP and [(18)F]BFP were applied, and their capacity to introduce fluorine-18 was compared with that of the established building block [(18)F]FBAM. Copper-assisted Huisgen 1,3-dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide- or alkyne-modified SNEW peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.

Therapeutic synergy between microRNA and siRNA in ovarian cancer treatment.

UNLABELLED: Development of improved RNA interference-based strategies is of utmost clinical importance. Although siRNA-mediated silencing of EphA2, an ovarian cancer oncogene, results in reduction of tumor growth, we present evidence that additional inhibition of EphA2 by a microRNA (miRNA) further "boosts" its antitumor effects. We identified miR-520d-3p as a tumor suppressor upstream of EphA2, whose expression correlated with favorable outcomes in two independent patient cohorts comprising 647 patients. Restoration of miR-520d-3p prominently decreased EphA2 protein levels, and suppressed tumor growth and migration/invasion both in vitro and in vivo. Dual inhibition of EphA2 in vivo using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes loaded with miR-520d-3p and EphA2 siRNA showed synergistic antitumor efficiency and greater therapeutic efficacy than either monotherapy alone. This synergy is at least in part due to miR-520d-3p targeting EphB2, another Eph receptor. Our data emphasize the feasibility of combined miRNA-siRNA therapy, and will have broad implications for innovative gene silencing therapies for cancer and other diseases. SIGNIFICANCE: This study addresses a new concept of RNA inhibition therapy by combining miRNA and siRNA in nanoliposomal particles to target oncogenic pathways altered in ovarian cancer. Combined targeting of the Eph pathway using EphA2-targeting siRNA and the tumor suppressor miR-520d-3p exhibits remarkable therapeutic synergy and enhanced tumor suppression in vitro and in vivo compared with either monotherapy alone.

Overexpression of EPH receptor B2 in malignant mesothelioma correlates with oncogenic behavior.

INTRODUCTION: Malignant pleural mesothelioma (MM) is an aggressive asbestos-associated malignancy with limited therapeutic options. This study describes the overexpression of Ephrin B2 receptor (EPHB2) in MM cell lines and tumors, and the effect of its manipulation on proliferative and invasive qualities of the disease. METHODS: Using expression arrays, we investigated EPHB2 in MM tumors compared with normal mesothelium. EPHB2 and downstream target expression were evaluated using reverse-transcriptase polymerase chain reaction and immunoblotting methods. The biological significance of EPHB2 in MM was evaluated using in vitro functional assays with and without targeting by EPHB2-short hairpin RNA or blocking peptide in two mesothelioma cell lines, HP-1 and H2595. RESULTS: EPHB2 is overexpressed in all MM cell lines, but not in benign mesothelial cells, and is significantly elevated in MM tumor tissue compared with matched normal peritoneum. Targeted knockdown of EPHB2 in HP-1 and H2595 cell lines reduced its expression and that of EPHB2 downstream targets such as matrix metalloproteinase (MMP-2) and vascular endothelial growth factor, whereas caspase 2 and caspase 8 had increased expression. Inhibition of EPHB2 resulted in a significant decrease in scratch closure (1.25-fold-1.8-fold), proliferation (1.5-fold), and invasion (1.7-fold-1.8-fold) compared with the controls. Most notably, however, EPHB2 silencing resulted in a significant increase in apoptotic proteins and activity. CONCLUSION: EPHB2 seems to play an important role in MM pathogenesis and these findings indicate that EPHB2 could serve as a potential novel therapeutic target for treatment of the disease.

EphB and Ephrin-B interactions mediate human mesenchymal stem cell suppression of activated T-cells.

Mesenchymal stromal/stem cells (MSC) express the contact-dependent erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinase family and their cognate ephrin ligands, which are known to regulate thymocyte maturation and selection, T-cell transendothelial migration, activation, co-stimulation, and proliferation. However, the contribution of Eph/ephrin molecules in mediating human MSC suppression of activated T-cells remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 are expressed by ex vivo expanded MSC, while the corresponding ligands, ephrin-B1 and EphB4, respectively, are highly expressed by T-cells. Initial studies demonstrated that EphB2-Fc and ephrin-B2-Fc molecules suppressed T-cell proliferation in allogeneic mixed lymphocyte reaction (MLR) assays compared with human IgG-treated controls. While the addition of a third-party MSC population demonstrated dramatic suppression of T-cell proliferation responses in the MLR, blocking the function of EphB2 or EphB4 receptors using inhibitor binding peptides significantly increased T-cell proliferation. Consistent with these observations, shRNA EphB2 or ephrin-B2 knockdown expression in MSC reduced their ability to inhibit T-cell proliferation. Importantly, the expression of immunosuppressive factors, indoleamine 2, 3-dioxygenase, transforming growth factor-ß1, and inducible nitric oxide synthase expressed by MSC, was up-regulated after stimulation with EphB4 and ephrin-B1 in the presence of interferon (IFN)-γ, compared with untreated controls. Conversely, key factors involved in T-cell activation and proliferation, such as interleukin (IL)-2, IFN-γ, tumor necrosis factor-α, and IL-17, were down-regulated by T-cells treated with EphB2 or ephrin-B2 compared with untreated controls. Studies utilizing signaling inhibitors revealed that inhibition of T-cell proliferation is partly mediated through EphB2-induced ephrin-B1 reverse signaling or ephrin-B2-mediated EphB4 forward signaling by activating Src, PI3Kinase, Abl, and JNK kinase pathways, activated by tyrosine phosphorylation. Taken together, these observations suggest that EphB/ephrin-B interactions play an important role in mediating human MSC inhibition of activated T cells.

EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion.

Eph/ephrin signaling has been implicated in various types of key cancer-enhancing processes, like migration, proliferation, and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase-activity profiling data published recently, we hypothesized a key role for the Eph/ephrin signaling system in medulloblastoma invasion. In primary medulloblastoma samples, a significantly higher expression of EphB2 and the ligand ephrin-B1 was observed compared with normal cerebellum. Furthermore, medulloblastoma cell lines showed high expression of EphA2, EphB2, and EphB4. Stimulation of medulloblastoma cells with ephrin-B1 resulted in a marked decrease in in vitro cell adhesion and an increase in the invasion capacity of cells expressing high levels of EphB2. The cell lines that showed an ephrin-B1-induced phenotype possessed increased levels of phosphorylated EphB2 and, to a lesser extent, EphB4 after stimulation. Knockdown of EphB2 expression by short hairpin RNA completely abolished ephrin ligand-induced effects on adhesion and migration. Analysis of signal transduction identified p38, Erk, and mTOR as downstream signaling mediators potentially inducing the ephrin-B1 phenotype. In conclusion, the observed deregulation of Eph/ephrin expression in medulloblastoma enhances the invasive phenotype, suggesting a potential role in local tumor cell invasion and the formation of metastases.

Preferential control of basal dendritic protrusions by EphB2.

The flow of information between neurons in many neural circuits is controlled by a highly specialized site of cell-cell contact known as a synapse. A number of molecules have been identified that are involved in central nervous system synapse development, but knowledge is limited regarding whether these cues direct organization of specific synapse types or on particular regions of individual neurons. Glutamate is the primary excitatory neurotransmitter in the brain, and the majority of glutamatergic synapses occur on mushroom-shaped protrusions called dendritic spines. Changes in the morphology of these structures are associated with long-lasting modulation of synaptic strength thought to underlie learning and memory, and can be abnormal in neuropsychiatric disease. Here, we use rat cortical slice cultures to examine how a previously-described synaptogenic molecule, the EphB2 receptor tyrosine kinase, regulates dendritic protrusion morphology in specific regions of the dendritic arbor in cortical pyramidal neurons. We find that alterations in EphB2 signaling can bidirectionally control protrusion length, and knockdown of EphB2 expression levels reduces the number of dendritic spines and filopodia. Expression of wild-type or dominant negative EphB2 reveals that EphB2 preferentially regulates dendritic protrusion structure in basal dendrites. Our findings suggest that EphB2 may act to specify synapse formation in a particular subcellular region of cortical pyramidal neurons.

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.