Cantharidin treatment inhibits hepatocellular carcinoma development by regulating the JAK2/STAT3 and PI3K/Akt pathways in an EphB4-dependent manner.

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. The tyrosine kinase receptor EphB4 promotes oncogenesis and tumor development and progression. Its inhibition is regarded as an effective strategy for the treatment of solid tumors. In the present study, we identified cantharidin as a novel EphB4 inhibitor for HCC treatment and evaluated the underlying molecular pharmacological mechanisms of action. We observed increased expression levels of EphB4 in HCC patients and a positive correlation between EphB4 and p-JAK2 levels in HCC patient samples. Knockdown of EphB4 using small interfering RNA decreased the expression levels of p-JAK2 and p-STAT3 in HCC cells, suggesting JAK2/STAT3 being a novel downstream signaling target of EphB4. Cell viability experiments revealed that the anti-cancer effect of cantharidin was positively correlated with EphB4 expression levels in HCC cell lines. We confirmed the potent antiproliferative activity of cantharidin on HepG2 cells with high expression of EphB4 and tumor xenograft. Molecular docking assay, immunoblotting assay and quantitative reverse transcription PCR assay indicated that cantharidin bound to EphB4, and thereby resulted in EphB4 suppression at mRNA and protein levels. Hep3B and SMMC-7721 cells were with low expression of EphB4. In EphB4-/HepG2, EphB4+/HepG2, and EphB4+/Hep3B cells, EphB4 knockdown alleviated the cantharidin-induced decrease in cell viability and colony formation ability and increase in apoptosis in HepG2 cells, while its overexpression exacerbated these effects in Hep3B cells and increased the apoptosis of HepG2 cells. In nude mouse models, cantharidin suppressed tumor growth more effectively in EphB4+/SMMC-7721 xenografts than in wild-type SMMC-7721 xenografts. Underlying mechanistic study showed that by targeting EphB4, cantharidin blocked a novel target, the downstream JAK2/STAT3 pathway, and the previously known target, the PI3K/Akt signaling, resulting in intrinsic apoptosis. These results indicated that cantharidin may be a potential candidate for HCC treatment by regulating the EphB4 signaling pathway.

Vascular Endothelial Receptor Tyrosine Phosphatase: Identification of Novel Substrates Related to Junctions and a Ternary Complex with EPHB4 and TIE2.

Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling.

EphrinB2-EphB4-RASA1 Signaling in Human Cerebrovascular Development and Disease.

Recent whole exome sequencing studies in humans have provided novel insight into the importance of the ephrinB2-EphB4-RASA1 signaling axis in cerebrovascular development, corroborating and extending previous work in model systems. Here, we aim to review the human cerebrovascular phenotypes associated with ephrinB2-EphB4-RASA1 mutations, including those recently discovered in Vein of Galen malformation: the most common and severe brain arteriovenous malformation in neonates. We will also discuss emerging paradigms of the molecular and cellular pathophysiology of disease-causing ephrinB2-EphB4-RASA1 mutations, including the potential role of somatic mosaicism. These observations have potential diagnostic and therapeutic implications for patients with rare congenital cerebrovascular diseases and their families.

Targeting receptor tyrosine kinase EphB4 in cancer therapy.

Eph receptors and their Eph receptor-interacting (ephrin) ligands together form an important cell communication system with diverse roles. Experimental evidence demonstrated Eph receptor bidirectional signaling with both tumor-suppressing and tumor-promoting activities in cancer cells. The tyrosine kinase EphB4, a member of the Eph receptor family, has been associated with tumor angiogenesis, growth and metastasis, thus making it a valuable and attractive target for drug design for therapeutic applications. In the past decade, many studies have focused on elucidating the structure and function of EphB4 in complex with its ligand ephrinB2 for their role in carcinogenesis. Meanwhile, an array of compounds targeting EphB4 have been studied and several selective inhibitors have been tested in clinical studies. This review discusses the structure and function of the EphB4 receptor, analyzes its potential as a target for anticancer therapy, and summarizes the information about inhibitors of EphB4 kinase activity. Conclusively, EphB4 is a challenging but promising therapeutic target in cancer.

Multivalent biomaterial platform to control the distinct arterial venous differentiation of pluripotent stem cells.

Vascular endothelial cells (ECs) differentiated from pluripotent stem cells have enormous potential to be used in a variety of therapeutic areas such as tissue engineering of vascular grafts and re-vascularization of ischemic tissues. To date, various protocols have been developed to differentiate stem cells toward vascular ECs. However, current methods are still not sufficient to drive the distinct arterial venous differentiation. Therefore, developing refined method of arterial-venous differentiation is critically needed to address this gap. Here, we developed a biomaterial platform to mimic multivalent ephrin-B2/EphB4 signaling and investigated its role in the early arterial and venous specification of pluripotent stem cells. Our results show immobilized ephrinB2 or EphB4 on hydrogel substrates have a distinct effect on arterial venous differentiation by regulating several arterial venous markers. When in combination with Wnt pathway agonist or BMP4 signaling, the ephrin-B2/EphB4 biomaterial platform can create diverging EC progenitor populations, demonstrating differential gene expression pattern across a wide range of arterial and venous markers, as well as phenotypic markers such as anti-thrombotic, pro-atherogenic and osteogenic genes, that are consistent with the in vivo expression patterns of arterial and venous ECs. Importantly, this distinct EC progenitor population cannot be achieved by current methods of applying soluble factors or hemodynamic stimuli alone, illustrating that fine-tuning of developmental signals using the biomaterial platform offers a new approach to better control the arterial venous differentiation of stem cells.

NVP-BHG712: Effects of Regioisomers on the Affinity and Selectivity toward the EPHrin Family.

Erythropoietin-producing hepatocellular (EPH) receptors are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates intracellular kinase activity. EPHs are key players in bidirectional intercellular signaling, controlling cell morphology, adhesion, and migration. They are increasingly recognized as cancer drug targets. We analyzed the binding of NVP-BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples turned out to be a regioisomer (NVPiso) of the inhibitor, initially described in a Novartis patent application. They only differ by the localization of a single methyl group on either one of two adjacent nitrogen atoms. The two compounds of identical mass revealed different binding modes. Furthermore, both in vitro and in vivo experiments showed that the isomers differ in their kinase affinity and selectivity.

Modeling EphB4-EphrinB2 protein-protein interaction using flexible docking of a short linear motif.

BACKGROUND: Many protein-protein interactions are mediated by a short linear motif. Usually, amino acid sequences of those motifs are known or can be predicted. It is much harder to experimentally characterize or predict their structure in the bound form. In this work, we test a possibility of using flexible docking of a short linear motif to predict the interaction interface of the EphB4-EphrinB2 complex (a system extensively studied for its significance in tumor progression). METHODS: In the modeling, we only use knowledge about the motif sequence and experimental structures of EphB4-EphrinB2 complex partners. The proposed protocol enables efficient modeling of significant conformational changes in the short linear motif fragment during molecular docking simulation. For the docking simulations, we use the CABS-dock method for docking fully flexible peptides to flexible protein receptors (available as a server at http://biocomp.chem.uw.edu.pl/CABSdock/ ). Based on the docking result, the protein-protein complex is reconstructed and refined. RESULTS: Using this novel protocol, we obtained an accurate EphB4-EphrinB2 interaction model. CONCLUSIONS: The results show that the CABS-dock method may be useful as the primary docking tool in specific protein-protein docking cases similar to EphB4-EphrinB2 complex-that is, where a short linear motif fragment can be identified.

EphrinB2/EphB4 pathway in postnatal angiogenesis: a potential therapeutic target for ischemic cardiovascular disease.

Ischemic cardiovascular disease remains one of the leading causes of morbidity and mortality in the world. Proangiogenic therapy appears to be a promising and feasible strategy for the patients with ischemic cardiovascular disease, but the results of preclinical and clinical trials are limited due to the complicated mechanisms of angiogenesis. Facilitating the formation of functional vessels is important in rescuing the ischemic cardiomyocytes. EphrinB2/EphB4, a novel pathway in angiogenesis, plays a critical role in both microvascular growth and neovascular maturation. Hence, investigating the mechanisms of EphrinB2/EphB4 pathway in angiogenesis may contribute to the development of novel therapeutics for ischemic cardiovascular disease. Previous reviews mainly focused on the role of EphrinB2/EphB4 pathway in embryo vascular development, but their role in postnatal angiogenesis in ischemic heart disease has not been fully illustrated. Here, we summarized the current knowledge of EphrinB2/EphB4 in angiogenesis and their interaction with other angiogenic pathways in ischemic cardiovascular disease.

EphB4 localises to the nucleus of prostate cancer cells.

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

N-(2,4)-dinitrophenyl-L-arginine Interacts with EphB4 and Functions as an EphB4 Kinase Modulator.

The erythropoietin-producing hepatocellular carcinoma receptor B4 is a receptor tyrosine kinase whose expression is preserved in various malignancies, including colon, gastric, and breast carcinoma. Hepatocellular carcinoma receptor B4 presence in tumor cells and involvement in cancer suppression makes it a potential therapeutic target for activating compounds. Moreover, modulators of its activity also have a strong potential to be used in diagnosis and therapy monitoring. We used virtual ligand screening to identify novel hepatocellular carcinoma receptor B4 kinase modulators for experimental testing. Three independent assay platforms confirmed that dinitrophenyl-L-arginine is likely to affect the kinase activity of hepatocellular carcinoma receptor B4. An enzyme-coupled spectrophotometric assay has been used to examine this possibility and may prove to be useful for assessing other novel kinase modulator candidates. Overall, our observations suggest that dinitrophenyl-L-arginine has an activating effect on hepatocellular carcinoma receptor B4 and, therefore, more efficient derivatives may have therapeutic effects in tumors where hepatocellular carcinoma receptor B4 exhibits antimalignant properties. The hepatocellular carcinoma receptor B4-activating effect is discussed with respect to previously described mechanisms, using predicted and experimental structures for docked ligands. As a novel kinase activity modulator, dinitrophenyl-L-arginine may provide new insights into molecular mechanisms by which kinases are activated or regulated, and may serve as a lead compound for the generation of novel hepatocellular carcinoma receptor B4-activating therapeutic compounds.

Pyrrolo[3,2-b]quinoxaline derivatives as types I1/2 and II Eph tyrosine kinase inhibitors: structure-based design, synthesis, and in vivo validation.

The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I inhibitors previously discovered in silico (compounds A and B) were used to design type I1/2 and II inhibitors. Chemical synthesis of about 25 derivatives culminated in the discovery of compounds 11d (type I1/2), 7b, and 7g (both of type II), which have low-nanomolar affinity for Eph kinases in vitro and a good selectivity profile on a panel of 453 human kinases (395 nonmutant). Surface plasmon resonance measurements show a very slow unbinding rate (1/115 min) for inhibitor 7m. Slow dissociation is consistent with a type II binding mode in which the hydrophobic moiety (trifluoromethyl-benzene) of the inhibitor is deeply buried in a cavity originating from the displacement of the Phe side chain of the so-called DFG motif as observed in the crystal structure of compound 7m. The inhibitor 11d displayed good in vivo efficacy in a human breast cancer xenograft.

Completing the structural family portrait of the human EphB tyrosine kinase domains.

The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X-ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild-type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand-binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis-phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme-selective kinase inhibitors for mechanistic studies and therapeutic applications.

Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library.

The inability to generate soluble, correctly folded recombinant protein is often a barrier to successful structural and functional studies. Access to affordable synthetic genes has, however, made it possible to design, make and test many more variants of a target protein to identify suitable constructs. We have used rational design and gene synthesis to create a controlled randomised library of the EphB4 receptor tyrosine kinase, with the aim of obtaining soluble, purifiable and active catalytic domain material at multi-milligram levels in Escherichia coli. Three main parameters were tested in designing the library--construct length, functional mutations and stability grafting. These variables were combined to generate a total of 9720 possible variants. The screening of 480 clones generated a 3% hit rate, with a purifiable solubility of up to 15 mg/L for some EphB4 constructs that was largely independent of construct length. Sequencing of the positive clones revealed a pair of hydrophobic core mutations that were key to obtaining soluble material. A minimal kinase domain construct containing these two mutations exhibited a +4.5°C increase in thermal stability over the wild-type protein. These approaches will be broadly applicable for solubility engineering of many different protein target classes. Atomic coordinates and structural factors have been deposited in PDB under the accession 2yn8 (EphB4 HP + staurosporine).

EphB4 cellular kinase activity assayed using an enzymatic protein interaction system.

Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.

Optimization of inhibitors of the tyrosine kinase EphB4. 2. Cellular potency improvement and binding mode validation by X-ray crystallography.

Inhibition of the tyrosine kinase erythropoietin-producing human hepatocellular carcinoma receptor B4 (EphB4) is an effective strategy for the treatment of solid tumors. We have previously reported a low nanomolar ATP-competitive inhibitor of EphB4 discovered in silico by fragment-based high-throughput docking combined with explicit solvent molecular dynamics simulations. Here we present a second generation of EphB4 inhibitors that show high inhibitory potency in both enzymatic and cell-based assays while preserving the appealing selectivity profile exhibited by the parent compound. In addition, respectable levels of antiproliferative activity for these compounds have been obtained. Finally, the binding mode predicted by docking and molecular dynamics simulations is validated by solving the crystal structures of three members of this chemical class in complex with the EphA3 tyrosine kinase whose ATP-binding site is essentially identical to that of EphB4.

Biophysical characterization and stabilization of the recombinant albumin fusion protein sEphB4-HSA.

The use of albumin fusion proteins as therapeutic drug candidates is an attractive approach to design novel biopharmaceuticals with increased circulation time in vivo. The purpose of this work was to characterize and stabilize the fusion protein sEphB4-human serum albumin (HSA), a 120 kDa protein containing the extracellular domain of EphB4 and HSA, and to identify stabilizing excipients for storage. Comparative biophysical studies combined with empirical phase diagram analysis show that both structural integrity and conformational stability of sEphB4 and sEphB4-HSA are best maintained above pH 5 and below 50 °C, with different structural phases observed outside this range. A major physical degradation pathway for sEphB4-HSA is formation of soluble aggregates. Excipient studies using size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and fluorescence spectroscopy identified disaccharide sugars (e.g., sucrose and trehalose) as effective stabilizers against protein aggregation, and NaCl as an effective stabilizer for protecting overall conformational stability. A combination of biophysical studies with sEphB4-HSA and its individual component proteins (sEphB4 and HSA), along with correlation analysis of SEC and DSC stability data in the presence of different excipients suggest that the aggregation pathway of the albumin fusion protein is primarily mediated by the sEphB4 rather than the HSA component.

Controllable delivery of small-molecule compounds to targeted cells utilizing carbon nanotubes.

Carbon nanotubes (CNTs) have emerged as a new alternative and efficient tool for transporting molecules with biotechnological and biomedical applications, because of their remarkable physicochemical properties. Encapsulation of functional molecules into the hollow chambers of CNTs can not only stabilize encapsulated molecules but also generate new nanodevices. In this work, we have demonstrated that CNTs can function as controllable carriers to transport small-molecule compounds (SMCs) loaded inside their hollow tunnels onto targeted cells. Using indole as model compound, CNTs can protect indole molecules during transportation. Labeling indole-loaded CNTs (indole@CNTs) with EphB4-binding peptides generates cell-homing indole@CNTs (CIDs). CIDs can selectively target EphB4-expressing cells and release indole onto cell surfaces by near-infrared (NIR) irradiation. Released indole molecules exhibit significant cell-killing effects without causing local overheating. This establishes CNTs as excellent near-infrared controllable delivery vehicles for SMCs as selective cell-killing agents.

Novel EphB4 monoclonal antibodies modulate angiogenesis and inhibit tumor growth.

EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis.

Library screening by fragment-based docking.

We review our computational tools for high-throughput screening by fragment-based docking of large collections of small molecules. Applications to six different enzymes, four proteases, and two protein kinases, are presented. Remarkably, several low-micromolar inhibitors were discovered in each of the high-throughput docking campaigns. Probable reasons for the lack of submicromolar inhibitors are the tiny fraction of chemical space covered by the libraries of available compounds, as well as the approximations in the methods employed for scoring, and the use of a rigid conformation of the target protein.

Structure-based optimization of potent and selective inhibitors of the tyrosine kinase erythropoietin producing human hepatocellular carcinoma receptor B4 (EphB4).

The tyrosine kinase EphB4 is an attractive target for drug design because of its recognized role in cancer-related angiogenesis. Recently, a series of commercially available xanthine derivatives were identified as micromolar inhibitors of EphB4 by high-throughput fragment-based docking into the ATP-binding site of the kinase domain. Here, we have exploited the binding mode obtained by automatic docking for the optimization of these EphB4 inhibitors by chemical synthesis. Addition of only two heavy atoms, methyl and hydroxyl groups, to compound 4 has yielded the single-digit nanomolar inhibitor 66, with a remarkable improvement of the ligand efficiency from 0.26 to 0.37 kcal/(mol per non-hydrogen atom). Compound 66 shows very high affinity for a few other tyrosine kinases with threonine as gatekeeper residue (Abl, Lck, and Src). On the other hand, it is selective against kinases with a larger gatekeeper. A 45 ns molecular dynamics (MD) simulation of the complex of EphB4 and compound 66 provides further validation of the binding mode obtained by fragment-based docking.