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The Small Molecule Specific Ephb4 Kinase Inhibitor NVP-BHG712

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The Small Molecule Specific Ephb4 Kinase Inhibitor NVP-BHG712 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Crossref Angiogenesis (2010) 13:259–267 DOI 10.1007/s10456-010-9183-z ORIGINAL PAPER The small molecule specific EphB4 kinase inhibitor NVP-BHG712 inhibits VEGF driven angiogenesis Georg Martiny-Baron • Philipp Holzer • Eric Billy • Christian Schnell • Joseph Brueggen • Mireille Ferretti • Niko Schmiedeberg • Jeanette M. Wood • Pascal Furet • Patricia Imbach Received: 13 July 2010 / Accepted: 5 August 2010 / Published online: 29 August 2010 Ó The Author(s) 2010. This article is published with open access at Springerlink.com Abstract EphB4 and its cognitive ligand ephrinB2 play kinases in biochemical as well as in cell based assays. an important role in embryonic vessel development and Furthermore, NVP-BHG712 shows excellent pharmacoki- vascular remodeling. In addition, several reports suggest netic properties and potently inhibits EphB4 autophos- that this receptor ligand pair is also involved in pathologic phorylation in tissues after oral administration. In vivo, vessel formation in adults including tumor angiogenesis. NVP-BHG712 inhibits VEGF driven vessel formation, Eph/ephrin signaling is a complex phenomena character- while it has only little effects on VEGF receptor (VEGFR) ized by receptor forward signaling through the tyrosine activity in vitro or in cellular assays. The data shown here kinase of the receptor and ephrin reverse signaling through suggest a close cross talk between the VEGFR and EphR various protein–protein interaction domains and phos- signaling during vessel formation. In addition to its phorylation motifs of the ephrin ligands. Therefore, inter- established function in vascular remodeling and endothelial fering with EphR/ephrin signaling by the means of targeted arterio-venous differentiation, EphB4 forward signaling gene ablation, soluble receptors, dominant negative appears to be an important mediator of VEGF induced mutants or antisense molecules often does not allow to angiogenesis since inhibition of EphB4 forward signaling discriminate between inhibition of Eph/ephrin forward and is sufficient to inhibit VEGF induced angiogenesis. reverse signaling. We developed a specific small molecular weight kinase inhibitor of the EphB4 kinase, NVP- Keywords Angiogenesis Á EphB4 Á EphrinB2 Á VEGF Á BHG712, which inhibits EphB4 kinase activity in the low Protein kinase inhibitor nanomolar range in cellular assays showed high selectivity for targeting the EphB4 kinase when profiled against other Introduction G. Martiny-Baron (&) Formation of the vascular network is tightly controlled by a Novartis Institutes for Biomedical Research, Expertise Platform variety of growth factors and their appropriate receptors. Kinases, Novartis Campus, Fabrikstrasse 16, WJS152.1.72.2, Thus, receptor tyrosine kinases which are expressed on 4056 Basel, Switzerland endothelial cells (ECs) have been shown to be crucial for e-mail: [email protected] embryonic vasculogenesis and angiogenesis (for review see P. Holzer Á N. Schmiedeberg Á P. Furet Á P. Imbach [1]). While VEGF (vascular endothelial growth factor) Novartis Institutes for Biomedical Research, Global Discovery receptors activated by VEGF mainly contribute to very Chemistry, Basel, Switzerland early steps of vasculogenesis and angiogenesis including E. Billy Á C. Schnell Á J. Brueggen Á M. Ferretti differentiation of EC precursors and formation of a prim- Novartis Institutes for Biomedical Research, Oncology, Basel, itive vascular network, two other receptor tyrosine kinases Switzerland and their ligands, namely the angiopoietin/TIE-2 (tyrosine kinase with Ig like loops and EGF like repeats 2) and Present Address: J. M. Wood ephrinB2/EphB4 respectively, contribute to later steps of S*BIO Pte Ltd, Singapore, Singapore vascular development like vessel branching and vessel 123 260 Angiogenesis (2010) 13:259–267 maturation and the recruitment of EC associated pericytes/ receptor inhibits angiogenesis in a VEGF driven in vivo smooth muscle cells. model, which has been used to characterize small molecule The Eph receptors (receptor cloned from erythropoietin- inhibitors of the VEGFR2 kinase [29]. Therefore, we tested producing hepatocellular carcinoma) are the largest sub- whether the selective inhibitor of the EphB4 kinase NVP- family of receptor tyrosine kinases and are activated by BHG712 would mimic the effect of soluble EphB4 in this their cell surface bound ligands of the equally large ephrin VEGF driven angiogenesis model. To our knowledge, this family (Eph receptor interacting protein). Both, receptors is the first report demonstrating that a selective small and ligands are divided into an A- and B-class; nine EphA molecule kinase inhibitor of the EphB4 kinase is sufficient receptors bind to five A-ligands and five EphB receptors to inhibit VEGF driven angiogenesis in vivo. bind to three ephrinB ligands. Like for other receptor tyrosine kinases (RTKs), receptor signaling is initiated by oligomerization and tyrosine-phosphorylation of the Methods receptor. Unlike other ligand/RTK pairs, also ephrin ligands can transmit signals upon receptor engagement, a Synthesis of NVP-BHG712 phenomenon which is called bi-directional signaling. EphrinB-ligands, which possess a transmembrane and Synthesis of NVP-BHG712 was performed in the labora- cytoplasmatic domain, get tyrosine-phosphorylated upon tories of Novartis Institutes for Biomedical Research and receptor binding which induces ephrin reverse signaling in has been described in the patent application WO2007/ addition to the receptor forward signaling (for review see 062805 [30]. [2]). Although quite promiscuous interactions between EphRs and their ligands the ephrins have been observed, In vitro kinase assays ephrinB2 is the only known ligand for EphB4. EphB4 and ephrinB2 were the first genes discovered to be differen- All in vitro kinase assays were performed with recombi- tially expressed in arterial and venous endothelium and nant purified kinases either purchased from external ven- genetic ablation of either EphB4 or ephrinB2 are embry- dors or produced in house. To estimate kinase activity both, TM onic lethal due to impaired vascular remodeling and arrest TR-FRET-based LanthaScreen and Caliper mobility shift of endothelial differentiation [3, 4]. were used. The assays have been described previously Over-expression of EphB4 has been reported for several [31, 32]. TM tumor types including breast, colon, bladder, endometrium In brief, the LanthaScreen assay technology is based on and ovary [5–10]. The role of EphB4 in cancer is not fully the discrimination between the unphosphorylated substrate elucidated. On the one side, EphB4 expressed on tumor and the phosphorylated product by a phospho-specific cells can interact with ephrinB2 of the endothelium and antibody, binding only to the phosphorylated version of the thereby promotes tumor angiogenesis and growth [11], on substrate. Both, antibody and substrate, carry fluorescent the other hand EphB4 has been described to either promote labels and the close proximity of the labels in the formed or suppress tumor growth, depending on the tumor type and complex allows the measurement of a fluorescence reso- tumor model which has been looked at [5, 12–15]. The nance energy transfer (FRET) signal. Reading of the FRET paradoxes of EphB4 signaling in cancer has been recently signal in a time-dependent/time-gated manner further discussed [16]. improves the assay performance by reducing background Despite this, EphB4 and ephrinB2 signaling has been fluorescence [33]. For dose–response measurements NVP- proven to be involved in sites of adult angiogenesis by the BHG712 was pre-diluted in 90% DMSO and 50 nL of use of soluble EphB4 [17, 18]. In addition, monoclonal compound solutions were dispensed directly into the empty inhibitory antibodies and peptides, blocking EphB4-eph- assay plate using a HummingBird nanodispenser (Zinsser rinB2 interaction have been developed to study the role of Analytic). The kinase reactions were started by addition of EphB4/ephbrinB2 signaling [19–21]. Further tools which 4.5 lL ATP solution (4 lM ATP, 20 mM Tris/HCL, 1 mM have been developed in the context of EphB4/ephrinB2 DTT, 0.03% Tween20, 0.01 mM Na3VO4) and 4.5 lL signaling include engineered mouse models [3, 4, 22, 23], enzyme/substrate mix (100 nM fluorescein poly-GAT; soluble receptors [5, 17, 24] and peptides blocking ligand Invitrogen), 0.5% bovine serum albumin, 20 mM Tris/ receptor interaction [19, 20]. HCL, 1 mM DTT, 0.03% Tween20, 0.01 mM Na3VO4). Small molecule inhibitors of the EphB4 kinase have Further components of the enzyme/substrate mix were the been described recently by others with different degree of enzymes as well as MgCl2/MnCl2 which were adjusted specificity against other kinases [25–28]. So far, no efficacy specifically to the requirements of the individual enzyme. data from in vivo studies for these compounds have been After incubation for 60 min at r.t. the kinase reactions were reported. In our studies we noticed, that soluble EphB4 stopped by addition of 4.5 lL stop solution (50 mM EDTA 123 Angiogenesis (2010) 13:259–267 261 pH 8.0, 0.04% NP-40, 20 mM Tris/HCl pH 7.4) followed proteins were immunoprecipated using either anti-V5-epi- by 4.5 lL of detection mix (1.72 lg/mL Tb-PY20 anti- tope or anti-myc-epitope antibodies and analyzed by wes- body; Invitrogen), 1% bovine serum albumin, 20 mM Tris/ tern using the mab 4G10 anti-phospho-tyrosine antibody. HCl, 1 mM DTT, 0.03% Tween20,
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