Discovery and structural analysis of Eph receptor tyrosine kinase inhibitors

Abstract

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC 50 s of 40 nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the "DFG-out" inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.The Eph/ephrin family is the largest among tyrosine kinases and is unique in that the ligands and receptors are both membrane bound providing the possibility for bidirectional cell-cell signaling. Genome analysis reveals that there are 14 Eph receptors and eight ephrin ligands. Eph receptor signaling is responsible for arguably the most diverse set of biological phenomena of any tyrosine kinase family including organ development, tissue remodeling, neuronal signaling, insulin secretion, and bone metabolism. 1, 2 Not surprisingly, deregulation of ephrindependent signaling has been implicated in pathological conditions related to all of these systems. 3 The involvement of Eph/ephrin-signaling in tumorigenesis has been the most extensively investigated due to frequent upregulation of Eph receptor or ligand expression in numerous tumor types. [4][5][6] The emerging picture is complicated by the diversity of biological function that is associated with individual receptors and ligands, including oncogenic or tumor suppressor functions. The possibility of targeting Eph/ephrins therapeutically may be the most straightforward in the context of inhibiting Eph/ephrin-signaling in the vasculature as a means of preventing tumor angiogenesis. 2,6 Currently only a few small molecule Eph kinase © 2009 Elsevier Ltd. All rights reserved. * Corresponding Authors: Sirano Dhe-Paganon, Tel: 416-946-3876, E-mail: sirano.dhepaganon(@)utoronto.ca Nathanael Gray, Tel: 617-582-8590, E-mail: nathanael_gray(@)dfci.harvard.edu . † These authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ¶ Once inside PyMol, press the play button (at the bottom right) in order to start the 3D movie NIH Public Access To date the majority of investigation into functions of Eph receptor tyrosine kinases has been accomplished using genetic and biochemical methods. Pharmacological app...

Overexpression of Ephrin A2 receptors in cancer stromal cells is a prognostic factor for the relapse of gastric cancer

“…Recently, overexpression of EphA2 or A4 was also reported to be associated with poor prognosis in patients with gastric cancer [25]. We observed expression of EphA2 in cancer cells from 65 of 107 (60.7 %) patients with gastric cancer, suggesting that EphA2 may be a good molecular target in gastric cancer, [26,27], siRNA/oligonucleotides as inhibitors of Eph expression [28,29], peptides/mAb that inhibit Eph-ephrin interactions [30,31], cytotoxic mAbs and mAb conjugates [32,33], and nanoparticles/mAb as imaging agents [34][35][36]. Most of these molecules were found to target cancer cells but not stromal cells, and their effects on tumor progression involving cancer stromal cell interactions were unclear.…”

Pharmacological inhibition of Eph receptors enhances glucose-stimulated insulin secretion from mouse and human pancreatic islets

“…Both Eph RTK inhibitors and their control compounds, including IM, are type II kinase inhibitors [12,16]. As there are no reported Eph inhibitors that are selective for a single Eph RTK family member, these compounds are pan-Eph inhibitors, although they exhibit substantially reduced affinity for EphA1, EphA6, EphA7 and EphB1 [12]. Importantly, IM and the two control compounds do not affect EphA5 receptor phosphorylation and consistently do not affect GSIS from both mouse and human islets.…”

“…In addition, the data reveal that small molecular weight Eph inhibitors can increase glucose-induced plasma insulin levels and improve glucose tolerance in mice. Both Eph RTK inhibitors and their control compounds, including IM, are type II kinase inhibitors [12,16]. As there are no reported Eph inhibitors that are selective for a single Eph RTK family member, these compounds are pan-Eph inhibitors, although they exhibit substantially reduced affinity for EphA1, EphA6, EphA7 and EphB1 [12].…”

“…1a), compound 9 (ESM Fig. 1b) and controls were synthesised as previously described [12]. Based on 1 H nuclear magnetic resonance (NMR) spectroscopy, their purity was estimated to be 95-99% (ESM Figs 2 and 3).…”

EPH-EPHRIN in human gastrointestinal cancers

“…Choi et al [86] reported the discovery of EPHB2 receptor kinase inhibitors. They also performed crystallographic analyses of EPHA3 and EPHA7 in complex with their inhibitors and discussed the possibility of generating new inhibitors using a structure-based design [86] .…”