Small Molecules Can Selectively Inhibit Ephrin Binding to the EphA4 and EphA2 Receptors
ABSTRACT 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.
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ABSTRACT: Fragment-based ligand design (FBLD) approaches have become more widely used in drug discovery projects from both academia and industry, and are even often preferred to traditional high-throughput screening (HTS) of large collection of compounds (>10(5)). A key advantage of FBLD approaches is that these often rely on robust biophysical methods such as NMR spectroscopy for detection of ligand binding, hence are less prone to artifacts that too often plague the results from HTS campaigns. In this article, we introduce a screening strategy that takes advantage of both the robustness of protein NMR spectroscopy as the detection method, and the basic principles of combinatorial chemistry to enable the screening of large libraries of fragments (>10(5) compounds) preassembled on a common backbone. We used the method to identify compounds that target protein-protein interactions.Chemistry & biology 01/2013; 20(1):19-33. DOI:10.1016/j.chembiol.2012.10.015 · 6.59 Impact Factor
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ABSTRACT: EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1-based cues from their neighbors to facilitate entry into a terminal differentiation pathway.Molecular biology of the cell 11/2010; 21(22):3902-14. DOI:10.1091/mbc.E10-03-0242 · 5.98 Impact Factor
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ABSTRACT: Various thoracic inhomogeneities affect the measured surface ECG signal. Similarly when the conductivities of these inhomogeneities change the ECG may change as well. Conductivity of tissue may change due to variation in body water and electrolyte balance e.g. due to many diseases. Furthermore, there exits uncertainty regarding the correct values of tissue conductivities and their inter- and intrapatient alterations. Thus the effects of these changes on the ECG signal parameters and on the forward and inverse ECG problem may be of importance. An accurate thorax model based on finite difference method featuring the anatomy of the Visible Human Man was developed. The effects of conductivity changes were determined by increasing the conductivity by 10%-a change that can be of physiological origin. X, Y and Z components of a current dipole source located at the center of the heart were energized and corresponding ECG signals were determined. Results indicated that physiological changes of tissue conductivity produced marked changes in ECG signal, which should be considered in modelling and in ECG analysis. The results manifested the importance of the correct conductivity values. When constructing accurate models of human thorax as a volume conductor the correct tissue conductivities may be more important than the accuracy of anatomical features.19th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 1997., Chicago, Illinois; 02/1997