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.
- SourceAvailable from: Bainan Wu
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- "For instance, the APY, KYL, and VTM peptides (which were named based on the first three amino acids of their sequences) bind to EphA4 tightly with K d values in the low micromolar range (Lamberto et al., 2012; Murai et al., 2003). In addition, a few small molecular weight compounds that inhibit ephrin binding to EphA4 at low micromolar concentration have also been reported from HTS campaigns (Giorgio et al., 2011; Noberini et al., 2008, 2011a, 2011b; Qin et al., 2008). However, their detailed mechanism of action remains unclear and likely complex, possibly involving compound oxidations or covalent binding, which are typical issues encountered in traditional HTS hits (Baell and Holloway , 2010; Noberini et al., 2008, 2011a, 2011b). "
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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- "EphA2 border localization was disrupted by a function-blocking antibody that interferes with E-cadherin–mediated adhesion (Supplemental Figure S1A). Moreover, contact-dependent activation of EphA2 was perturbed by a 2,5 dimethylpyrrolyl benzoic acid (DMBA) derivative (Supplemental Figure S1B) that inhibits ephrin-A1 binding to EphA2 (Noberini et al., 2008), providing more direct evidence that ligands and not other kinases trigger EphA2 phosphorylation in response to cell– cell contact. However, long-term treatment with DMBA also altered EphA1 expression levels, limiting its utility for further studies. "
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: The zebrafish is an in vivo model system originally used to study development of vertebrates. Over the last years it has gained a more important role in studies focusing on diseases such as cancer and thrombosis. Platelets play an important role in thrombosis and therefore drugs inhibiting platelet enzymes and receptors are developed to prevent thrombosis. The most effective drugs inhibit the COX enzymes (aspirin) or the platelet activating receptor P2Y-12 (clopidogrel among others). Lack of clarity exists about the P2Y12 receptor. In T2DM patients, there is speculation that there is less inhibition by 16 antagonists. Also the current insight in P2Y12 signaling is limited to suppression of production of cAMP (a platelet inhibitor) and activation of protein kinase B/Rap1b (which stimulate aggregation). Aims of the thesis: (1) To evaluate the position of the zebrafish as a model system in research on platelet function and thrombus; (2) Discuss the main problems the currently most used anti-platelet drugs (aspirin and clopidogrel) possess; (3) To study the inhibition of the P2Y-12 receptor in diabetes patients and to demonstrate which secondary signalling molecules are activated by P2Y-12