Small Molecules Can Selectively Inhibit Ephrin Binding to the EphA4 and EphA2 Receptors

Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2008; 283(43):29461-72. DOI: 10.1074/jbc.M804103200
Source: PubMed


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.


Available from: Roberta Noberini, Nov 18, 2015
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    • "Hippocampal neurons were treated with either AβO or AβO-FITC (3 or 5 μM, as described in the respective figures) alone, or they were co-treated with the KYL peptide (KYLPYWPVLSSL; 30–60 μM, GenScript, Piscataway, NJ, USA Inc.) [28], [33], [34] or STI (1–5 μM, Novartis, Basel, Switzerland) [9]. The expression vector encoding Flag-EphA4 was a kind gift from Drs. Fumitoshi Irie and Yu Yamaguchi (Sanford-Burnham Medical Research Institute, USA). "
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    ABSTRACT: The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-β oligomers (AβOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AβOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AβOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AβOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AβOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AβOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AβOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AβOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AβOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.
    PLoS ONE 03/2014; 9(3):e92309. DOI:10.1371/journal.pone.0092309 · 3.23 Impact Factor
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    • "Therefore our findings suggest that ephrinB1 does not control learning and memory through direct modulation of synaptic transmission or synaptic plasticity, but instead plays a role in postnatal cortical maturation. Given that ephrinB2 and ephrinB3 have been shown to directly modulate hippocampal synaptic plasticity and learning and memory [50], [51], our findings show that ephrinB1 impacts learning and memory through a mechanism that is distinct from other ephrinBs. Further analyses of EfnB1Y/− adult brain showed a significant increase in the number of neurons only in the cortex of EfnB1Y/− mice. "
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    ABSTRACT: Eph receptors and their ephrin ligands play critical roles in the development of the nervous system, however, less is known about their functions in the adult brain. Here, we investigated the function of ephrinB1, an ephrinB family member that is mutated in CranioFrontoNasal Syndrome. We show that ephrinB1 deficient mice (EfnB1(Y/-) ) demonstrate spared spatial learning and memory but exhibit exclusive impairment in non-spatial learning and memory tasks. We established that ephrinB1 does not control learning and memory through direct modulation of synaptic plasticity in adults, since it is not expressed in the adult brain. Rather we show that the cortex of EfnB1(Y/-) mice displayed supernumerary neurons, with a particular increase in calretinin-positive interneurons. Further, the increased neuron number in EfnB1(Y/-) mutants correlated with shorter dendritic arborization and decreased spine densities of cortical pyramidal neurons. Our findings indicate that ephrinB1 plays an important role in cortical maturation and that its loss has deleterious consequences on selective cognitive functions in the adult.
    PLoS ONE 02/2014; 9(2):e88325. DOI:10.1371/journal.pone.0088325 · 3.23 Impact Factor
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    • "Several different synthetic peptides and small molecules have been studied for this purpose; the advantage of these artificial ligands is that they can show a much higher selectivity compared to the physiological ephrin ligands. A number of linear peptides and small molecules have been reported to act as artificial ligands of Eph receptors: KYL and other linear peptides for EphA4 [25]; TNYL-RAW peptide for EphB4 [26]; SWL and other peptides for EphA2 [27]; and dimethyl-pyrrole derivatives for EphA2 and EphA4 [28]. "
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    ABSTRACT: The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. We used a combination of phage peptide display and computer modeling/docking approaches and discovered a novel cyclic nonapeptide, now designated TYY. This peptide selectively inhibits the binding of the ephrinA5 ligand with EphA4 and significantly blocks angiogenesis in a 3D matrigel culture system. Molecular docking reveals that TYY recognizes the same binding pocket on EphA4 that the natural ephrin ligand binds to and that the Tyr3 and Tyr4 side chains of TYY are both critical for the TYY/EphA4 interaction. The discovery of TYY introduces a valuable probe of EphA4 function and a new lead for EphA4-targeted therapeutic development.
    PLoS ONE 11/2013; 8(11):e80183. DOI:10.1371/journal.pone.0080183 · 3.23 Impact Factor
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