Abstract
Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.
Highlights
The Eph receptor tyrosine kinases belong to the largest family of tyrosine kinase receptors
We describe the discovery of lithocholic acid (LCA), a secondary bile acid, as a novel competitive, reversible antagonist of the Eph-ephrin system
All the compounds of the chemical collection were incubated for 1 hour at the concentration of 200 mM and only lithocholic acid resulted to significantly reduce EphA2-ephrinA1 binding
Summary
The Eph receptor tyrosine kinases belong to the largest family of tyrosine kinase receptors. Eph-ephrin binding within the same class is highly promiscuous and inter-class binding examples have been reported [2,3]. The membrane-bound protein nature of ephrin ligands gives particular features to this system. Cell-cell contact is needed to activate the system, even if ephrins A released or cleaved from cells retain the ability to activate Eph receptors [4,5,6]. Bidirectional signals are generated by Ephephrin interaction: forward signals into the cells expressing Eph receptors go along with reverse signals into the cells bearing ephrin ligands. Increasing evidence shows that Eph receptors and ephrins can transmit signals independently of their interaction, through crosstalk with other signalling pathways [7]
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