Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

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.

Δ5-Cholenoyl-amino acids as selective and orally available antagonists of the Eph–ephrin system

“…The synthesized compounds 5e21 were evaluated for their ability to disrupt the binding of biotinilated-ephrin-A1 to the EphA2 receptor, using a previously validated ELISA binding protocol [17]. The pIC 50 values for the newly synthesized compounds are reported in Table 1, together with the corresponding standard error of the mean (SEM).…”

“…Among them, derivatives of lithocholic acid (compound 1, Fig. 1) were discovered as competitive and reversible antagonists of the EphA2 receptor [16], active in prostate cancer [17] and cardiac muscle cells [18] at non-cytotoxic concentration. Other small molecules interfering with the Epheephrin system reported so far include i) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-2-hydroxybenzoic acid (also know as CPD-1, Fig.…”

Perturbation of the EphA2–EphrinA1 System in Human Prostate Cancer Cells by Colonic (Poly)phenol Catabolites

“…29 To perform binding assays, all of the compounds were dissolved in PBS as 1 mM stock solution and immediately tested to assess their interference with EphA2−ephrinA1 binding as previously described. 22 Briefly, the EphA2-Fc ectodomain was immobilized on ELISA plates (Corning 9018), and binding of biotinylated-ephrin-A1-Fc was detected using a standard colorimetric reaction developed by streptavidin−HRP and tetramethylbenzidine. Selectivity and specificity of the assay were previously assessed using nonbiotinylated ephrinA1-Fc as a ligand of the EphA2-Fc receptor.…”

“…Furthermore, Fc alone (AG714, Millipore) did not interfere with the binding process in the range of concentrations tested. 22 All of the compounds were tested three times, incubating at 100 μM for 1 h before the addition of biotinylated ephrinA1-Fc at a concentration corresponding to its K D (100 ng/mL) (Figure 2A). Compounds displacing >40% binding, namely, gallic acid, 3′,4′-dihydroxyphenylacetic acid, protocatechuic acid, pyrogallol, and pyrocatechol, were further studied to calculate the inhibitory concentration that reduced binding by 50% (IC 50 ).…”

Eph receptor tyrosine kinases in cancer stem cells

“…Another study has found lithocholic acid as a reversible competitive inhibitor of EphA2, which inhibits ephrin binding to several Eph receptors and subsequent receptor tyrosine phosphorylation (64). Although Eph receptor signaling in tumor cells often appears to be ligand-independent, interfering with ligand-receptor interactions may suppress tumor angiogenesis and signaling in the other components of the tumor microenvironment.…”