Discovery, development and pharmacological characterization of small molecules interfering with Eph-ephrin system

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands are involved in many biological processes as cell migration and morphology, axon guidance, synaptic plasticity and angiogenesis both during embryogenesis and in adult tissues. Alterations of this system have been found in human cancers and above all, EphA2 and EphB4 overexpression is often correlated with aggressive tumor phenotypes and poor prognosis. Based on this evidence Eph-ephrin system represents a promising target in cancer field. Through an ELISA-based binding screening, we recently identified lithocholic acid (LCA), a secondary bile acid able to modulate Eph-ephrin activity showing a Ki value of 49 µM. LCA resulted a novel specific, reversible, not-selective antagonist of Eph receptors, able to dose-dependently inhibit Eph receptor phosphorylation induced by ephrin ligand in different cell lines and able to inhibit ephrin-A1-induced PC3 cell rounding. Moreover, Surface Plasmon Resonance analysis (SPR) confirmed the specific and reversible binding of LCA to EphA2 receptor, excluding its interaction with ephrin-A1 ligand. Therefore, LCA scaffold was used to design and synthesize a new series of derivatives analyzing the structure-activity relationship and describing the pharmacophoric structure. In particular, we found that the concurrent presence of a large hydrophobic region (cyclopenta[a]perhydrophenantrene scaffold) and an anionic hydrogen bond acceptor group (carboxylate functionality) are essential for the inhibition of EphA2–ephrin-A1 binding. Cholanic acid (Ki = 5.1 µM) and isolithocholic acid (Ki = 25 µM) emerged from this series. Moreover, we observed that the conjugation of the lateral acidic chain with glycine generated a compound (glycolithocholic acid, Ki= 38.5 µM) endowed with an activity similar to LCA. Starting from this discovery, a second series of derivatives, where LCA structure is coupled with amino acids was generated and a new compound emerged: L-Trp LCA (Ki= 1.2 µM), which is structurally formed by LCA structure conjugated with L-Tryptophan. As a result, the most active and promising compounds (cholanic acid, isolithocholic acid and L-Trp-LCA) were further pharmacologically characterized in functional assays. Investigations on PC3, T47D ad HUVE cells revealed that all the compounds able to disrupt EphA2–ephrin-A1 binding in the ELISA assay, were specific, reversible Eph receptors antagonist that dose-dependently inhibited Eph phosphorylation induced by ephrin ligands. Surprisingly, isolithocholic acid showed similar potency values in EphA2 antagonism compared to cholanic acid and L-Trp LCA, due to its activity on EphA2 kinase that is not shared by the other derivatives. Compounds were also able to inhibit PC3 cell rounding and retraction induced by ephrin-A1 ligand, and L-Trp LCA was particularly active in blocking these cell morphology changes. Moreover, through the tube formation assay performed in HUVECs, the antiangiogenic properties of the compounds have been evaluated. Among the active compounds, cholanic acid resulted the most potent in the inhibition of vessels reticulation, at not-cytotoxic concentrations. Beside LCA, in our ELISA binding based screening we identified also nine plant extracts, rich of polyphenols, commonly used as food supplements. In displacement studies they were able to reversibly inhibit EphA2–ephrin-A1 binding showing IC50 values between 0.83-24 µg/ml. The active extracts were further characterized and showed also specific antagonistic properties toward EphA2 receptor. All together, these findings could be useful to better characterize the Eph-ephrin system in its complexity as well as to figure out the role played by this system in physiological and pathological conditions. Finally, the identification of a pharmacophoric scaffold could be an interesting starting point for a rational chemical synthesis of new compounds with better affinity and improved physicochemical properties.