Discovery and development of Eph-ephrin antagonists endowed with antiangiogenic properties

Eph-ephrin system is involved in many biological processes including cell migration and morphology, axon guidance, synaptic plasticity and angiogenesis both during embryogenesis and in adult tissues. Many reports correlates a deregulation of Eph-ephrin system to aggressive tumor phenotypes in a number of human cancers and, in particular EphA2 and EphB4 overexpression, is often associate with a poor prognosis. This evidence indicates Eph-ephrin system as a new promising target in cancer field. By means of 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. Further investigations brought out LCA as a novel specific reversible 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. Despite the specific properties of antagonist related to the Eph-ephrin system, LCA was not very potent and was endowed with adverse PK parameters (i.e. solubility), thus, the research of small molecules showing better profile was necessary. Using LCA scaffold as a track, first of all we could identify the structural elements of the bile acid construct important for binding to Eph receptors, allowing the extrapolation of the pharmacophoric group and, on the other hand, we could design and synthesize a first new series of derivatives analyzing the structure-activity relationship. If the 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, the hydroxy group in -3 can be modified: the inversion or the absence of this group produced, respectively, isolithocholic acid (Ki= 25 µM) and cholanic acid (Ki= 5 µM), compounds with better affinity compared to LCA. Furthermore, considering that the conjugation of LCA with a glycine generated a still active compound (glycoLCA Ki=38 µM) we have dealt with the synthesis of a new series of derivatives whose structure was functionalized with amino acids. Among this new series, the LCA conjugate with a L-Trp (PCM126) emerged as specific antagonist, able to reversibly disrupt EphA2–ephrin-A1 binding in the ELISA assay, showing a Ki value of 1μM. The improved activity shown by PCM126 could be due to the facilitated interaction between the carboxilyc function of the amino acid conjugate and the essential amino acid residue Arg 103 of the LBD of EphA2. Starting from this observation, we tried to even further improve the binding affinity to Eph receptors reinforcing this crucial interaction and synthetisizing the superior homolog of PCM126 (the homo-L-Trp-conjugated of LCA), namely PCM 129. PCM129 resulted the best compound of the series, not only in binding assays, showing a ki value of 330nm 330nM towards the EphA2-ephrin-A1 interaction but also in functional assays: in fact PCM 129 is endowed with a good activity in disrupting EphA2 phosphorylation in PC3 prostate cancer cells and in HUVEC cells, was able to inhibit PC3 cell rounding and retraction induced by ephrin-A1 ligand, and showed high potency in blocking in vitro angiogenesis on HUVE cells, suggesting promising applications as antiangiogenic compound. Moreover, PCM129 is endowed not only with better potency in Eph-ephrin antagonism but also with an improvement of the tolerability and toxicity pointing out this compound as a good candidate for further development.