Identification and pharmacological characterization of novel EPH/EPHRIN antagonists

Altered function and expression of Eph receptors and ephrin ligands have been found in different tumors and associated with poor prognosis of patients, development of metastasis and tumor angiogenesis. For these reasons, the Eph/ephrin system has been suggested as new pharmacological target in oncology and our research group has focused its attention on the development of small molecules inhibiting the Eph/ephrin interaction. Lithocholic acid was found to be a reversible and weak competitive antagonist of the EphA2 receptor and an improvement of potency was obtained by conjugating its carboxylic group with L-homo tryptophan. However, the resulting compound, UniPR129 was not oral bioavailable in mice. To overcome this limit, structural modifications on UniPR129 scaffold were made and two series of compounds were obtained from UniPR129. The first resulted from the substitution of indole of the L-homo tryptophan with other aromatic substituents and the second from the introduction of more hydrophilic substituents instead of the hydroxylic group in 3α position. This last strategy was also applied on Lithocholic acid in order to evaluate the impact of such structural modifications on the inhibitory potency. This work provided the pharmacological characterization of UniPR129 derivates together with new Lithocholic acid derivates, with the aim to identify the best bioavailable compounds in mice and to evaluate their anti-tumor activity. APO184 emerged as the most potent compound among the class of Lithocholic acid derivates, showing an IC50 of 3 µM in displacement studies and an IC50 of 15.8µM when it was evaluated for its ability to inhibit the EphA2 activation in PC3 cells. Binding studies were also performed onto UniPR129 derivates. UniPR139 was obtained by modifying the aromatic substituent of the tryptophan, whereas UniPR502 and PCM505 were synthesized by inserting a different carbamoyloxy group on 3α position. UniPR139, UniPR502 and PCM505 inhibited the binding between EphA2 and ephrin-A1 ligand in reversible manner with IC50 of 1.8µM, 0.8µM, 0.95µM respectively, retaining the same potency of the parental compound. UniPR139 and UniPR502 did not discriminate between Eph receptor subclasses whereas PCM505 preferentially inhibited the binding within the EphA-subfamily. In in vitro functional studies, all these compounds acted as EphA2 antagonists, preventing ephrin-A1-induced EphA2 phosphorylation in PC3 cells in the low micromolar range but UniPR502 was the least active, displaying an IC50 of 6μM. In vitro anti-angiogenic properties were observed for UniPR139 and PCM505, which were able to inhibit the formation of tubular structures on HUVEC cells at concentrations ranging from 30 to 3μM, without non-specific cytotoxic effects. All the compounds were inactive toward EGFR, whereas UniPR502 and PCM505 partially interfered with VEGFR2 activation. Contrary to UniPR129, UniPR139 and UniPR502 were bioavailable after oral administration in mice. Since over-expression of EphA2 has been related to the process of prostatic carcinogenesis, it was evaluated the anti-tumor activity of two compounds on PC3-xenografted nude mice. Nevertheless, the oral administration of 30mg/Kg/die/os of UniPR139 and UniPR502 did not significantly reduce the tumor growth. Finally, the anti-tumor activity of UniPR129 was investigated on ApcMin/J mice and despite the lack of oral bioavailability, the compound reduced the size and the number of adenomas in the ileum without side effects. These preliminary in vivo results suggest UniPR129 as novel pharmacological tool and confirm, once more, Eph receptors as valid target in cancer therapy. This study demonstrated as the introduction of more hydrophilic groups on Lithocholic acid allowed to increase the inhibitory potency but not the inhibitory activity toward EphA2. On the contrary, potent and bioavailable Eph antagonists as UniPR139 and UniPR502 have been obtained by modifying the UniPR129 structure. Although they did not show anti-tumor efficacy in a murine model of prostate cancer, their anti-tumor activity should be evaluated in other types of cancers. The heterogenous role of Eph receptors, tumor angiogenesis and tumor microenvironment are factors which should be considered in the data analysis and which could negatively influence the effect of compounds. For these reasons, further in vivo investigations would be recommended for the Eph antagonists UniPR139 and UniPR502