Identification and pharmacological characterization of novel Eph-ephrin antagonists using a multi-strategy approach

The Eph-ephrin system is an appealing target for cancer therapy, but developing novel small molecules is challenging. This thesis examines three different approaches used for the identification and pharmacological characterization of novel small molecules acting as Eph-ephrin interaction inhibitors. In the first chapter, the target hopping approach results in the identification of Cilofexor, an agonist of the farnesoid X receptor, as a potential EphA2-ephrin-A1 interaction inhibitor. Cilofexor acts as a competitive and reversible EphA2 antagonist by preventing the phosphorylation and morphological changes induced by ephrin-A1 in PC3 prostate cancer cells. The second chapter illustrates an in vitro screening of natural compounds and phytochemicals previously predicted in silico studies. Contrary to expectations, this approach was not useful in identifying novel antagonists of the Eph-ephrin system. Nevertheless, these results underscore the importance of validating in silico results through proper biochemical and biological assays. The last chapters discuss a multidisciplinary approach that combines molecular modeling, chemical exploration, and pharmacological characterization of UniPR1331 derivatives. This strategy resulted in the identification of two novel classes of small molecules with improved potency and selectivity toward the EphA2 receptor. UniPR1454 emerges as a promising selective and competitive EphA2 antagonist displaying anti-proliferative activity in U251 glioblastoma cells and in a model of glioma stem cells. Moreover, the nanoparticle formulation of UniPR1454 shows a promising pharmacokinetic profile in vivo. Lastly, PIE-FCCS measurements suggest that UniPR1454 disrupts the head-tail interface of the EphA2 receptors, providing new insights into the modulation of its non-canonical signaling. The second class of 3β-carbamoyloxy derivatives displays improved binding affinity compared to the parental compound UniPR1331. Overall, this multidisciplinary approach results in the identification of novel potential Eph-ephrin interaction inhibitors as promising pharmacological tools.