Development of an orally bioavailable small molecule targeting Eph-ephrin system: in vitro and in vivo anticancer activity.

The Eph receptors represent the largest family of receptor tyrosine kinases (RTK) in humans. They are divided in 2 classes, EphA and EphB, based on the sequence homology of the extracellular domain and on their affinity for their ligands, the ephrins, which are glycosylphosphatidyl-inositol membrane-linked proteins (ephrin-As) or transmembrane proteins (ephrin-Bs). Eph-ephrin signaling plays a key role during embryogenesis, where it regulates the morphogenetic processes of organs and tissues. Several evidences showed their deregulated expression and/or function may promote tumorigenesis and the development of aggressive and metastatic phenotypes in a large variety of solid tumours, in the adult. Moreover, this system has a prominent role in tumour angiogenesis and for these reasons is an emerging target for the development of novel antiangiogenic therapies. Research programs aimed at developing small molecules antagonists of the Eph receptors are at their initial stages and available compounds suffer for chemical (stability and solubility) or pharmacological (bioavailability) drawbacks, limiting their application both in vitro and in vivo. Here, we report the pharmacological characterization of a new class of Eph antagonists, devoid of stability and bioavailability troubles, based on the 3beta-hydroxy-D5-cholenic acid nucleus. In particular UniPR1331 inhibits EphA2-ephrin-A1 binding with a pIC50 value of 5.45 and a Ki value of 1.4μM, when tested via ELISA-binding assay. The compound was inactive when tested as enzymatic inhibitor of the EphA2 kinase domain confirming to be a protein-protein interaction inhibitor (i-PPI) and surface plasmon resonance (SPR) analysis directly confirmed the binding of UniPR1331 to the extracellular domain of EphA2. Functional studies on human prostate adenocarcinoma cells (PC3 cells) and human umbilical vein endothelial cells (HUVEC), showed the ability of the compound to inhibit EphA2 phosporylation upon ephrin-A1 stimulation and to inhibit HUVEC tubes formation in the low micromolar range. Notably, UniPR1331 does not affect cell viability highlighting, as the observed reduction in the phosphorylation levels was not due to a condition of cellular stress or damage. The promising pharmacokinetic profile in mice allowed us to test its efficacy in in vivo xenografts after oral administration. UniPR1331, 30mg/kg os per day, significantly reduced the growth of glioblastoma U87 xenografted cells and dramatically increased time to progression.