Structure-activity realtionships of new EphaA2 ligands

Background: The Eph receptor tyrosine kinases constitute the largest family of receptor tyrosine kinases. Their endogenous ligands, ephrins, are plasma-membrane-anchored proteins and they interact with the extracellular ligand-binding domain of the Eph receptors. Eph receptors and ephrins form an important cell communication system, with several biological functions. In particular, the Eph–ephrin system, composed by the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. Aims: We have previously identified Lithocholic acid (LCA, compound 1, Table 1) as a novel competitive ligand at EphA2 receptor; its steroid scaffold can be exploited to design new chemical entities able to modulate the EphA2 activity in cell systems. Here, we explore the structure-activity relationships of LCA derivatives by synthetizing analogues 2-16 (Table 1) and by testing their activity on EphA2 binding and phosphorylation assays. Methods: Compounds 1–16 were obtained from commercial source or synthetized as described in Tognolini et al, 2012 and characterized by 1H NMR and 13C NMR analysis using a Bruker Avance 400 spectrometer (400 MHz). The purity of the compounds was assessed by elemental analysis. Binding constants (Ki) were obtained by measuring the inhibition of EphA2-ephrinA1 interaction by means of ELISA binding assay. Phosphorylation studies were performed using PC3 human prostate adenocarcinoma cells, which endogenously express the EphA2 receptor. Results: In our search for novel EphA2 receptor modulators, we have recently screened an “in house” chemical library of naturally occurring compounds, identifying LCA as a antagonist of the Eph receptors. Molecular modelling investigation identified a putative binding mode for LCA within the high affinity ephrin-binding pocket of the EphA2 receptor (Figure 1). LCA occupies the space of ephrinA1 G-H loop domain, inserting its cyclopenta[a]perhydrophenantrene scaffold into a hydrophobic channel within the Eph receptor. At the same time, the carboxyl group of LCA forms a salt bridge with Arg103, mimicking the interaction undertaken by Glu119 of ephrinA1. Starting from this theoretical model, a focused set of LCA derivatives, commercially available or obtained by chemical synthesis, were examined for their ability to disrupt EphA2-ephrinA1 interaction. Compounds 2-6 were selected to explore the interaction between the lipophilic scaffold of lithocholic acid and the EphA2 binding site. Compounds 7-12 and 13-16 were selected to examine the role played by the two polar ends of lithocholic acid. The potencies for inhibition of EphA2-ephrin-A1 interaction, as indicated by the Ki values, revealed that the lithocholic acid derivatives are particularly sensitive to the modulation of polar groups on the cyclopenta[a]perhydro phenanthrene scaffold. In particular, while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. In fact, removal of the alpha hydroxyl group at position 3 leads to compound 12, Cholanic acid, which results the most potent compound of the series. Cholanic acid competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonace analysis indicates that cholanic acid binds specifically and reversibly the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 cell lines, two effects depending on EphA2 activation by ephrin-A1 ligand. Conclusions: We synthesized a series of LCA derivatives to identify the structural determinants of EphA2 binding and we found that:  Introduction of polar groups at 3,7, and 12 positions was detrimental for activity.  Removal of hydroxyl group in position 3 significantly increased binding affinity.  Modulation of the carboxylic group in position 23 led to less potent or inactive compounds. Our exploration led to the discovery of Cholanic acid as the most potent compound of the series.