Structure–Activity Relationships and Mechanism of Action of Eph–ephrin Antagonists: Interaction of Cholanic Acid with the EphA2 Receptor

Abstract

The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to 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 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions.

Amino Acid Conjugates of Lithocholic Acid As Antagonists of the EphA2 Receptor

“…Molecular modeling investigations previously performed by our group 22 suggested that LCA ( 1 ) can mimic the binding mode of ephrin-A1 to the EphA2 receptor 32 by inserting its cyclopenta[a]perhydrophenanthrene scaffold into the hydrophobic EphA2 receptor ligand-binding channel and forming a salt bridge with Arg103 (Figure 2A), a critical residue for ephrin-A1 recognition. 29 In agreement with this hypothesis, modifications of the carboxylic group of LCA, e.g.…”

“…The procedure is similar to that reported in references 21 and 22. Briefly, PC3 cells (4,000 cells per well) were plated in 96-well plates (Greiner Bio One, Frickenhausen Germany) and grown for 17 hours.…”

Inhibition of Eph receptor–ephrin ligand interaction by tea polyphenols

“…For instance, the salicylic acid-dimethylpyrrole derivatives and salicylic acid-furanyl derivative preferentially target EphA2 and EphA4 [26,39], while lithocolic acid inhibits with similar potency all the Eph receptors [40] and cholanic acid shows somewhat higher potency towards EphA than EphB receptors [41]. These results suggest that different compounds may interact in different ways with EphA4.…”

“…A few small molecules that antagonize ephrin-binding to Eph receptors at micromolar concentrations have also been identified. These include: (i) salycilic acid derivatives, which inhibit ligand binding to a subset of Eph receptors through non-classical mechanisms [26,38,39]; (ii) the bile acid lithocolic acid, a competitive reversible inhibitor that targets all the Eph receptors [40], and (iii) cholanic acid, which is related to lithocolic acid but shows some preference for the EphA compared to the EphB receptor class [41]. A number of plant extracts rich in polyphenols, including a green tea extract, and several polyphenol catabolites were also recently found to inhibit ephrin binding to the EphA2 receptor in vitro and ephrin-induced EphA2 tyrosine phosphorylation in PC3 prostate cancer cells [42,43].…”

Δ5-Cholenoyl-amino acids as selective and orally available antagonists of the Eph–ephrin system

“…Among them, derivatives of lithocholic acid (compound 1, Fig. 1) were discovered as competitive and reversible antagonists of the EphA2 receptor [16], active in prostate cancer [17] and cardiac muscle cells [18] at non-cytotoxic concentration. Other small molecules interfering with the Epheephrin system reported so far include i) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-2-hydroxybenzoic acid (also know as CPD-1, Fig.…”