The Eph-
ephrin system, including the
EphA2 receptor and the ephrinA1
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 that is able to inhibit
EphA2 receptor activation; it is therefore potentially useful as a novel
EphA2 receptor-targeting agent. Herein we explore the structure-activity relationships of a focused set of
lithocholic acid derivatives based on molecular modeling investigations and displacement binding assays. Our exploration shows that while the 3-α-hydroxy group of
lithocholic acid has a negligible role in recognition of the
EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrinA1 to EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic
acid (
cholanic acid) as a novel compound that competitively inhibits the EphA2-ephrinA1 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 (K(D) ) in the low micromolar range. Furthermore,
cholanic acid blocks the phosphorylation of EphA2 as well as cell retraction and rounding in PC3
prostate cancer cells, two effects that depend on EphA2 activation by the ephrinA1
ligand. These findings suggest that
cholanic acid can be used as a template structure for the design of effective EphA2 antagonists, and may have potential impact in the elucidation of the role played by this receptor in pathological conditions.