Abstract A126: Structure activity relationships of new EphA2 ligands.
Authors: Tognolini Massimiliano , Incerti Matteo , Mohamed Iftiin Hassan , Giorgio Carmine , Barocelli Elisabetta , Russo Simonetta , Lelli Barbara , Bracci Luisa , Lodola Alessio 
Published: June 2012

Introduction: Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or deregulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumor phenotype. In particular EphA2 upregulation is correlated with tumor stage and progression and the expression of EphA2 in non-transformed cells induces malignant transformation and confers tumorigenic potential.

We recently identified lithocholic acid (LCA) as a competitive and reversible antagonist inhibiting Eph kinases-ephrins binding and functionality. In order to investigate the Structure Activity Relationship at Eph-ephrin system we synthesized a series of new derivatives exploring the positions 6,7,12 and the opposite ends of LCA, represented by the hydroxyl group in position 3 and by the carboxyl moiety.

Methods: The ability of the compounds to interfere with Ephephrin system was tested both in an ELISA-like binding assay and in functional cellular assays (EphA2 phosphorylation, cytotoxicity, proliferation). Surface Plasmon Resonance was used to clarify whether the compounds bound to Eph kinase or to ephrin.

Results: LCA derivatives resulted to bind to Eph kinase (by SPR) and they were particularly sensitive to the modulation of the steroidal scaffold. The introduction of an -hydroxyl or keto group in position 6, 7 or 12 resulted detrimental for binding affinity. The oxidation of the -hydroxyl group in position 3 as well as its acetylation led to compounds characterized by a reduced affinity compared to LCA. By contrast, the inversion of the chiral centre in position 3 or its removal, led to compounds endowed with higher affinity and efficacy. However, when the beta hydroxyl group of isoLCA was replaced by a hindered substituent, the compound became inactive. Esterification or conjugation with amine derivatives of the carboxylic group of LCA, also gave inactive compounds. Finally, the reduction of LCA carboxyl group to the corresponding alcohol derivative gave a compound, which retained some affinity for EphA2 binding site. The crystal structure of EphA2 combined to molecular docking and dynamics simulations suggested the compounds occupy the space of the ephrin-A1 G-H loop domain, inserting its cyclopenta[a]perhydro phenanthrene scaffold into a hydrophobic Eph receptor channel. The pentanoic acid fragment, emerging from position 17 of the LCA core, forms a salt bridge with Arg103, mimicking the interaction undertaken by ephrin-A1 Glu119. Finally, LCA 3-hydroyxl group interacts with Arg159, usually involved in a hydrogen bond with Asp86 of ephrin-A1.

Conclusions: All together, these findings indicate that the hydrophobic core of LCA can mimic the G-H loop in its interaction with EphA2 binding site. SAR data and computational results also suggest that the hydroxyl group in position 3 is not essential for the activity, representing an interesting point for a further expansion of the series.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A126.