ABSTRACT
Title
Lithocholic acid is an Eph-ephrin ligand interfering with Eph-kinase activation
Authors
I. Hassan Mohamed1, C. Giorgio1, L. Flammini1, E. Barocelli1, M.Incerti2, A. Lodola2, M. Tognolini1
1Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, 2Dipartimento Farmaceutico, Università degli Studi di Parma, Italy.
1Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, 2Dipartimento Farmaceutico, Università degli Studi di Parma, Italy.
Abstract
Eph-ephrin system plays a central role in a large variety of human cancers. In fact, altered expression and/or de-regulated 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-trasformed cells induces malignant transformation and confers tumorigenic potential (Zelinski et al., 2001; Miyazaki et al., 2003; Kinch et al., 2003; Herrem et al., 2005).Based on these evidences our aim was to identify small molecules able to modulate Eph-ephrin activity through an ELISA-based binding screening.
We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 binding (Ki = 49µM). LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Functional studies on PC3 human prostate cancer cells revealed that LCA dose-dependently antagonized EphA2 phosphorylation induced by ephrinA1 and at the same time LCA dose-dependently antagonized EphB4 phosphorylation induced by ephrinB2 in T47D human breast cancer cells, without affecting cell viability or other receptor tyrosine-kinases (EGFR, VEGFR, IGFR1β, IRKβ) activity.
LCA did not inhibit the enzimatic kinase activity of EphA2 at 100µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells.
In order to clarify the modality of interaction between LCA and Eph-ephrin system we tested other naturally occurring bile acids and we synthesized a series of LCA derivatives exploring the positions 6,7,12 and the opposite ends of the molecule represented by the hydroxil group in position 3 and by the carboxyl moiety.
LCA derivatives resulted particularly sensitive to the modulation of the cyclopenta[a]perhydro phenantrene scaffold. Indeed the introduction of hydroxyl group in position 7 or 12, always produced inactive compounds, exemplified by the naturally occurring cholic, deoxycholic and chenodeoxycholic acid. Similarly the introduction of a 6- or 7- keto group resulted detrimental for the binding affinity. The oxydation of the alpha hydroxyl group in position 3 as well as acetylation led to compounds still able to interfere with Eph-ephrin system.
In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, our work could provide an interesting starting point for a rational chemical development of molecules targeting this new emerging system in the oncology field.
Zelinski DP et al. (2001). Cancer Res 61:2301-2306.
Miyazaki T et al. (2003). Int J Cancer 103:657-663.
Kinch MS et al. (2003). Clin Cancer Res 9:613-618.
Herrem CJ et al. (2005). Clin Cancer Res 11:226-231.
We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 binding (Ki = 49µM). LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Functional studies on PC3 human prostate cancer cells revealed that LCA dose-dependently antagonized EphA2 phosphorylation induced by ephrinA1 and at the same time LCA dose-dependently antagonized EphB4 phosphorylation induced by ephrinB2 in T47D human breast cancer cells, without affecting cell viability or other receptor tyrosine-kinases (EGFR, VEGFR, IGFR1β, IRKβ) activity.
LCA did not inhibit the enzimatic kinase activity of EphA2 at 100µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells.
In order to clarify the modality of interaction between LCA and Eph-ephrin system we tested other naturally occurring bile acids and we synthesized a series of LCA derivatives exploring the positions 6,7,12 and the opposite ends of the molecule represented by the hydroxil group in position 3 and by the carboxyl moiety.
LCA derivatives resulted particularly sensitive to the modulation of the cyclopenta[a]perhydro phenantrene scaffold. Indeed the introduction of hydroxyl group in position 7 or 12, always produced inactive compounds, exemplified by the naturally occurring cholic, deoxycholic and chenodeoxycholic acid. Similarly the introduction of a 6- or 7- keto group resulted detrimental for the binding affinity. The oxydation of the alpha hydroxyl group in position 3 as well as acetylation led to compounds still able to interfere with Eph-ephrin system.
In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, our work could provide an interesting starting point for a rational chemical development of molecules targeting this new emerging system in the oncology field.
Zelinski DP et al. (2001). Cancer Res 61:2301-2306.
Miyazaki T et al. (2003). Int J Cancer 103:657-663.
Kinch MS et al. (2003). Clin Cancer Res 9:613-618.
Herrem CJ et al. (2005). Clin Cancer Res 11:226-231.