Fragment based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase

Linden, Oscar P.J. van; Farenc, Carine; Zoutman, Willem H.; Hameetman, Liesbeth; Wijtmans, Maikel; Leurs, Rob; Tensen, Cornelis P.; Siegal, Gregg; Esch, Iwan J. P. de

doi:10.1016/j.ejmech.2011.11.020

Cited by 23 publications

(20 citation statements)

References 30 publications

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“…Due to its key role in blood vessel formation, the Eph‐ephrin system might be a target of anti‐angiogenic therapies and possibly to overcome resistance to anti‐VEGFR therapies. In the last decade, intense drug discovery efforts have produced several classes of small molecules able to inhibit the kinase domain of Eph receptors (Lafleur et al ., ; van Linden et al ., ). Among the large number of identified compounds, NVP‐BHG712 has emerged as the most promising inhibitor, being able to inhibit angiogenesis by targeting the EphB4 receptor (Martiny‐Baron et al ., ; Noberini et al ., ).…”

Section: Introductionmentioning

confidence: 97%

UniPR129 is a competitive small molecule Eph‐ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations

Hassan-Mohamed

Giorgio

Incerti

et al. 2014

British J Pharmacology

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Background and Purpose The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph‐ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph‐ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance. Experimental Approach UniPR129 (the L‐homo‐Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin‐A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2‐ephrin‐A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti‐angiogenic effect was tested in vitro using cultures of HUVECs. Key Results UniPR129 disrupted EphA2‐ephrin‐A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects. Conclusions and Implications The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph‐ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.

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Section: Introductionmentioning

confidence: 97%

UniPR129 is a competitive small molecule Eph‐ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations

Hassan-Mohamed

Giorgio

Incerti

et al. 2014

British J Pharmacology

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“…[16-19] Few examples of EphA2 inhibitors targeting the intracellular kinase-domain have been recently reported in the literature. [20] As these compounds block EphA2 activity by occupying the ATP binding pocket, they suffer from lack of selectivity, which limits their use as pharmacological tools in vivo.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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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.

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“…To assay whether the overexpressed EPHA4 in SS cells is activated, Western blot analyses was performed using a specific antibody against a phosphorylated Y779 in the EPHA4 kinase domain (EPHA4-pY779). The specificity of this antibody was verified by demonstrating detection of both commercial and in-house designed phosphorylated purified EPHA4 kinase domains [ 35 , 36 ] ( suppl. figure 1 ).…”

Section: Resultsmentioning

confidence: 99%

EPHA4 is overexpressed but not functionally active in Sézary syndrome

et al. 2015

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EPHA4 belongs to the largest subfamily of receptor tyrosine kinases. In addition to its function during development, overexpression of EPHA4 in tumors has been correlated with increased proliferation, migration and poor survival. Several genome-wide transcription profiling studies have demonstrated high EPHA4 expression in Sézary syndrome (SS), a leukemic variant of cutaneous CD4+ T-cell lymphoma (CTCL) with an aggressive clinical course and poor prognosis. In this study we set out to explore the functional role of EPHA4 in SS. Both high EPHA4 mRNA and protein expression was found in circulating SS-cells of patients compared to healthy CD4+ T-cells. However, using a phosphospecific EPHA4 antibody, phosphorylation of the EPHA4 kinase domain was not detected in either circulating or skin residing SS cells. Moreover, treatment with the phosphatase inhibitor pervanadate did not result in detectable phosphorylation of the EPHA4 kinase domain, in either SS cells or in healthy CD4+ T-cells. Thus, the results from our study confirm high EPHA4 expression in SS cells both on the mRNA and protein levels, making EPHA4 a good diagnostic marker. However, the overexpressed EPHA4 does not appear to be functionally active and its overexpression might be secondary to other oncogenic drivers in SS, like STAT3 and TWIST1.

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Fragment based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase

Cited by 23 publications

References 30 publications

UniPR129 is a competitive small molecule Eph‐ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations

UniPR129 is a competitive small molecule Eph‐ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations

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

EPHA4 is overexpressed but not functionally active in Sézary syndrome

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