Carmine Giorgio

Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (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 conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.

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

The Eph receptor-ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The l-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small-molecule antagonists of the EphA2 receptor.

Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

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 (KD) 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.

Therapeutic perspectives of Eph–ephrin system modulation

Eph receptors are the largest class of kinase receptors and, together with their ligands ephrins, they have a primary role in embryogenesis. Their expression has been found deregulated in several cancer tissues and, in many cases, abnormal levels of these proteins have been correlated to a poor prognosis. Recently, the Eph-ephrin system was found to be deregulated in other pathological processes, involving the nervous and cardiovascular systems. The increasing body of evidence supports the Eph-ephrin system as a target not only for the treatment of solid tumors, but also to face other critical diseases such as amyotrophic lateral sclerosis and diabetes driving current efforts toward the development of pharmacological tools potentially able to treat these pathologies.

Perturbation of the EphA2–EphrinA1 system in human prostate cancer cells by colonic (poly)phenol catabolites

The Eph tyrosine kinase receptors and their ephrin ligands play a central role in human cancer as their deregulated expression induces tumorigenesis with aggressive phenotypes. To evaluate their potential contribution to EphA2-ephrinA1 modulation, several colonic catabolites of dietary (poly)phenolics, known to be generated in vivo, were screened using an ELISA-based binding assay. Some of the catabolites inhibited the binding in a dose-dependent manner (IC(50) values from 0.26 to 43 μM). Functional studies on prostate adenocarcinoma cells revealed that pyrogallol and protocatechuic acid specifically antagonized ephrinA1-Fc-induced EphA2 phosphorylation at concentrations that were not cytotoxic. The active concentrations of pyrogallol appear to be close to what can be reached in vivo under physiological conditions. Finally, because of the roles played by the Eph-ephrin system not only in cancer development but also in neurodegeneration and diabetes, pyrogallol and protocatechuic acid are candidates for more detailed functional studies to elucidate their role in these pathophysiological processes.

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

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.

Polyphenol rich botanicals used as food supplements interfere with EphA2-ephrinA1 system

The Eph tyrosine kinase receptors and their ephrin ligands play a central role in several human cancers and their deregulated expression or function promotes tumorigenesis, inducing aggressive tumor phenotypes. Green tea extracts (GTE) have been recently found to inhibit Eph-kinase phosphorylation. In order to evaluate the potential contribution of edible and medicinal plants on EphA2-ephrinA1 modulation, 133 commercially available plant extracts used as food supplements, essential and fixed oils were screened with an ELISA-based binding assay. Nine plant extracts, rich of polyphenols, reversibly inhibited binding in a dose-dependent manner (IC₅₀ 0.83-24 μg/ml). Functional studies on PC3 prostate adenocarcinoma cells revealed that active extracts antagonized ephrinA1-Fc-induced EphA2-phosphorylation at non-cytotoxic concentrations (IC₅₀ 0.31-11.3 μg/ml) without interfering with EGF-induced EGFR activation, suggesting a specific effect. These findings could furnish an interesting starting point regarding the potential relationship between diet, edible plant secondary metabolites and Eph-ephrin system, suggesting their possible involvement in cancer development modulation.

The ellagitannin colonic metabolite urolithin D selectively inhibits EphA2 phosphorylation in prostate cancer cells

SCOPE The Eph-ephrin system comprises emerging proteins involved in many pathophysiological processes. The pharmacological activity of the main metabolites derived from the intake of some classes of (poly)phenolic compounds, such as caffeoylquinic acids, flavan-3-ols, and ellagitannins, on the Eph-ephrin interaction was evaluated at physiological concentrations. Functional studies to elucidate their role in prostate cancer were also performed. METHODS AND RESULTS Among the 21 phenolics screened by an ELISA-binding assay, just urolithin C, urolithin D, and ellagic acid succeeded to inhibit the EphA2-ephrin-A1 binding. Urolithin D, the most active, was a competitive and reversible antagonist of EphA receptors able to discriminate between EphA and EphB receptors, showing intra-classes selectivity. Molecular modeling and structure-activity relationships shed light on the binding mode and selective activity of urolithin D. This catabolite blocked EphA2 phosphorylation mediated by ephrin-A1, while lacking cytotoxicity and anti-proliferative effects, and was inactive on the EphA2 kinase assay. CONCLUSION The mechanisms behind the cancer preventive properties of foods rich in flavan-3-ols and caffeoylquinic acids are not associated with metabolic pathways directly linked to the Eph-ephrin system. However, the ellagitannin-derived colonic metabolite urolithin D was able to exert remarkable and selective EphA-ephrin-A inhibition, which might impact on prostate cancer prevention.

Synthesis and structure-activity relationships of amino acid conjugates of cholanic acid as antagonists of the EphA2 receptor.

The Eph–ephrin system plays a critical role in tumor growth and vascular functions during carcinogenesis. We had previously identified cholanic acid as a competitive and reversible EphA2 antagonist able to disrupt EphA2-ephrinA1 interaction and to inhibit EphA2 activation in prostate cancer cells. Herein, we report the synthesis and biological evaluation of a set of cholanic acid derivatives obtained by conjugation of its carboxyl group with a panel of naturally occurring amino acids with the aim to improve EphA2 receptor inhibition. Structure-activity relationships indicate that conjugation of cholanic acid with linear amino acids of small size leads to effective EphA2 antagonists whereas the introduction of aromatic amino acids reduces the potency in displacement studies. The β-alanine derivative 4 was able to disrupt EphA2-ephrinA1 interaction in the micromolar range and to dose-dependently inhibit EphA2 activation on PC3 cells. These findings may help the design of novel EphA2 antagonists active on cancer cell lines.

1-Methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/cycloalkyl/phenyl/naphthylureido)-1H-pyrazole-4-carboxylic acid ethyl esters as potent human neutrophil chemotaxis inhibitors

In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains and in position 5 with a series of 3-substituted urea groups. These compounds were designed as development of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-OMe-stimulated neutrophil chemotaxis. The most active compounds in the fMLP-OMe induced chemotaxis test showed IC(50) in the range 0.19 nM-2 microM; but we observed a very strong inhibition in the IL8-induced chemotaxis test, having the most active compounds IC(50) at pM concentrations. In vivo compounds 2e and 2f, although to a lesser extent, at 50mg/kg os decreased granulocyte infiltration in zymosan-induced peritonitis in mice.