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

Scoperta, sviluppo e caratterizzazione farmacologica di piccole molecole in grado di interferire con Eph-ephrin system

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands are involved in many biological processes as cell migration and morphology, axon guidance, synaptic plasticity and angiogenesis both during embryogenesis and in adult tissues. Alterations of this system have been found in human cancers and above all, EphA2 and EphB4 overexpression is often correlated with aggressive tumor phenotypes and poor prognosis. Based on this evidence Eph-ephrin system represents a promising target in cancer field. Through an ELISA-based binding screening, we recently identified lithocholic acid (LCA), a secondary bile acid able to modulate Eph-ephrin activity showing a Ki value of 49 µM. LCA resulted a novel specific, reversible, not-selective antagonist of Eph receptors, able to dose-dependently inhibit Eph receptor phosphorylation induced by ephrin ligand in different cell lines and able to inhibit ephrin-A1-induced PC3 cell rounding. Moreover, Surface Plasmon Resonance analysis (SPR) confirmed the specific and reversible binding of LCA to EphA2 receptor, excluding its interaction with ephrin-A1 ligand. Therefore, LCA scaffold was used to design and synthesize a new series of derivatives analyzing the structure-activity relationship and describing the pharmacophoric structure. In particular, we found that the concurrent presence of a large hydrophobic region (cyclopenta[a]perhydrophenantrene scaffold) and an anionic hydrogen bond acceptor group (carboxylate functionality) are essential for the inhibition of EphA2–ephrin-A1 binding. Cholanic acid (Ki = 5.1 µM) and isolithocholic acid (Ki = 25 µM) emerged from this series. Moreover, we observed that the conjugation of the lateral acidic chain with glycine generated a compound (glycolithocholic acid, Ki= 38.5 µM) endowed with an activity similar to LCA. Starting from this discovery, a second series of derivatives, where LCA structure is coupled with amino acids was generated and a new compound emerged: L-Trp LCA (Ki= 1.2 µM), which is structurally formed by LCA structure conjugated with L-Tryptophan. As a result, the most active and promising compounds (cholanic acid, isolithocholic acid and L-Trp-LCA) were further pharmacologically characterized in functional assays. Investigations on PC3, T47D ad HUVE cells revealed that all the compounds able to disrupt EphA2–ephrin-A1 binding in the ELISA assay, were specific, reversible Eph receptors antagonist that dose-dependently inhibited Eph phosphorylation induced by ephrin ligands. Surprisingly, isolithocholic acid showed similar potency values in EphA2 antagonism compared to cholanic acid and L-Trp LCA, due to its activity on EphA2 kinase that is not shared by the other derivatives. Compounds were also able to inhibit PC3 cell rounding and retraction induced by ephrin-A1 ligand, and L-Trp LCA was particularly active in blocking these cell morphology changes. Moreover, through the tube formation assay performed in HUVECs, the antiangiogenic properties of the compounds have been evaluated. Among the active compounds, cholanic acid resulted the most potent in the inhibition of vessels reticulation, at not-cytotoxic concentrations. Beside LCA, in our ELISA binding based screening we identified also nine plant extracts, rich of polyphenols, commonly used as food supplements. In displacement studies they were able to reversibly inhibit EphA2–ephrin-A1 binding showing IC50 values between 0.83-24 µg/ml. The active extracts were further characterized and showed also specific antagonistic properties toward EphA2 receptor. All together, these findings could be useful to better characterize the Eph-ephrin system in its complexity as well as to figure out the role played by this system in physiological and pathological conditions. Finally, the identification of a pharmacophoric scaffold could be an interesting starting point for a rational chemical synthesis of new compounds with better affinity and improved physicochemical properties.I recettori tirosina kinasi Eph e i rispettivi ligandi, le efrine, sono entrambi localizzati sulla membrana plasmatica e sono coinvolti in diversi processi biologici sia durante lo sviluppo embrionale, sia nella vita adulta. Tali processi comprendono: migrazione e variazioni della morfologia cellulare, guida assonale, plasticità sinaptica e angiogenesi. Alterazioni di questo sistema sono state individuate in diverse tipologie di tumore nell’uomo e soprattutto la sovraespressione dei recettori EphA2 ed EphB4 è stata sempre più spesso associata a fenotipi tumorali maggiormente aggressivi e ad una prognosi infausta. Tali evidenze hanno messo in luce il sistema Eph-ephrin come target promettente in campo oncologico. Attraverso uno screening basato su un sistema di saggi di legame ELISA, abbiamo recentemente individuato l’acido litocolico (LCA), un acido biliare secondario in grado di interferire con il sistema Eph-ephrin evidenziando un valore di Ki pari a 49 µM. LCA si è dimostrato un antagonista specifico, reversibile e non selettivo dei recettori Eph, in grado di inibire in maniera dose-dipendente la fosforilazione dei recettori Eph indotta dai rispettivi ligandi in differenti linee cellulari ed in grado anche di inibire l’arrotondamento delle cellule PC3 stimolate dal ligando ephrin-A1. Oltretutto, l’analisi condotta con la Plasmon Resonance (SPR) ha confermato che LCA si lega in maniera specifica e reversibile ad EphA2 escludendo interazioni con il ligando ephrin-A1. Di conseguenza, la struttura dell’acido litocolico è stata utilizzata come modello per lo sviluppo e la sintesi di una nuova serie di derivati di cui è stata analizzata la relazione struttura- attività ed è stato ricavato il gruppo farmacoforico. In particolare, di questa serie abbiamo osservato che la presenza contemporanea di una grande regione idrofobica (la struttura ciclopenta[a]peridrodrofenantrenica) e di un gruppo anionico accettore di elettroni (funzione carbossilica) sono essenziali per l’attività inibitoria del legame EphA2–ephrin-A1. Due composti sono emersi da questa prima serie: l’acido colanico (Ki= 5.1 µM) e l’acido isolitocolico (Ki= 25 µM). Inoltre, abbiamo osservato che la coniugazione della catena acida laterale con una glicina è in grado di generare un composto (acido glicolitocolico , Ki=38.5 µM) che presenta attività simile all’acido litocolico. Partendo da questo dato, è stata prodotta una seconda serie di derivati, in cui la struttura di LCA è accoppiata ad aminoacidi. Tra questi è emerso un nuovo composto, L-Trp LCA (Ki= 1.2 µM), in cui la struttura di LCA è stata derivatizzata con un L-Triptofano. A questo punto, i composti più attivi e promettenti (acido colanico, acido isolitocolico e L-Trp LCA) sono stati farmacologicamente caratterizzati in saggi funzionali. Esperimenti effettuati su PC3, T47D e HUVEC hanno rivelato che tutti i composti che sono stati in grado di inibire il legame EphA2–ephrin-A1 nei saggi ELISA,sono degli antagonisti specifici, reversibili per i recettori Eph che inibiscono in maniera dose-dipendente la fosforilazione dei recettori Eph indotta dai ligandi efrina. Sorprendentemente, l’acido isolitocolico ha mostrato valori di potenza comparabili a LCA e L-Trp LCA nell’antagonismo sul recettore EphA2, e ciò è dovuto alla sua attività anche a livello kinasico, non mostrata invece dagli altri composti. Infine, i derivati sono stati in grado di inibire l’arrotondamento e la retrazione indotta da ephrin-A1 delle cellule PC3 e L-Trp LCA si è mostrato particolarmente attivo nel bloccare tali cambiamenti morfologici delle cellule. Attraverso il tube formation assay effettuato in HUVEC, sono state valutate anche le proprietà antiangiogeniche dei composti attivi. Fra quelli considerati, l’acido colanico si è dimostrato il più potente nel bloccare la formazione dei vasi, a concentrazioni non tossiche. Accanto ad LCA, nel nostro screening iniziale abbiamo identificato anche nove estratti naturali derivati da piante, ricchi di polifenoli, usati comunemente come supplementi nella dieta. In studi di spiazzamento sono stati in grado di inibire reversibilmente il legame EphA2–ephrin-A1, mostrando valori di IC50 compresi tra 0.83-24 µg/ml. Gli estratti attivi sono stati dunque caratterizzati in dettaglio e si sono rivelati specifici antagonisti per il recettore EphA2. Nel loro insieme, le valutazioni qui elencate, possono essere utili non solo per caratterizzare il sistema Eph-ephrin nella sua complessità ma anche per comprendere al meglio il ruolo giocato dal sistema in condizioni fisiologiche e patologiche. Infine, l’identificazione del gruppo farmacoforico può essere considerato un importante punto di partenza per una sintesi chimica razionale dei nuovi composti con valori di affinità migliorati e caratteristiche chimico-fisiche più maneggevoli

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

Discovery and development of Eph-ephrin antagonists endowed with antiangiogenic properties

Eph-ephrin system is involved in many biological processes including cell migration and morphology, axon guidance, synaptic plasticity and angiogenesis both during embryogenesis and in adult tissues. Many reports correlates a deregulation of Eph-ephrin system to aggressive tumor phenotypes in a number of human cancers and, in particular EphA2 and EphB4 overexpression, is often associate with a poor prognosis. This evidence indicates Eph-ephrin system as a new promising target in cancer field. By means of an ELISA-based binding screening, we recently identified lithocholic acid (LCA), a secondary bile acid able to modulate Eph-ephrin activity showing a Ki value of 49 µM. Further investigations brought out LCA as a novel specific reversible antagonist of Eph receptors, able to dose-dependently inhibit Eph receptor phosphorylation induced by ephrin ligand in different cell lines and able to inhibit ephrin-A1-induced PC3 cell rounding. Despite the specific properties of antagonist related to the Eph-ephrin system, LCA was not very potent and was endowed with adverse PK parameters (i.e. solubility), thus, the research of small molecules showing better profile was necessary. Using LCA scaffold as a track, first of all we could identify the structural elements of the bile acid construct important for binding to Eph receptors, allowing the extrapolation of the pharmacophoric group and, on the other hand, we could design and synthesize a first new series of derivatives analyzing the structure-activity relationship. If the large hydrophobic region (cyclopenta[a]perhydrophenantrene scaffold) and an anionic hydrogen bond acceptor group (carboxylate functionality) are essential for the inhibition of EphA2–ephrin-A1 binding, the hydroxy group in -3 can be modified: the inversion or the absence of this group produced, respectively, isolithocholic acid (Ki= 25 µM) and cholanic acid (Ki= 5 µM), compounds with better affinity compared to LCA. Furthermore, considering that the conjugation of LCA with a glycine generated a still active compound (glycoLCA Ki=38 µM) we have dealt with the synthesis of a new series of derivatives whose structure was functionalized with amino acids. Among this new series, the LCA conjugate with a L-Trp (PCM126) emerged as specific antagonist, able to reversibly disrupt EphA2–ephrin-A1 binding in the ELISA assay, showing a Ki value of 1μM. The improved activity shown by PCM126 could be due to the facilitated interaction between the carboxilyc function of the amino acid conjugate and the essential amino acid residue Arg 103 of the LBD of EphA2. Starting from this observation, we tried to even further improve the binding affinity to Eph receptors reinforcing this crucial interaction and synthetisizing the superior homolog of PCM126 (the homo-L-Trp-conjugated of LCA), namely PCM 129. PCM129 resulted the best compound of the series, not only in binding assays, showing a ki value of 330nm 330nM towards the EphA2-ephrin-A1 interaction but also in functional assays: in fact PCM 129 is endowed with a good activity in disrupting EphA2 phosphorylation in PC3 prostate cancer cells and in HUVEC cells, was able to inhibit PC3 cell rounding and retraction induced by ephrin-A1 ligand, and showed high potency in blocking in vitro angiogenesis on HUVE cells, suggesting promising applications as antiangiogenic compound. Moreover, PCM129 is endowed not only with better potency in Eph-ephrin antagonism but also with an improvement of the tolerability and toxicity pointing out this compound as a good candidate for further development

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

Biochemical characterization of EphA2 antagonists with improved physico-chemical properties by cell-based assays and surface plasmon resonance analysis

Amino acid conjugates of lithocholic acid (LCA) have been recently described as effective disruptors of the EphA2-ephrin-A1 interaction able to inhibit EphA2 phosphorylation in intact cells and thus able to block prometastatic responses such as cellular retraction and angiogenesis. However, these LCA-based compounds were significantly more potent at disrupting the EphA2-ephrin-A1 interaction than at blocking phenotype responses in cells, which might reflect an unclear mechanism of action or a metabolic issue responsible for a reduction of the compound concentration at the cell's surface. Through the synthesis of new compounds and their examination by a combination of cell-based assays and real-time interaction analysis by surface plasmon resonance, we showed at molecular level that l-tryptophan conjugates of lithocholic acid disrupt EphA2-ephrin-A1 interaction by targeting the EphA 2 receptor and that the presence of a polar group in position 3 of steroid scaffold is a key factor to increase the effective concentration of the compounds in cancer cell lines

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

BACKGROUND AND PURPOSES: 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 tumors. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different facets 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 appear crucial to obtain 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 human umbilical vein endothelial cells (HUVEC). KEY RESULTS: UniPR129 disrupts 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 to 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