Signaling by EphrinB1 and Eph Kinases in Platelets Promotes Rap1 Activation, Platelet Adhesion, and Aggregation via Effector Pathways that Do Not Require Phosphorylation of EphrinB1

We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1m and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized firinogen via αIIbβ3. Adhesion occurs more slowly than with adenosine diphosphate (ADP) abd requires phosphatidylinositol 3 (PI3)—kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca++ ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca++; (2) this is accomplished in part by activating Rap1; and (3) these effects require not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain

A photosensitizing fusion protein with targeting capabilities

Abstract The photodynamic treatment for antimicrobial applications or anticancer therapy relies on reactive oxygen species generated by photosensitizing molecules after absorption of visible or near-infrared light. If the photosensitizing molecule is in close vicinity of the microorganism or the malignant cell, a photocytotoxic action is exerted. Therefore, the effectiveness of photosensitizing compounds strongly depends on their capability to target microbial or cancer-specific proteins. In this study, we report on the preparation and preliminary characterization of human recombinant myoglobin fused to the vasoactive intestinal peptide to target vasoactive intestinal peptide receptor (VPAC) receptors. Fe-protoporphyrin IX was replaced by the photosensitizing compound Zn-protoporphyrin IX. Taking advantage of the fluorescence emission by Zn-protoporphyrin IX, we show that the construct can bind prostate cancer cells where the VPAC receptors are expressed

Metadynamics for perspective drug design: Computationally driven synthesis of new protein-protein interaction inhibitors targeting the EphA2 receptor

Metadynamics (META-D) is emerging as a powerful method for the computation of the multidimensional freeenergy surface (FES) describing the protein-ligand binding process. Herein, the FES of unbinding of the antagonist N-(3α-hydroxy-5β-cholan-24-oyl)-L-β-homotryptophan (UniPR129) from its EphA2 receptor was reconstructed by META-D simulations. The characterization of the free-energy minima identified on this FES proposes a binding mode fully consistent with previously reported and new structure-activity relationship data. To validate this binding mode, new N-(3α-hydroxy-5β-cholan-24-oyl)-L-β-homotryptophan derivatives were designed, synthesized, and tested for their ability to displace ephrin-A1 from the EphA2 receptor. Among them, two antagonists, namely compounds 21 and 22, displayed high affinity versus the EphA2 receptor and resulted endowed with better physicochemical and pharmacokinetic properties than the parent compound. These findings highlight the importance of free-energy calculations in drug design, confirming that META-D simulations can be used to successfully design novel bioactive compounds

UniPR1331, a small molecule targeting Eph/ephrin interaction, prolongs survival in glioblastoma and potentiates the effect of antiangiogenic therapy in mice

Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to standard therapeutic approaches that combine surgery, radiotherapy, and chemotherapy. As opposed to healthy tissues, EphA2 has been found highly expressed in specimens of glioblastoma, and increased expression of EphA2 has been shown to correlate with poor survival rates. Accordingly, agents blocking Eph receptor activity could represent a new therapeutic approach. Herein, we demonstrate that UniPR1331, a pan Eph receptor antagonist, possesses significant in vivo anti-angiogenic and anti-vasculogenic properties which lead to a significant anti-tumor activity in xenograft and orthotopic models of GBM. UniPR1331 halved the final volume of tumors when tested in xenografts (p<0.01) and enhanced the disease-free survival of treated animals in the orthotopic models of GBM both by using U87MG cells (40 vs 24 days of control, p<0.05) or TPC8 cells (52 vs 16 days, p<0.01). Further, the association of UniPR1331 with the anti-VEGF antibody Bevacizumab significantly increased the efficacy of both monotherapies in all tested models. Overall, our data promote UniPR1331 as a novel tool for tackling GBM

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

Targeting the Eph-ephrin system with protein-protein interaction (PPI) inhibitors

Eph-ephrin system is emerging as a new potential target in several diseases including cancer, diabetes, neurodegenerative diseases and inflammation. In the last decade, several efforts have been made to develop small molecule antagonists of Eph receptors. Both natural and synthetic compounds were discovered with (poly) phenol and steroidal derivatives on one side and the α1 agonist doxazosin, 2,5-dimethylpyrrol- 1-yl-benzoic acids and amino acid conjugates of lithocholic acid on the other. In the present paper we critically present available data for these compounds and discuss their potential usefulness as pharmacological tools or as candidates for a lead-optimization program

Are we using the right pharmacological tools to target EphA4?

The EphA4 receptor has been proposed to be a key actor in neurodegenerative diseases. In the last years, several research groups focused their efforts on the discovery of small molecules capable of blocking EphA4 activity by binding its extracellular domain. However, none of the compounds so far identified possess adequate chemical and/or pharmacological profiles to assess the "druggability" of EphA4 in animal models. New efforts are required to deliver a new generation of suitable pharmacological tools