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    Acknowledgments This work was supported by Progetto PON - “Ricerca e Competitività 2007–2013” - PON01_01802: “Sviluppo di molecole capaci di modulare vie metaboliche intracellulari redox-sensibili per la prevenzione e la cura di patologie infettive, tumorali, neurodegenerative e loro delivery mediante piattaforme nano tecnologiche” and PON01_02512: “Ricerca e sviluppo di bioregolatori attivi sui meccanismi epigenetici dei processi infiammatori nelle malattie croniche e degenerative.” The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
    Introduction The erythropoietin-producing hepatoma (Eph) family is the largest class of receptor tyrosine kinases (RTKs). Eph receptors are type I transmembrane proteins, which transduce bidirectional intracellular signals via interacting with their membrane-bound ligands (ephrins) [1]. Ephrin stimulation induces the dimerization and autophosphorylation of the Eph receptor driving the activation of down-stream signaling molecules which include Src family kinases, PI3K, MAP kinases, and so on [2,3]. The down-stream signaling molecules contributed to the growth, survival and migration of cancer SecinH3 [4]. The overexpression of Eph receptors is found in many kinds of tumors, including colon, prostate, breast, and ovarian cancers [5]. Based on their interactions with ephrin ligand, Eph receptors are divided into Eph A and Eph B receptors. The EphB subfamily is the largest one of receptor tyrosine kinases, which comprises of six members from EphB1 to 6. EphB4 receptor has distinctive specificity for ephrinB2 and weak binding affinity to ephrinB1 and ephrinB3 [6]. The interaction between EphB4 and ephrin-B2 requires direct cell-cell contact and induces forward signaling of the receptor and reverse signaling of the ligand [7]. Once the normal balance of the Eph-ephrin is changed, it will disrupt the signaling and may drive tumourigenesis. Usually, the EphB4/ephrin-B2 balance is disrupted by over-expression of the EphB4 receptor in many types of tumor [8]. Recent studies show that EphB4 is over-expressed in several tumor types including prostate, colon, breast, ovarian, melanoma, endometrial, pancreatic, and lung cancers [8,9]. Physiologically, EphB4 plays critical roles in tumor growth, invasiveness, chemoresistance, adhesion, migration and tumor angiogenesis [[10], [11], [12]]. Therefore, EphB4 is a potential therapeutic target, screening and finding the small molecule inhibitors on EphB4 are full of promise for the targeted therapy of overexpressed EphB4 tumors. Study on the interactions between EphB4 and their specific ligands plays an important role in the design and discovery of new lead compounds. Affinity chromatography (AC) provides a powerful tool for screening drug candidates and potential binding partners for biological targets by chromatographic separation and bioactivity evaluation [13,14]. AC is a type of liquid chromatography which utilizes an immobilized biologically-related ligand (affinity ligand) as the stationary phase [15,16]. Based on highly specific intermolecular interactions (such as between enzymes and substrates, antibodies and antigens, proteins and specific ligands, drugs and receptors), this technique has been widely used in various studies, such as drug-protein binding interactions, sample preparations and separation, binding affinity screening of ligands from pooled compounds [[17], [18], [19], [20], [21], [22], [23]]. The advantages of this method include its high selectivity, rapid analysis, high precision, ease of automation, and ability to work with small amounts of a target solute or binding agent [15,16,22,24,25].
    Material and methods
    Results and discussion
    Conflicts of interest
    Acknowledgements This work was supported by National Natural Science Foundation of China (grant numbers 81773772, 81603148), the China Postdoctoral Science Foundation (grant number 2016M592806).