The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. lead for EphA4-targeted therapeutic development. Introduction The Eph receptors belong to the subfamily of receptor tyrosine kinases (RTKs) [1-4]. To date, 16 Eph receptors have been identified; these fall into two subclasses that show similar overall structure but have different binding affinities for the ephrin ligands [2,3]. The EphA4 receptor has a number of important functions in the vascular and neural development occurring during embryogenesis, postnatal tissue repair, and tumorigenesis [3,5-7]. It is expressed in neurons, where it promotes the repulsive guidance of developing axons [8,9]. Mice lacking the EphA4 exhibit pronounced axonal regeneration and functional recovery following spinal cord injury [9,10]. Inhibition of EphA4 by soluble protein inhibitors such as ephrin-A5-Fc, EphA4-Fc, or peptides induces axonal regeneration following spinal cord injury and reduces ischemia-induced apoptotic neuronal cell death [11,12]. In humans with amyotrophic lateral sclerosis (ALS), EphA4 expression inversely correlates with disease onset and survival. Knockdown of EphA4 is able to rescue the axonopathy induced by proteins that cause ALS . Therefore, inhibition of the EphA4-ephrin interaction can be a useful strategy for promoting axon regeneration in the nervous system [14,15]. A potential role for EphA4 in human cancer is also receiving increasing attention. Altered expression patterns of EphA4/ephrins are correlated with tumor behaviors, such as invasiveness, vascularization, and metastatic potential. Overexpression of EphA4 Gefitinib (Iressa) IC50 is observed in many human cancers, including prostate, breast, gastric, and pancreatic tumors [16-20]. High levels of EphA4 RNA expression are correlated significantly with reduced overall survival of cancer patients . EphA4 binds to both type A ephrins and most type B ephrins, but it also forms a hetero receptor complex with fibroblast growth factor receptor (FGFR). Signaling pathways mediated by EphA4 and FGFR play critical roles in promoting the proliferation and migration of embryonic neural stem cells and of cancer cells [22,23]. A promising strategy for anti-angiogenic cancer therapies is to antagonize Eph-ephrin interactions by interfering with the Eph receptor/ephrin system . Several different synthetic peptides and small molecules have been studied for this purpose; the advantage of these artificial ligands is that they can show a much higher selectivity compared to the physiological ephrin ligands. A number of linear peptides and small molecules have been reported to act as artificial ligands of Eph receptors: KYL and additional linear peptides for EphA4 ; TNYL-RAW peptide for EphB4 ; SWL and additional peptides for EphA2 ; and dimethyl-pyrrole derivatives for EphA2 and EphA4 . Compared with these linear peptides, cyclic peptides display more restricted conformations that often result in higher affinity, selectivity, and stability [29,30]. As a result, cyclic peptides are more desired as probes of receptor functions and prospects for therapeutic development. In this statement, we identify a new cyclic nonapeptide, TYY, [c(CTYYWPLPC)], from the M13 phage display library. TYY binds to EphA4, but not to EphB4, and selectively inhibits the binding of ligand ephrinA5 to EphA4. At a low micromolar concentration, TYY shows pronounced anti-angiogenesis activity inside a 3D tradition system. A molecular docking study further identifies a putative structural model for the TYY peptide bound to EphA4. This is the 1st cyclic peptide to be reported in the literature as a new lead for the development of conformationally restrained and enzymatically stable agents focusing on EphA4. Materials and Methods Phage display A pre-made library consisting of a disulfide-constrained nonapeptide Gefitinib (Iressa) IC50 (Ph.D.-C7C) Gefitinib (Iressa) IC50 library (New England Biolabs, Beverly, MA) was utilized for panning about EphA4. Recombinant mouse EphA4 Fc Chimera protein (R&D Systems, Minneapolis, MN) was incubated over night at 4C in 96-well ELISA plate wells at concentrations of 10 g/ml in covering buffer (0.1 M NaHCO3). Wells were clogged for 1 h with obstructing buffer (0.1 M NaHCO3, 5 mg/ml BSA, 0.02%NaN3), and rinsed with CC2D1B TBST (0.1% Tween20 in TBS). In round 1 of panning, 51010 plaque-forming models (PFUs) of the phage library in 100 l TBST were incubated for 1h at space heat in EphA4 receptor-coated wells. After washing, the remaining bound phage were eluted with 100 l of 0.2 M glycine-HCl (pH 2.2), and then neutralized by 15 l 1M Tris-HCl (pH9.1). The entire eluate was used to infect early-log phase ER2738 host bacteria and amplified (4.5 h, 37 C). The phage were concentrated and stored according to the manufacturers recommendations. In rounds 2, 3, and 4, we added 4.91010, 1.811011, 1.951011 PFUs of the.