Structure−Activity Relationship Analysis of Peptides Targeting the EphA2 Receptor

Abstract

The EphA2 receptor tyrosine kinase has emerged as a promising new therapeutic target in cancer due to its high expression in tumors. EphA2-specific antibodies have been used to deliver drugs and toxins to tumor cells, leading to inhibition of tumor growth and metastatic dissemination. We previously identified two related peptides, YSA and SWL, that selectively bind to the ligandbinding domain of EphA2 but not other Eph receptors, and could therefore be useful as selective targeting agents. Here we characterize the two peptides and a series of derivatives. Based on systematic amino acid replacements, only 5 YSA residues appear to be critical for high affinity receptor binding. Furthermore, a peptide comprising only the first 5 residues of YSA retains selectivity for EphA2. Similar to ephrin-A1, the physiological ligand for EphA2, both YSA and SWL activate EphA2 and inhibit downstream oncogenic signaling pathways in PC3 cancer cells. The two peptides and derivatives are quite stable in conditioned cell culture medium and show promise for delivering drugs and imaging agents to EphA2-expressing tumors.The EphA2 receptor tyrosine kinase, a member of the large Eph receptor family, is a promising therapeutic target in cancer because it is widely overexpressed in many cancer types, including breast, ovarian, prostate, pancreatic and lung cancer (1-3). EphA2 is present not only in the tumor cells but also in the tumor vasculature, while it is undetectable in normal quiescent vasculature (4,5). Furthermore, high EphA2 levels have been associated with a poor clinical prognosis (1-3) and with the more malignant, basal type of breast and prostate cancers (6,7). EphA2 overexpression has indeed been shown to induce oncogenic transformation and invasiveness of cultured mammary epithelial cells (8), and EphA2 downregulation with siRNA or anti-sense oligonucleotides has a negative impact on tumor growth and metastasis in mouse cancer models (9,10). Interestingly, EphA2 is tyrosine phosphorylated (activated) at low to undetectable levels in most tumors, suggesting an oncogenic role that is independent of ligand-mediated activation (11-13).Five glycosylphosphatidylinositol (GPI)-linked ligands (ephrin-A1 to -A5) can induce EphA2 tyrosine phosphorylation and activation in mammalian cells (14). A number of † This work was supported by NIH grant CA82713 (to EBP), a grant from MedImmune (to EBP), and Department of Defense Breast Cancer Research Program grant W81XWH-07-1-0462 (to ZH and EBP), Prostate Cancer Research Program grant W81XWH-06-1-0077 (to EBP) and Postdoctoral Fellowship DAAMD17-01-1-0168 (to MK). (11,13,15). Therefore, EphA2 functions as a tumor suppressor when its signaling ability is activated by ephrin ligands, whereas its tumor promoting effects may be ligand-independent (3,12,13,16).A number of EphA2-targeting agents have been developed. Several agonistic monoclonal antibodies and ephrin-A1 Fc, a soluble form of ephrin-A1, have been shown to decrease tumor growth and metastasis in mouse models (17)(18)(19)(20...

Design, Synthesis and Bioevaluation of an EphA2 Receptor‐Based Targeted Delivery System

“…[26] EphA2 activation by the ephrin-A1 ligand results in receptor internalization and subsequent degradation at least in part through a lysosomal pathway. [22, 27] Similar to the ephrin-A1 Fc ligand, YSA-L2-PTX and dYNH-L2-PTX cause EphA2 internalization into PC3M cells and receptor colocalization with the lysosomal marker Lamp1 (Figure 4A). On the contrary, the scrambled DYP-L2-PTX fails to trigger EphA2 internalization (Figure 4A).…”

“…[6] The amino acid sequence YSAYPDSVPMMS (YSA), identified by using a phage display technique, has been shown to bind the extracellular domain of EphA2 and promote receptor activation and internalization in several cancer cell types. [22] …”

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

“…These peptides interact with the ephrin-binding pocket in the ligand-binding domain of EphA2 and compete with ephrin ligands for binding (40, 42). Computer modeling of peptide binding to the ephrin-binding pocket of EphA2 and structure-activity relationship studies have revealed a 1:1 binding stoichiometry (42, 43), which has been supported by ITC data (44, 45). …”

Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells

“…These peptides induce receptor endocytosis and activate EphA2 phosphorylation leading to downstream signaling (Koolpe et al, 2002; Mitra et al; Pasquale, 2005). Other EphA2-targeting approaches have been reported including small molecule EphA2 targeting agents (Chang et al, 2008; Noberini et al, 2008), viral vectors expressing soluble ephrin-A1 protein (Brantley-Sieders et al, 2008) and RNA-interference blocking EphA2 expression (Carles-Kinch et al, 2002; Duxbury et al, 2004; Kikawa et al, 2002).…”