PEGylation potentiates the effectiveness of an antagonistic peptide that targets the EphB4 receptor with nanomolar affinity.

Abstract

The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.

• Authors: Roberta Noberini, Sayantan Mitra, Ombretta Salvucci, Fatima Valencia, Srinivas Duggineni, Natalie Prigozhina, Ke Wei, Giovanna Tosato, Ziwei Huang, Elena B Pasquale
• Journal: PloS one (PLoS One) Vol. 6 Issue 12 Pg. e28611 ( 2011) ISSN: 1932-6203 [Electronic] United States
• PMID: 22194865 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Culture Media
• Ephrin-B2
• Peptides
• TNYL-RAW peptide
• Phosphotyrosine
• Polyethylene Glycols
• Receptor, EphB4

Topics

• Animals
• Cell Line
• Coculture Techniques
• Culture Media (pharmacology)
• Ephrin-B2 (metabolism)
• Epithelial Cells (cytology, drug effects, metabolism)
• Half-Life
• Human Umbilical Vein Endothelial Cells (cytology, drug effects, metabolism)
• Humans
• Mice
• Microchemistry
• Neovascularization, Physiologic (drug effects)
• Peptides (blood, pharmacology)
• Pericardium (cytology)
• Phosphorylation (drug effects)
• Phosphotyrosine (metabolism)
• Polyethylene Glycols (metabolism)
• Protein Binding (drug effects)
• Protein Stability (drug effects)
• Receptor, EphB4 (antagonists & inhibitors, metabolism)