PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

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

Molecular modeling of mono- and bis-quaternary ammonium salts as ligands at the α4β2 nicotinic acetylcholine receptor subtype using nonlinear techniques

The neuronal nicotinic acetylcholine receptor (nAChR) has been a target for drug development studies for over a decade. A series ofmono- andbis-quaternary ammonium salts, known to be antagonists at nAChRs, were separated into 3 structural classes and evaluated using both self-organizing map (SOM) and genetic functional approximation (GFA) algorithm models. Descriptors from these compounds were used to create several nonlinear quantitative structure-activity relationships (QSARs). The SOM methodology was effective in appropriately grouping these compounds with diverse structures and activities. The GFA models were also able to predict the activities of these molecules. Charge distribution and the hydrophobic free energies were found to be important indicators of bioactivity for this particular class of molecules. These QSAR approaches may be a useful to screen and selectin silico new drug candidates from larger compound libraries to be further evaluated in in vitro biological assays