ABSTRACT
Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.
Subject(s)
Angiogenesis Inhibitors , Drug Delivery Systems , Human Umbilical Vein Endothelial Cells , Neovascularization, Pathologic , Signal Transduction , Vascular Endothelial Growth Factor A , Allosteric Regulation/drug effects , Allosteric Site , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Crystallography, X-Ray , HEK293 Cells , Heterografts , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Human Umbilical Vein Endothelial Cells/transplantation , Humans , Mice , Mice, SCID , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Protein Binding , Signal Transduction/drug effects , Signal Transduction/genetics , Swine , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.