Characterization of a drug-targetable allosteric site regulating vascular endothelial growth factor signaling
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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.
KeywordsAngiogenesis VEGF DARPin VEGFR-2 KDR Inhibition Receptor downregulation
Andreas Cornelius, Johan Abram and Nicole Bassler of Molecular Partners are acknowledged for production and characterization of D4b and HD4b. We thank Drs. Richard Kammerer and Roger Benoit for designing the receptor D4–5 construct, Dr. Aurélien Rizk for advice with Squassh analysis, and Mayanka Asthana for assistance in protein purification. We also thank staff at beamline X06SA at the Swiss Light Source in Villigen Switzerland for help with X-ray data acquisition. This work was supported by grants from Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_152908 to KB-H) and Oncosuisse (KLS-3569-02-2015 to KB-H).
Experiments were conceived, designed and interpreted by HKB, SMM, CLP, DA and KB-H. Experiments were performed by KMT, CLP, DA and SMM. The manuscript was written by KMT, CLP, DA, SMM, HKB and KB-H.
Compliance with ethical standards
Conflict of interest
HKB is shareholder of Molecular Partners AG commercializing the DARPin® Technology. The other authors do not declare competing financial interests.
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