Identification of Receptor Tyrosine Kinase Inhibitors Using Cell Surface Biotinylation and Affinity Isolation

The mammalian vascular endothelial growth factor receptor tyrosine kinases (VEGFRs) bind circulating growth factors and regulate the process of angiogenesis. The discovery of new small molecules that target the enzymatic activity of the VEGFR family as potential antiangiogenic drugs is of much commercial interest in the pharmaceutical sector. Here, we describe the use of a combined cell surface biotinylation and affinity isolation procedure to monitor ligand-stimulated VEGFR trafficking in endothelial cells, in which novel VEGFR inhibitors from chemical libraries can be identified by their ability to inhibit receptor internalization. Unlike a traditional cell-free enzyme activity assay, such a cell-based approach provides a physiologically relevant readout of inhibitor activity. In this example, we use the VEGF-A–VEGFR-2 axis and the well-characterized tyrosine kinase inhibitor sunitinib as a working model; however this technique is highly applicable for the identification of inhibitors to other receptor tyrosine kinases.