TY - JOUR
T1 - Identification of receptor tyrosine kinase inhibitors using cell surface biotinylation and affinity isolation
AU - Latham, Antony M.
AU - Kankanala, Jayakanth
AU - Fishwick, Colin W.G.
AU - Ponnambalam, Sreenivasan
PY - 2015/1/1
Y1 - 2015/1/1
N2 - 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.
AB - 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.
KW - Cell surface biotinylation
KW - Drug design
KW - Endothelial cells
KW - Receptor tyrosine kinase
KW - Tyrosine kinase inhibitor
KW - VEGF-A
KW - VEGFR2
UR - http://www.scopus.com/inward/record.url?scp=84939511859&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84939511859&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-2917-7_8
DO - 10.1007/978-1-4939-2917-7_8
M3 - Article
C2 - 26285749
AN - SCOPUS:84939511859
SN - 1064-3745
VL - 1332
SP - 121
EP - 131
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -