Objective

Receptor tyrosine kinases (RTK) are transmembrane receptors that translate stimulation by growth factors into regulation of cell growth, proliferation, cell death and differentiation. Consequently, aberrant RTK-signalling is implicated in cancer, making it a popular anti-cancer drug target. Development of pharmaceutical RTK-inhibitors requires assays that characterize receptor-ligand interaction and that identify inhibitors thereof.

A novel method labelled the vascular endothelial growth factor (VEGF) at a single site with the fluorophore TMR. The labelled ligand was used in combination with HEK293 cells expressing the RTK VEGF-receptor 2 (VEGFR2) fused to the NanoLuc® luciferase. Upon interaction of ligand and receptor, bioluminescence resonance energy transfer (BRET) between luciferase and fluorophore takes place.

The signal of luciferase and fluorophore were acquired simultaneously with the PHERAstar microplate reader. This way, not only the pKi of a VEGF-inhibitor was determined, but also the time-course of binding and inhibition was monitored.