Characterisation of tyrosine kinase inhibitor-receptor interactions at VEGFR2 using sunitinib-red and nanoBRET

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, proliferation and migration of vascular endothelial cells. It is well known that cardiovascular safety liability for a wide range of small molecule tyrosine kinase inhibitors (TKIs) can result from interference with the VEGFR2 signalling system. In this study we have developed a ligand-binding assay using a fluorescent analogue of sunitinib (sunitinib-red) and full length VEGFR2 tagged on its C-terminus with the bioluminescent protein nanoluciferase to monitor ligandbinding to VEGFR2 using bioluminescence resonance energy transfer (BRET). This NanoBRET assay is a proximity-based assay (requiring the fluorescent and bioluminescent components to be within 10 nm of each other) that can monitor the binding of ligands to the kinase domain of VEGFR2. Sunitinib-red was not membrane permeable but was able to monitor the binding affinity and kinetics of a range of TKIs in cell lysates. Kinetic studies showed that sunitinib-red bound rapidly to VEGFR2 at 25 degrees C and that cediranib had slower binding kinetics with an average residence time of 111 min. Comparison between the log Ki values for inhibition of binding of sunitinib-red and log IC50 values for attenuation of VEGF165a-stimulated NFAT responses showed very similar values for compounds that inhibited sunitinib-red binding. However, two compounds that failed to inhibit sunitinib-red binding (dasatinib and entospletinib) were still able to attenuate VEGFR2-mediated NFAT signalling through inhibition of downstream signalling events. These results suggest that these compounds may still exhibit cardiovascular liabilities as a result of interference with downstream VEGFR2 signalling.

Automated summary

Vascular endothelial growth factor (VEGF) is crucial for angiogenesis and vascular endothelial cell functions. The study developed a ligand-binding assay to monitor the binding affinity and kinetics of different tyrosine kinase inhibitors (TKIs) to VEGF receptor 2 (VEGFR2). The results showed that compounds inhibiting the binding of a fluorescent analogue of sunitinib (sunitinib-red) also attenuated VEGFR2-mediated signaling, suggesting potential cardiovascular liabilities.