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

Van Daele, Marieke; Kilpatrick, Laura E.; Woolard, Jeanette; Hill, Stephen J.

Abstract

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 ligand-binding 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 °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.

Citation

Van Daele, M., Kilpatrick, L. E., Woolard, J., & Hill, S. J. (2023). Characterisation of tyrosine kinase inhibitor-receptor interactions at VEGFR2 using sunitinib-red and nanoBRET. Biochemical Pharmacology, 214, Article 115672. https://doi.org/10.1016/j.bcp.2023.115672

• Journal Article Type: Article
• Acceptance Date: Jun 26, 2023
• Online Publication Date: Jul 3, 2023
• Publication Date: 2023-08
• Deposit Date: Jul 4, 2023
• Publicly Available Date: Jul 4, 2023
• Journal: Biochemical Pharmacology
• Print ISSN: 0006-2952
• Electronic ISSN: 1873-2968
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 214
• Article Number: 115672
• DOI: https://doi.org/10.1016/j.bcp.2023.115672
• Keywords: VEGFR2, NanoBRET, sunitinib-red, tyrosine kinase inhibitors, NFAT, ligand-binding
• Public URL: https://nottingham-repository.worktribe.com/output/22713561
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0006295223002630?via%3Dihub