Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, UK.
Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, UK; Division of Bimolecular Science and Medicinal Chemistry, School of Pharmacy, Biodiscovery Institute, University of Nottingham, NG7 2RD, UK.
Biochem Pharmacol. 2023 Aug;214:115672. doi: 10.1016/j.bcp.2023.115672. Epub 2023 Jul 3.
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 K values for inhibition of binding of sunitinib-red and log IC values for attenuation of VEGFa-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.
血管内皮生长因子（VEGF）是血管内皮细胞增殖、迁移和血管生成的重要介质。众所周知，广泛的小分子酪氨酸激酶抑制剂（TKI）的心血管安全性风险可能是由于干扰 VEGFR2 信号系统所致。在这项研究中，我们使用荧光类似物舒尼替尼（舒尼替尼红）和全长带有生物发光蛋白纳米荧光素酶标签的 VEGFR2 构建了一种配体结合测定法，使用生物发光共振能量转移（BRET）监测配体与 VEGFR2 的结合。这种 NanoBRET 测定法是一种基于邻近性的测定法（需要荧光和生物发光成分彼此之间的距离在 10nm 以内），可以监测配体与 VEGFR2 激酶结构域的结合。舒尼替尼红不可渗透细胞膜，但能够监测细胞裂解物中一系列 TKI 的结合亲和力和动力学。动力学研究表明，舒尼替尼红在 25°C 时迅速与 VEGFR2 结合，而西地尼布的结合动力学较慢，平均停留时间为 111 分钟。舒尼替尼红结合抑制的 log K 值与 VEGFa 刺激 NFAT 反应抑制的 log IC 值之间的比较表明，抑制舒尼替尼红结合的化合物具有非常相似的值。然而，两种未能抑制舒尼替尼红结合的化合物（达沙替尼和恩杂鲁胺）仍能通过抑制下游信号事件来减弱 VEGFR2 介导的 NFAT 信号转导。这些结果表明，这些化合物可能仍会因干扰下游 VEGFR2 信号而产生心血管不良反应。
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