Novel BRET approaches to unravel the molecular pharmacology of VEGFR2 receptors: Insights into ligand binding, allosterism and signalling bias

揭示 VEGFR2 受体分子药理学的新 BRET 方法：深入了解配体结合、变构和信号偏倚

• 批准号: BB/L019418/1
• 负责人: Stephen Hill
• 金额: $ 51.77万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL019418%2F1
• 关键词: Novel; BRET; approaches; unravel; molecular

The way in which cells communicate with each other and mediate cellular responses is an integral part of all life and controls the inner workings of organs within the body allowing them to respond, adapt and survive. This cellular communication is largely chemically based and messenger molecules can be both small (e.g. adrenaline) and large (e.g. many growth factors including vascular endothelial growth factor, VEGF, which is the subject of this proposal). VEGF is released from cells in response to low oxygen and during wound healing and has an important role in the development and expansion of the microcirculation (growth of new blood vessels from a pre-existing vasculature; angiogenesis) in diseases such as cancer. VEGF mediates its physiological roles by interacting with a receptor on the cell surface of endothelial cells that then undergoes a conformational change to cause tyrosine residues on its intracellular surfaces to become phosphorylated. This sets in motion a chain of events that leads to the activation of a range of different intracellular signalling proteins. These mediate a variety of changes such as cell motility, protein expression and cell survival. VEGF has three different cell surface receptors that it interacts with but it is VEGFR2 which is the most important for angiogenesis.A complication of VEGF signalling is that there are multiple isoforms of VEGF that differ in size and may have markedly different abilities to bind to VEGFR2 and trigger responses. Furthermore, VEGFR2 normally functions as a homodimer (i.e. a complex of one VEGFR2 molecule bound to a second molecule of VEGFR2). However it is also known that VEGFR2 can also interact with the other two VEGF receptors (VEGFR1 and VEGFR3) to form heterodimers. In addition, co-receptors for VEGFR2 exist on the cell membrane (e.g. Neuropilin-1, NRP1) that can either enhance the binding of VEGF to VEGFR2 or can modulate subsequent functional responses. It is unknown, however, how different VEGF isoforms bind to the receptor in these complexes. Quantitative evaluation of ligand-receptor interactions has been the cornerstone of the drug discovery process, particularly in the case of other types of cell surface receptor (G protein-coupled receptors) which are the target for over 40% of all known drugs. However, binding of VEGF isoforms to VEGFR2 has not received the same degree of analysis because of the protein nature of the growth factor and the complexity of its interactions. This proposal aims to exploit the detailed and quantitative analytical skills of the PI and CI to unravel ligand-binding and signalling characteristics (molecular pharmacology) of specific VEGF isoforms utilizing new biotechnology approaches (developed by the industrial partner) to the study of ligand-receptor and receptor-protein interactions in living cells. This uses measurement of luminescence (biologically generated light output) of different colours. The industrial partner has engineered a novel bioluminescent protein (NanoLuc) that was originally isolated from a deep-sea shrimp. This is blue, bright, stable and visible to the naked eye. It can be attached to extracellular or intracellular terminals of VEGFR2 or its interacting proteins without loss of function. The light produced is of an appropriate wavelength to excite a neighbouring fluorescent molecule if it is in very close proximity to NanoLuc and thus can then generate light of a different colour (e.g. red). This strategy will be used to monitor in living cells the binding of fluorescently-labelled VEGF to VEGFR2 and to establish the detailed molecular pharmacology of each VEGF isoform in binding to specific VEGFR2 dimers, VEGFR2-NRP1 complexes and to determine whether they are capable of selectively triggering specific intracellular signalling pathways leading to signalling bias. This will provide considerable insight into VEGF function and provide new opportunities for drug discovery.

细胞相互沟通和介导细胞反应的方式是所有生命的组成部分，并控制着体内器官的内部运作，使它们能够做出反应，适应和生存。这种细胞通讯主要是基于化学的，信使分子可以是小的（例如肾上腺素）和大的（例如许多生长因子，包括血管内皮生长因子，VEGF，这是本提案的主题）。VEGF响应于低氧和在伤口愈合期间从细胞中释放，并且在诸如癌症的疾病中的微循环的发展和扩张（从预先存在的脉管系统生长新血管;血管生成）中具有重要作用。VEGF通过与内皮细胞的细胞表面上的受体相互作用来介导其生理作用，所述受体然后经历构象变化以引起其细胞内表面上的酪氨酸残基变得磷酸化。这启动了一系列事件，导致一系列不同的细胞内信号蛋白的激活。这些介导各种变化，如细胞运动性，蛋白质表达和细胞存活。VEGF有三种不同的细胞表面受体与之相互作用，但VEGFR 2对血管生成是最重要的。VEGF信号传导的复杂性在于存在多种大小不同的VEGF同种型，并且可能具有显著不同的结合VEGFR 2和触发反应的能力。此外，VEGFR 2通常作为同二聚体（即，一个VEGFR 2分子与第二个VEGFR 2分子结合的复合物）起作用。然而，还已知VEGFR 2也可以与其他两种VEGF受体（VEGFR 1和VEGFR 3）相互作用以形成异源二聚体。此外，细胞膜上存在VEGFR 2的共受体（例如神经纤毛蛋白-1，NRP 1），其可以增强VEGF与VEGFR 2的结合或可以调节随后的功能反应。然而，不同的VEGF亚型如何与这些复合物中的受体结合尚不清楚。配体-受体相互作用的定量评价一直是药物发现过程的基石，特别是在其他类型的细胞表面受体（G蛋白偶联受体）的情况下，这些受体是超过40%的所有已知药物的靶点。然而，由于生长因子的蛋白质性质及其相互作用的复杂性，VEGF同种型与VEGFR 2的结合尚未得到相同程度的分析。该提案旨在利用PI和CI的详细和定量分析技能，利用新的生物技术方法（由工业合作伙伴开发）来研究活细胞中的配体-受体和受体-蛋白质相互作用，以揭示特定VEGF亚型的配体结合和信号传导特征（分子药理学）。这使用不同颜色的发光（生物产生的光输出）的测量。该工业合作伙伴设计了一种新型生物发光蛋白（NanoLuc），最初从深海虾中分离出来。这是蓝色的，明亮，稳定，肉眼可见。它可以连接到VEGFR 2或其相互作用蛋白的细胞外或细胞内末端而不丧失功能。所产生的光具有适当的波长，以激发邻近的荧光分子，如果它非常接近NanoLuc，因此可以产生不同颜色的光（例如红色）。该策略将用于监测活细胞中荧光标记的VEGF与VEGFR 2的结合，并建立每种VEGF亚型与特定VEGFR 2二聚体、VEGFR 2-NRP 1复合物结合的详细分子药理学，并确定它们是否能够选择性触发导致信号传导偏倚的特定细胞内信号传导途径。这将为VEGF功能提供相当多的见解，并为药物发现提供新的机会。

项目成果

Effects of receptor tyrosine kinase inhibitors on VEGF165 a- and VEGF165 b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2.

• DOI: 10.1111/bph.13116
• 发表时间: 2015-06
• 期刊: British journal of pharmacology
• 影响因子: 7.3
• 作者: Carter JJ;Wheal AJ;Hill SJ;Woolard J
• 通讯作者: Woolard J

Comparison of the ligand-binding properties of fluorescent VEGF-A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET.

使用 NanoBRET 比较活细胞和膜制剂中荧光 VEGF-A 亚型与 VEGF 受体 2 的配体结合特性。

• DOI: 10.1111/bph.14755
• 发表时间: 2019
• 期刊: British journal of pharmacology
• 影响因子: 7.3
• 作者: Peach CJ

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

• DOI: 10.1016/j.chembiol.2018.06.012
• 发表时间: 2018-10-18
• 期刊: Cell chemical biology
• 影响因子: 8.6
• 作者: Peach CJ;Kilpatrick LE;Friedman-Ohana R;Zimmerman K;Robers MB;Wood KV;Woolard J;Hill SJ
• 通讯作者: Hill SJ

The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors.

荧光相关光谱的使用来表征荧光标记的G蛋白偶联受体的分子迁移率。

• DOI: 10.1042/bst20150285
• 发表时间: 2016-04-15
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Kilpatrick LE;Hill SJ
• 通讯作者: Hill SJ

Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2.

• DOI: 10.3390/ijms19041264
• 发表时间: 2018-04-23
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Peach CJ;Mignone VW;Arruda MA;Alcobia DC;Hill SJ;Kilpatrick LE;Woolard J
• 通讯作者: Woolard J