Tyrosine Kinases in G Protein Mediated Signaling

说明书(申请人提供)：G蛋白偶联受体(GPCR)药物开发传统上侧重于正构体激动剂和拮抗剂的概念，其中受体结构决定下游信号的性质，配基效力决定其强度。在过去的十年里，新的“多维效能”和“功能选择性”范式已经证实，GPCRs通过参与经典的G蛋白依赖和新的G蛋白非依赖的信号网络来介导生物效应，并且配体结构可以通过稳定活性受体状态而不同于天然配体的比例来“偏向”信号传递。这种“偏向激动剂”是一种新的药理实体，能够定性地改变gpr信号，实际上创造了“由配基结构驱动的具有不同疗效的新受体”。偏向激动剂的前景在于这种产生“混合”效应的能力，在本项目的当前期限内的工作已经完成 证明了arrestin途径选择性激动剂可以在体内产生常规激动剂或拮抗剂无法达到的潜在有益效果。同时，我们的数据表明，以非自然的方式激活GPCRs可能会导致不可预测的下游生物学后果，特别是当使用选择性激活迄今尚未得到充分表征的arrestin依赖的信号网络的配体时。因此，由于无法根据芳香素偏向配体的体外疗效来预测其体内作用，因此开发偏向疗法的努力受到了阻碍。这一建议的中心假设是，体内结果依赖于arrestin的信号产生于一组相对离散和保守的生物反应的激活，这些生物反应可以在体外使用基于细胞的分析来描述，这些细胞分析适合于药物发现研究。在下一学期，我们计划使用最先进的生物物理和生物化学方法结合体内偏向配体作用的特征来检验这一假说。使用分子内闪光Bret传感器监测受体激活后拦阻蛋白的构象变化，Aim 1将检查决定arrestin的因素 ‘构象特征’，并在体外确定arrestin构象与受体/配体效力之间的关系。利用多维疗效分析和基于SILAC的信号网络全细胞磷蛋白质组分析，AIM 2将确定在体外可获得的偏向配体效应的范围，并测试不同GPCR之间依赖Arrestin的信号网络是否保守。然后，Aim 3将通过确定arrestin构象、体外配基效率和体内暴露于arrestin选择性偏向激动剂相关的生物学表型之间的关系来“闭合循环”。这些实验将在小鼠的骨形成模型中使用偏向的PTH1甲状旁腺激素受体配体。该项目的完成将有助于建立一个合理的框架，以便 体外配基对Arrestin途径选择性偏向的GPCR配基的体内作用的有效性，为开发新的偏向疗法提供关键的信息和工具。

DESCRIPTION (provided by applicant): G protein-coupled receptor (GPCR) drug development has traditionally focused on the concepts of orthosteric agonism and antagonism, in which receptor structure determines the nature of the downstream signal and ligand efficacy determines its intensity. Over the past decade, the newer paradigms of 'pluridimensional efficacy' and 'functional selectivity' have established that GPCRs mediate biological effects by engaging both classical G protein-dependent and novel G protein-independent signaling networks, and that ligand structure can 'bias' signaling by stabilizing active receptor states in different proportions than the native ligand. Such 'biased agonists' are novel pharmacological entities with the ability to qualitatively change GPCR signaling, in effect creating 'new receptors with distinct efficacy profiles driven by ligand structure. The promise of biased agonism resides with this ability to engender 'mixed' effects, and work during the current term of this project has demonstrated that arrestin-pathway selective agonism can produce potentially beneficial effects in vivo that are not attainable with conventional agonists or antagonists. At the same time, our data suggest that activating GPCRs in 'unnatural' ways can lead to unpredictable downstream biological consequences, especially when using ligands that selectively activate as-yet poorly characterized arrestin-dependent signaling networks. Efforts to develop biased therapeutics are thus hampered by an inability to predict the in vivo actions of arrestin-biased ligands based on their in vitro efficacy. The central hypothesis of this proposal is that the in vivo consequences arrestin-dependent signaling arises from activation of a relatively discrete and conserved set of biological responses that can be profiled in vitro using cell-based assays that are amenable to drug discovery research. During the next term, we plan to test this hypothesis using state-of-the-art biophysical and biochemical approaches combined with in vivo characterization of biased ligand action. Using intramolecular FlAsH BRET sensors that monitor conformational changes in arrestins following receptor activation, Aim 1 will examine the factors that determine the arrestin 'conformational signature' and determine the relationship between arrestin conformation and receptor/ligand efficacy in vitro. Using multi-dimensional efficacy profiling and SILAC-based whole cell phosphoproteomic analysis of signaling networks, Aim 2 will determine the range of biased ligand effects attainable in vitro and test whether the arrestin-dependent signaling network is conserved between different GPCRs. Aim 3 will then 'close the loop' by determining the relationship between arrestin conformation, in vitro ligand efficacy, and the biological phenotype associated with in vivo exposure to arrestin-selective biased agonists. These experiments will employ biased PTH1 parathyroid hormone receptor ligands in a murine model of bone formation. Completion of this project will help establish a rational framework for relating in vitro ligand efficacy to the in vivo actions of arrestin pathway-selective biased GPCR ligands, providing information and tools that are critical to the development of novel biased therapeutics.