Understanding biased agonism in receptor tyrosine kinases to devise new modalities for their targeting in cancer

了解受体酪氨酸激酶的偏向激动作用，以设计其靶向癌症的新方式

• 批准号: 10324584
• 负责人: Krishna Chaitanya Mudumbi
• 金额: $ 9.6万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-08 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324584
• 关键词: Adaptor Signaling Protein; Address; Affect; Affinity; Agonist; Amphiregulin; Area; Award; Biochemical; Biological Assay; Biophysics; Cancer Biology; Cell Proliferation; Cell surface; Cells; Dimerization; Environment; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epiregulin; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Fostering; Goals; Growth; Growth Factor; Heparin Binding; In Vitro; Institutes; Intervention; Kinetics; Laboratories; Life; Ligand Binding; Ligands; Malignant Neoplasms; Mediating; Mentorship; Methods; Microscopy; Modality; Modeling; Mutation; Nature; Normal Cell; Oncogenic; Outcome; Pharmacology; Phase Transition; Phosphorylation; Play; Positioning Attribute; Process; Protein Tyrosine Kinase; Protein phosphatase; Proteins; Receptor Activation; Receptor Inhibition; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Research; Resolution; Resources; Signal Transduction; Signaling Molecule; Site; Specificity; Structure; System; Tail; Techniques; Testing; Therapeutic; Time; Training; Transforming Growth Factors; Work; base; betacellulin; cancer cell; career; design; dimer; drug development; epigen; faculty research; in vivo; insight; light microscopy; medical schools; novel therapeutic intervention; particle; ras Proteins; receptor; recruit; research study; response; single molecule; skills; stoichiometry; success; therapeutic target

PROJECT SUMMARY/ABSTRACT This project takes a unique dynamic view of signaling by receptor tyrosine kinases (RTKs), testing the hypothesis that signaling specificity is kinetically defined, and that modulating dynamics might underlie a new therapeutic approach. Advancing with these questions will require new microscopy-based approaches in living cells – exploiting techniques that I have focused on throughout my career and taking advantage of the Lemmon lab’s biochemical expertise. Despite decades of study, and their importance as therapeutic targets, RTKs remain poorly understood mechanistically. Most RTKs dimerize upon ligand binding, and this is still believed to be the key step in their activation. The prevailing simple ‘on/off’ view is inconsistent, however, with the fact that RTKs can respond differentially to their multiple distinct activating ligands – displaying biased agonism or functional selectivity. Recent work in the Lemmon lab suggests that this selectivity is kinetically defined, with the life-time of the RTK’s activated state differing from ligand to ligand and defining the nature of the signaling outcome. Testing this new hypothesis requires single-molecule analysis of receptor activation kinetics in relevant cellular contexts. To date, kinetic arguments have only been inferred from structural and indirect signaling studies. My proposal focuses on directly observing the kinetics of RTK signaling in living cells. In particular, I will study the lifetime of different activated dimeric RTK states – and the resulting signaling kinetics – for the epidermal growth factor receptor (EGFR) when bound to its 7 different activating ligands. These studies will exploit advanced single-molecule fluorescence microscopy techniques in living cells that I have been developing, and will also correlate the results with structural and signaling work. My career goal is to obtain a research faculty position at a leading institute where I will continue to dissect the mechanisms of RTK dimerization and signaling. My successful transition to independence in this field would be significantly bolstered by augmenting my microscopy expertise with other biophysical and structural techniques in both in vitro and in vivo systems. It is with these acquired skills that I will be able to investigate how receptor dimerization dynamics define signaling specificity, and how they might be modulated pharmacologically. The success of this project will be greatly enhanced by the outstanding collaborators that I have assembled to advise me throughout my transition to independence. In addition, the exceptional research environment at the Cancer Biology Institute and the Yale Medical school area has all the necessary resources required for the proposed training and research studies. The K99/R00 would provide me with the protected time needed for this advanced training and allow me to continue to foster my growth under the mentorship of Dr. Mark Lemmon. I expect that the time provided by this award will allow me to elucidate the relationship between receptor dimerization dynamics and signal specificity, and will illuminate new avenues for pharmacological intervention.

项目摘要/摘要 该项目通过受体酪氨酸激酶（RTK）对信号传导独特的动态视图，测试 信号特异性是动力学定义的假设，并且调节动力学可能是新的 治疗方法。解决这些问题将需要新的基于显微镜的方法 牢房 - 利用我在整个职业生涯中都关注的技术，并利用 Lemmon Lab的生化专业知识。尽管研究了数十年，及其作为治疗目标的重要性，但 RTK的机械理解水平仍然很差。大多数RTK二聚在配体绑定上，这仍然是 被认为是激活的关键步骤。但是，普遍的简单“开/关”视图与 RTK可以对其多种不同的激活配体的反应不同的事实 - 表现出偏见 Lemmon Lab的最新工作表明，这种选择性在动力学上是 定义，随着RTK激活状态的终身时间从配体到配体的区别，并定义了的性质 信号结果。测试这一新假设需要单分子的受体激活分析 相关细胞环境中的动力学。迄今为止，动力学论证仅是从结构和 间接信号研究。我的建议重点是直接观察RTK信号的动力学 细胞。特别是，我将研究不同激活的二聚体RTK状态的寿命以及所得的信号 动力学 - 与表皮生长因子受体（EGFR）结合到其7种不同的活化配体时。 这些研究将利用活细胞中的晚期单分子荧光显微镜技术I 一直在发展，还将将结果与结构和信号工作相关联。 我的职业目标是在领先的研究所获得研究教师职位 解剖RTK二聚和信号传导的机制。我成功地过渡到独立性 通过在其他生物物理和其他生物物理和 体外和体内系统中的结构技术。正是这些获得的技能，我将能够 研究受体二聚化动力学如何定义信号特异性，以及如何调制它们 药理学。杰出的合作者将大大提高该项目的成功 在整个过渡到独立性的过程中，已经组装了建议。此外，杰出研究 癌症生物学研究所和耶鲁医学院地区的环境拥有所有必要的资源 拟议的培训和研究所必需的。 K99/r00会为我提供受保护的 这项高级培训所需的时间，让我继续以 马克·莱蒙（Mark Lemmon）博士。我希望该奖项提供的时间将使我能够阐明关系 受体二聚化动力学和信号特异性之间，并将阐明新的途径 药理干预。