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.

项目概要/摘要 该项目采用受体酪氨酸激酶 (RTK) 信号传导的独特动态视角，测试 假设信号特异性是动力学定义的，并且调节动力学可能是新的基础 治疗方法。解决这些问题需要新的基于显微镜的生活方法 细胞——利用我在整个职业生涯中关注的技术，并利用 莱蒙实验室的生化专业知识。尽管经过数十年的研究，以及它们作为治疗靶点的重要性， RTK 在机制上仍然知之甚少。大多数 RTK 在配体结合后形成二聚体，这仍然是 据信这是激活它们的关键步骤。然而，流行的简单“开/关”观点与 事实上，RTK 可以对其多种不同的激活配体做出不同的反应 - 显示出偏见 激动或功能选择性。莱蒙实验室最近的工作表明，这种选择性在动力学上是 定义，RTK 激活状态的寿命因配体而异，并定义了 信号结果。测试这一新假设需要对受体激活进行单分子分析 相关细胞环境中的动力学。迄今为止，动力学论证仅从结构和 间接信号传导研究。我的建议侧重于直接观察生活中 RTK 信号的动力学 细胞。特别是，我将研究不同激活二聚体 RTK 状态的寿命 - 以及由此产生的信号 动力学 – 表皮生长因子受体 (EGFR) 与其 7 种不同的激活配体结合时的动力学。 这些研究将在活细胞中利用先进的单分子荧光显微镜技术 一直在开发中，并将把结果与结构和信号工作联系起来。 我的职业目标是在领先的研究所获得研究教职，我将继续在那里 剖析 RTK 二聚化和信号传导机制。我在这方面成功过渡到独立 通过增强我的显微镜专业知识与其他生物物理和 体外和体内系统的结构技术。凭借这些获得的技能，我将能够 研究受体二聚化动力学如何定义信号特异性，以及如何调节它们 药理学上。我所领导的杰出合作者将极大地促进该项目的成功 在我向独立过渡的整个过程中，大家聚集在一起为我提供建议。此外，卓越的研究 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需的。 K99/R00 将为我提供受保护的 这次高级培训需要时间，让我能够在导师的指导下继续促进我的成长 马克·莱蒙博士。我希望这个奖项提供的时间能让我阐明其中的关系 受体二聚化动力学和信号特异性之间的关系，并将阐明新的途径 药物干预。