Targeting differential kinase domain dimerization of EGFR mutants

靶向 EGFR 突变体的差异激酶结构域二聚化

基本信息

批准号：
10201967
负责人：
Yuko Tsutsui
金额：
$ 16.75万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10201967
关键词：
Address Binding Binding Sites Biochemical Biological Assay Biology Cancer Patient Cell physiology Chemicals Clinic Complement Crystallization Crystallography Deuterium Dimerization Disulfides Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Exhibits Exons Extracellular Domain Family Generations Growth Factor Hydrogen In Vitro Induced Mutation Kinetics Lasers Libraries Ligands Link Malignant neoplasm of lung Maps Mass Spectrum Analysis Metabolism Molecular Motivation Mutate Mutation Oncogenic Phosphotransferases Process Proteins Reaction Receptor Activation Receptor Inhibition Receptor Signaling Research Scanning Scheme Signal Transduction Structure Techniques Therapeutic Tyrosine Kinase Inhibitor Validation Variant X ray diffraction analysis analytical ultracentrifugation base biochemical tools biophysical analysis biophysical techniques cell motility computerized tools design dimer inhibitor/antagonist insight interest light scattering monomer mutant novel novel strategies novel therapeutic intervention protein protein interaction real time monitoring receptor screening small molecule

项目摘要

In this R03 application, the PI will develop an independent project in the Lemmon lab aimed at understanding the relative importance of mutation-induced dimerization (versus direct activation) of the kinase domain of the Epidermal Growth Factor Receptor (EGFR) in lung cancer patients. The EGFR regulates critical cellular processes such as cell motility, metabolism, proliferation and differentiation, and is normally activated by ligand- induced dimerization. In a subset of lung cancer patients, kinase domain mutations activate EGFR in the absence of ligand. Traditionally, these mutations (such as L858R and exon 19 deletions) have been thought to bias the monomeric kinase domain to its active state – independent of dimerization. However, some recent studies suggest that kinase domain dimerization is key to mutational activation of EGFR. Moreover, several rare EGFR mutations – such as those in exon 18, or which cause kinase domain duplication (KDD) – seem to activate EGFR primarily by dimerizing its kinase domain. In this proposal, we ask whether/how common EGFR mutations promote kinase domain dimerization, and whether they are capable of ‘super-activating’ a kinase domain dimer. In addition, we address the ‘dimerization only’ activation mechanisms of exon 18 and KDD mutations. By understanding these different modes of activation, we hope to gain insight into new therapeutic approaches to inhibit EGFR by targeting the dimerization interface – which could be mutant-specific in detail. Through in vitro kinase assay approaches to establish reaction kinetics, as well as a host of biophysical methods, structural analysis, and computational tools (validated experimentally) we propose three Specific Aims: Aim 1: Understanding relative contribution of kinase domain dimerization to activation of EGFR by oncogenic mutations. Using a synthetic disulfide-linked EGFR kinase domain dimer, we will study the elusive asymmetric EGFR kinase domain dimer biochemically. We will ask whether common EGFR mutations further enhance activity of this dimer, and will compare activities of dimers induced by exon 18 and KDD mutations. Aim 2: Structural analysis and ‘in-solution’ dynamics of the EGFR kinase domain harboring exon 18 mutations or kinase domain duplications (KDD) that promote ligand-independent dimerization. Protein crystallography will be pursued to visualize the interfaces of the different kinase domain dimers – to ask whether they are identical or mutant-specific. In parallel, hydrogen-deuterium exchange mass spectrometry (HDX-MS) will be employed to map interfaces and their stability. These studies will be complemented by other biophysical studies to gain a view of dimer stability and dynamics for dimers induced by different means and mutations. Aim 3: Targeting the kinase domain dimerization interface by small molecules as disruptors of protein- protein interactions (PPIs). Finally, we will identify structural motifs that can bind to the EGFR kinase domain dimerization interface and disrupt allosteric activation in an uncompetitive or non-competitive fashion by screening libraries of macrocyclic small molecules in the Yale Center for Molecular Discovery.

在此R03应用程序中，PI将在旨在理解的Lemmon Lab中开发一个独立的项目突变诱导的二聚化（与直接激活）的相对重要性的相对重要性肺癌患者的表皮生长因子受体（EGFR）。诸如细胞运动，新陈代谢，繁殖和分化等过程，并从配体激活在肺癌患者的子群中诱导的二聚化，激酶结构域在没有的情况下激活EGFR 传统上，这些突变（例如L858R和外显子19删除）被认为是偏见单体激酶结构域至其活性状态 - 独立于二聚化。建议Tomain Dimerization是EGFR的关键玩具玩具玩具玩具玩具。突变 - 例如外显子18或引起激酶结构域重复（KDD）的突变似乎激活了EGFR 我们主要通过在此提案中降低其激酶结构域。促进激酶结构域二聚体，以及它们是否能够“超级激活”激酶结构域二聚体。此外，我们仅处理外显子18和KDD突变的“二聚化”激活机制了解不同的激活方式，我们希望能够深入了解新的新疗法通过靶向二聚化界面来抑制EGFR - 通过体外可以详细介绍激酶测定方法建立反应动力学以及许多生物物理方法的结构分析和计算工具（经过验证的实验）我们提出了三个具体目的： AIM 1：了解激酶结构域二聚化对EGFR的EGFR对EGFR的EGFR的EGFR EGFR的EGFR EGFR eGFR eGFR eGFR eGFR eGFR由EGFR By EGFR eGFR由EGFR By EGFR By EGFR由EGFR By EGFR By EGFR By EGFR由EGFR By EGFR By EGFR By EGFR By EGFR的EGFR对EGFR的EGFR进行了EGFR的EGFR的EGFR的EGFR的EGFR的EGFR的EGFR的EGFR的EGFR，EGFR由EGFR由EGFR由EGFR由EGFR由EGFR由EGFR 使用合成二硫键EGFR激酶结构域的致癌突变。非对称EGFR激酶域二聚体生化。增强该二聚体的活性，并将比较外显子18和KDDD突变的二聚体二聚体的活性。 AIM 2：EGFR激酶Domaing Exon 18的结构分析和“解决方案”动力学突变或激酶结构域的重复（KDD），促进了配体依赖性二聚体晶体学将被追捕以可视化不同激酶二聚体的界面 - 询问是否是否它们是相同或突变的特异性的。将用于绘制界面和稳定性。为了获得不同手段和静态的二聚体的二聚体稳定性和动力学的研究。 AIM 3：通过小分子作为蛋白质破裂的小分子靶向激酶结构域二聚化界面蛋白质相互作用（PPI）。二聚化界面和以非竞争或非竞争方式破坏变构激活在耶鲁分子发现中心中的大环小分子的筛选文库。