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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R 03申请中，PI将在Lemmon实验室开发一个独立项目，旨在了解突变诱导的二聚化（相对于直接激活）的激酶结构域的相对重要性，肺癌患者的表皮生长因子受体（EGFR）。EGFR调节关键细胞过程如细胞运动、代谢、增殖和分化，并且通常由配体激活，诱导二聚化。在肺癌患者的一个亚组中，激酶结构域突变在EGFR缺失的情况下激活EGFR。的配体。传统上，这些突变（如L 858 R和外显子19缺失）被认为会偏向于基因突变。单体激酶结构域到其活性状态-不依赖于二聚化。然而，最近的一些研究表明激酶结构域二聚化是EGFR突变激活关键。此外，一些罕见的EGFR 突变-如外显子18中的突变，或引起激酶结构域重复（KDD）的突变-似乎激活EGFR 主要通过二聚化其激酶结构域。在这项提案中，我们询问EGFR突变是否/有多常见促进激酶结构域二聚化，以及它们是否能够“超活化”激酶结构域二聚体。此外，我们解决了外显子18和KDD突变的“仅二聚化”激活机制。通过了解这些不同的激活模式，我们希望深入了解新的治疗方法，通过靶向二聚化界面来抑制EGFR-这可能是详细的muplatin特异性的。通过体外建立反应动力学的激酶测定方法，以及许多生物物理方法，结构分析方法，分析和计算工具（实验验证），我们提出了三个具体目标：目的1：了解激酶结构域二聚化对EGFR活化的相对贡献，致癌突变使用合成的二硫键连接的EGFR激酶结构域二聚体，我们将研究难以捉摸的不对称EGFR激酶结构域二聚体。我们将进一步探讨常见的EGFR突变是否增强该二聚体的活性，并将比较外显子18和KDD突变诱导的二聚体的活性。目的2：携带外显子18的EGFR激酶结构域的结构分析和“溶液中”动力学突变或激酶结构域重复（KDD），其促进配体非依赖性二聚化。蛋白将采用晶体学来观察不同激酶结构域二聚体的界面，以询问是否它们是相同的或多核苷酸特异性的。同时，氢氘交换质谱（HDX-MS）将用于映射接口及其稳定性。这些研究将得到其他生物物理研究的补充。研究以获得由不同手段和突变诱导的二聚体的二聚体稳定性和动力学的观点。目的3：通过小分子作为蛋白质的破坏剂靶向激酶结构域二聚化界面- 蛋白质相互作用（PPI）。最后，我们将确定可以结合EGFR激酶结构域的结构基序二聚化界面，并以非竞争性或非竞争性方式破坏变构活化，在耶鲁分子发现中心筛选大环小分子文库。