Targeting differential kinase domain dimerization of EGFR mutants

在此R03应用程序中，PI将在Lemmon实验室开发一个独立项目，旨在了解 突变诱导的蛋白激酶结构域二聚化(相对于直接激活)的相对重要性 肺癌患者表皮生长因子受体的检测EGFR调节关键细胞 细胞运动、新陈代谢、增殖和分化等过程，通常由配体激活- 诱导二聚化。在一组肺癌患者中，激酶结构域突变在缺失的情况下激活了EGFR 配位体。传统上，这些突变(如L858R和外显子19缺失)被认为会使 单体激酶结构域到其活性状态--不依赖于二聚作用。然而，最近的一些研究 提示激酶结构域二聚化是EGFR突变激活的关键。此外，几种罕见的EGFR 突变--例如外显子18上的突变，或者引起激酶域复制(KDD)的突变--似乎激活了EGFR 主要是通过将其激活域二聚化。在这项建议中，我们询问是否/如何常见的EGFR突变 促进激活域的二聚化，以及它们是否能够‘超激活’一个激活域二聚体。 此外，我们还讨论了外显子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，我们希望能洞察到新的治疗方法 通过靶向二聚化界面来抑制EGFR--这可能是突变特有的详细信息。通过体外试验 建立反应动力学的激酶分析方法，以及许多生物物理方法，结构 分析和计算工具(经实验验证)，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 生化上不对称的EGFR激酶结构域二聚体。我们会问常见的EGFR突变是否会进一步 增强该二聚体的活性，并将比较由外显子18和KDD突变诱导的二聚体的活性。 目的2：含有外显子18的EGFR激酶结构域的结构分析和‘溶液中’动力学 促进配体非依赖性二聚化的突变或激酶域复制(KDD)。蛋白 我们将继续进行结晶学研究，以可视化不同的激酶结构域二聚体的界面--询问是否 它们是相同的或突变特有的。氢-氚交换质谱仪(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。 研究获得由不同方式和突变引起的二聚体的稳定性和动力学的观点。 目的3：以小分子作为蛋白质的干扰物靶向激活域二聚化界面- 蛋白质相互作用(PPI)。最后，我们将确定可以与EGFR激酶结构域结合的结构基序 二聚界面并以非竞争性或非竞争性的方式破坏变构活化 耶鲁大学分子发现中心大环小分子文库的筛选。

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.