Novel Kinase Allostery FRET Assays for Mechanistic Studies and Drug Design

项目摘要/摘要。蛋白激酶的信号转导控制细胞的许多方面 发展和扩散。解除对激酶的调控与许多癌症有关，而激酶抑制剂 是一类重要的化疗药物。然而，目前大多数可用的抑制剂效果不佳。 选择性(因为它们针对保守的ATP结合位点)，一年内临床耐药近 万能的。因此，对新的、更具特异性的激酶抑制剂的需求越来越大。的关注点 大多数Ser/Thr激酶的调控是在激活状态和自抑制状态之间的构象转换。 每一种激酶都有一组独特的变构介体参与这种转变，包括小分子配体，后... 激酶的翻译修饰，以及蛋白质与蛋白质的相互作用。活动状态和自动禁止状态包括 以一些保守的结构特征的构象来区分，包括激酶激活环。 在变构活化中，根据X射线结晶学，活化环残基移动了几个纳米， 解锁底物多肽结合部位，定位用于催化的保守的天冬氨酸残基。然而，非常 关于这些构象变化在溶液中是如何发生的，以及它们在激酶活性中的作用，我们知之甚少，因为 目前还没有对该激酶的结构状态和动力学进行实时分析。 在这项建议中，我们描述了一种新的测定方法的使用 人极光A激酶(Aura)的激活环，它是有丝分裂的关键调节因子，已参与 癌症发病机制。我使用定点突变将两个荧光探针整合到一个光环中 分子，然后我用纳米级的测量技术测量它们之间的距离 Förster共振能量转移(FRET)。我将使用这一化验，结合活动性化验，点 关键残基的突变，以及最先进的时间分辨FRET和动力学技术，以定义 支配灵气激活的构象集合和结构元素(目标1)。我也会用这个 使用FRET鉴定和筛选AURA变构抑制剂药物的方法(目标2)。我会澄清一下 现有抑制剂的结合模式，并将改进目前可用的高通量药物筛选 变构抑制剂的方法，它特异性地结合在激酶活性部位之外。我们预计我们的 FRET检测将在药物发现和了解变构调节方面取得重大进展。 根据这个奖项，我将在明尼苏达大学接受为期三年的博士后培训，与 激酶结构生物学专家尼古拉斯·莱文森博士和荧光专家大卫·托马斯博士 光谱学和药物发现方法。在他们的双重指导下，我将发展写作、演讲、 以及生物物理学、结构生物学和药物开发方面的实验室技术。这 培训将使我成为一名研究蛋白质变构及其机制的独立研究员。 药物设计申请。

PROJECT SUMMARY/ABSTRACT. Signal transduction by protein kinases controls many aspects of cell development and proliferation. Deregulation of kinases has been linked to many cancers, and kinase inhibitors are an important class of chemotherapeutic drugs. However, most currently available inhibitors are poorly selective (because they target the conserved ATP binding site), and clinical resistance within one year is nearly universal. There is thus an increasing demand for new and more specific kinase inhibitors. The focus of regulation for most Ser/Thr kinases is on the conformational transition between active and autoinhibited states. Each kinase has a unique set of allosteric mediators of this transition, including small molecule ligands, post- translational modifications of the kinase, and protein-protein interactions. Active and autoinhibited states are distinguished by the conformations of a few conserved structural features, including the kinase activation loop. In allosteric activation, according to x-ray crystallography, activation loop residues move several nanometers, unblocking the substrate peptide binding site and positioning a conserved Asp residue for catalysis. Yet, very little is known about how these conformational changes occur in solution and their role in kinase activity because there are currently no real-time assays for the structural state and dynamics of the kinase. In this proposal, we describe the use of a novel assay determining the conformation of the activation loop of human Aurora A kinase (AurA), a key regulator of mitosis which has been implicated in cancer pathogenesis. I use site-directed mutagenesis to incorporate two fluorescent probes into a single AurA molecule, and I then measure the distance between them using the nanometer-scale measurement technique of Förster resonance energy transfer (FRET). I will use this assay, in conjunction with activity assays, point mutation of critical residues, and state-of-the-art time-resolved FRET and kinetics techniques, to define the conformational ensembles and structural elements governing AurA activation (Aim 1). I will also use this assay to characterize and screen AurA allosteric inhibitor drugs using FRET (Aim 2). I will elucidate the binding modes of existing inhibitors and will improve upon currently available high-throughput drug screening methods for allosteric inhibitors which bind specifically outside the kinase active site. We anticipate that our FRET assay will lead to major advances in drug discovery and the understanding of allosteric regulation. Under this award, I will train for three years as a postdoctoral scholar at the University of Minnesota with Dr. Nicholas Levinson, an expert in kinase structural biology, and Dr. David Thomas, an expert in fluorescence spectroscopy and drug discovery methods. Under their dual mentorship, I will develop skills in writing, presenting, and collaboration, as well as laboratory techniques in biophysics, structural biology, and drug development. This training will prepare me to be an independent researcher studying mechanisms of protein allostery and its applications for drug design.