Novel Kinase Allostery FRET Assays for Mechanistic Studies and Drug Design

项目总结/摘要。通过蛋白激酶的信号转导控制细胞的许多方面 发展和扩散。激酶的失调与许多癌症有关，激酶抑制剂 是一类重要的化疗药物。然而，大多数目前可用的抑制剂是差的， 选择性（因为它们靶向保守的ATP结合位点），一年内的临床耐药率接近 普遍的。因此，对新的和更特异性的激酶抑制剂的需求不断增加。的焦点 大多数Ser/Thr激酶的调节是在活性和自抑制状态之间的构象转变上。 每种激酶都有一套独特的这种转变的变构介质，包括小分子配体，后 激酶的翻译修饰和蛋白质-蛋白质相互作用。主动和自动抑制状态是 区别在于一些保守结构特征的构象，包括激酶激活环。 在变构激活中，根据X射线晶体学，激活环残基移动了几纳米， 解除底物肽结合位点的封闭并定位保守的Asp残基用于催化。然而， 关于这些构象变化如何在溶液中发生以及它们在激酶活性中的作用知之甚少， 目前还没有对激酶的结构状态和动力学的实时测定。 在这个提议中，我们描述了一种新的测定方法的使用， 人Aurora A激酶（AurA）的激活环，AurA是有丝分裂的关键调节因子， 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AurA中 然后我用纳米尺度的测量技术测量它们之间的距离 Förster共振能量转移（FRET）我将使用该测定，结合活性测定， 关键残基的突变，以及最先进的时间分辨FRET和动力学技术，以确定 构象系综和控制AurA激活的结构元件（Aim 1）。我也会用这个 使用FRET（Aim 2）表征和筛选AurA变构抑制剂药物的测定。我将阐明 现有抑制剂的结合模式，并将改善目前可用的高通量药物筛选 用于特异性结合激酶活性位点外的变构抑制剂的方法。我们预计， FRET分析将导致药物发现和了解变构调节的重大进展。 在这个奖项下，我将在明尼苏达大学作为博士后学者接受三年的培训， 博士激酶结构生物学专家尼古拉斯·莱文森和荧光学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，我将培养写作、演讲、 和合作，以及生物物理学，结构生物学和药物开发的实验室技术。这 培训将使我准备成为一名独立的研究人员，研究蛋白质变构的机制及其 用于药物设计。

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.