RNA Aptamer-Based Screen for Selective Inhibitors of GRK2

基于 RNA 适体的 GRK2 选择性抑制剂筛选

基本信息

批准号：
7929294
负责人：
John Tesmer
金额：
$ 3.86万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7929294
关键词：
ADRBK1 gene Active Sites Adrenal Glands Affinity Arrestins Beta-Adrenergic Receptor Kinase 1 Binding Binding Sites Biological Assay Biological Sciences Cardiac Cardiovascular Diseases Cardiovascular Physiology Catecholamines Cell Membrane Permeability Cell physiology Cells Chemicals Collaborations Complex Disease Engineering Exhibits Family Flow Cytometry Fluorescence G Protein-Coupled Receptor Kinase Family G protein coupled receptor kinase G-Protein-Coupled Receptors Genomics Goals Heart Diseases Heart failure Heterotrimeric GTP-Binding Proteins Institutes Kinetics Link Lobe Michigan Molecular Molecular Bank Neuraxis Nucleotides Peptides Permeability Phosphotransferases Physiological Play Production Protein Kinase Protein Kinase C Proteins RNA RNA Binding Reporting Role Signal Transduction Site Stimulus Structure Surface Tail Testing Therapeutic Therapeutic Agents Transgenic Organisms Universities aptamer base counterscreen design human disease improved inhibitor/antagonist interest mouse model mutant novel overexpression prevent public health relevance receptor sangivamycin small molecule small molecule libraries statistics therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): A small family of G protein-coupled receptor (GPCR) kinases (GRKs) negatively regulates heterotrimeric G protein signaling by phosphorylating multiple sites in the cytoplasmic loops and tails of activated GPCRs. Through this process, cells adapt to persistent stimuli that act at GPCRs and protect themselves from damage incurred by sustained signaling. GRKs can also play maladaptive roles in human disease. GRK2 is overexpressed during heart failure, which not only uncouples cardiac receptors from the central nervous system, but also promotes the release of excessive amounts of catecholamines from the adrenal gland. Inhibition of GRK2 by transgenic peptides prevents cardiac failure in mouse models, suggesting that GRK2 is an excellent target for the treatment of heart disease. However, selective small molecule inhibitors of GRKs have not been reported, perhaps due to high homology among the active sites of GRKs and other AGC kinases. Over the last six years, our lab has made significant progress in understanding the structure and function of GRKs, and we are currently investigating the molecular basis for the selective inhibition of GRK2 by a high affinity RNA aptamer. Our preliminary crystallographic studies of this complex demonstrate that the aptamer binds primarily to the large lobe of the kinase domain, where it blocks the entrance to the nucleotide binding site of the kinase domain. We hypothesize that this RNA aptamer can be used in a displacement assay to identify small molecules that bind to regions on GRK2 outside of its active site that are also critical for activity. We have designed improved versions of the original RNA aptamer for use in a robust flow cytometry protein interaction assay to screen for compounds that compete with RNA binding to GRK2. In collaboration with the Center for Chemical Genomics at the University of Michigan, we have conducted a preliminary HTS of ~40,000 compounds with excellent statistics. Using activity-based secondary screens, we will confirm which hits derived from this screen and those from screens conducted at a Molecular Libraries Probe Production Center bind directly to GRK2 and inhibit kinase activity. These compounds will be further characterized to establish membrane permeability, their mode of inhibition, and their selectivity for GRK2. Although all active molecules are of interest, small molecules that do not exhibit competitive inhibition with ATP are of particular importance because they would likely represent novel and selective therapeutic leads for the treatment of heart disease. PUBLIC HEALTH RELEVANCE: GRK2 is strongly linked to cardiovascular physiology and disease. Our flow cytometry protein interaction assay will allow us to rapidly screen large libraries of small molecules with the goal of identifying compounds that interfere with a high-affinity RNA aptamer that selectively binds to the kinase domain of GRK2. These compounds have the potential to interact with novel sites on the surface of the kinase domain and thus serve as selective inhibitors of GRK2.

描述（由申请人提供）：一小部分G蛋白偶联受体（GPCR）激酶（GRKS）通过磷酸化活性GPCR的细胞质环和尾巴中的多个位点来负调节异三聚体G蛋白信号传导。通过此过程，细胞适应对GPCR作用的持续刺激，并保护自己免受持续信号传导造成的损害。 GRK还可以在人类疾病中发挥不良适应性作用。在心力衰竭期间，GRK2过表达，这不仅取消了中枢神经系统的心脏受体，而且还促进了从肾上腺中释放过多的儿茶酚胺。通过转基因肽对GRK2的抑制可防止小鼠模型中的心脏衰竭，这表明GRK2是治疗心脏病的绝佳靶标。但是，尚未报道GRK的选择性小分子抑制剂，这可能是由于GRK和其他AGC激酶的活性位点的同源性很高。在过去的六年中，我们的实验室在理解GRK的结构和功能方面取得了重大进展，我们目前正在研究高亲和力RNA适体对GRK2选择性抑制的分子基础。我们对该复合物的初步晶体学研究表明，适体主要与激酶结构域的大叶结合，在那里它阻断了激酶结构域的核苷酸结合位点的入口。我们假设该RNA适体可以用于位移测定中，以鉴定与其活性位点外GRK2区域结合的小分子，这对活动也至关重要。我们已经设计了用于在强大的流式细胞仪蛋白相互作用测定中使用的原始RNA适体的改进版本，以筛选与RNA与GRK2结合竞争的化合物。与密歇根大学化学基因组学中心合作，我们进行了约40,000种具有出色统计数据的HTS的HTS。使用基于活动的二级筛选，我们将确认哪些筛选源是从该筛选中得出的命中，以及在分子文库下进行的筛选探针生产中心直接与GRK2结合并抑制激酶活性。这些化合物将进一步表征以建立膜渗透性，它们的抑制方式及其对GRK2的选择性。尽管所有活性分子都很有意义，但不表现出对ATP表现出竞争性抑制的小分子特别重要，因为它们可能代表了治疗心脏病的新颖和选择性治疗铅。公共卫生相关性：GRK2与心血管生理学和疾病密切相关。我们的流式细胞仪蛋白相互作用分析将使我们能够快速筛选大型小分子的库，其目的是识别干扰高亲和力RNA适体的化合物，该化合物选择性地结合了GRK2的激酶结构域。这些化合物具有与激酶结构域表面上的新位点相互作用的潜力，从而充当GRK2的选择性抑制剂。