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）：G蛋白偶联受体（GPCR）激酶（GRK）的一个小家族通过磷酸化激活的GPCR的细胞质环和尾部中的多个位点来负调节异三聚体G蛋白信号传导。通过这个过程，细胞适应作用于 GPCR 的持续刺激，并保护自己免受持续信号传导造成的损害。 GRK 还可在人类疾病中发挥适应不良作用。 GRK2在心力衰竭期间过度表达，这不仅使心脏受体与中枢神经系统解偶联，而且还促进肾上腺释放过量的儿茶酚胺。转基因肽对 GRK2 的抑制可预防小鼠模型中的心力衰竭，这表明 GRK2 是治疗心脏病的绝佳靶点。然而，GRKs 的选择性小分子抑制剂尚未见报道，这可能是由于 GRKs 和其他 AGC 激酶的活性位点具有高度同源性。在过去的六年里，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，目前我们正在研究高亲和力 RNA 适体选择性抑制 GRK2 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。我们假设这种 RNA 适体可用于置换测定，以识别与 GRK2 活性位点之外的区域结合的小分子，这些区域对活性也至关重要。我们设计了原始 RNA 适体的改进版本，用于强大的流式细胞术蛋白质相互作用测定，以筛选与 RNA 与 GRK2 结合竞争的化合物。我们与密歇根大学化学基因组学中心合作，对约 40,000 种化合物进行了初步 HTS，并具有出色的统计数据。使用基于活性的二次筛选，我们将确认来自该筛选的命中以及来自分子库探针生产中心进行的筛选的命中直接与 GRK2 结合并抑制激酶活性。这些化合物将被进一步表征以确定膜通透性、它们的抑制模式以及它们对 GRK2 的选择性。尽管所有活性分子都令人感兴趣，但不表现出与 ATP 竞争性抑制的小分子尤其重要，因为它们可能代表治疗心脏病的新颖且选择性的治疗先导化合物。公共卫生相关性：GRK2 与心血管生理学和疾病密切相关。我们的流式细胞术蛋白质相互作用测定将使我们能够快速筛选大型小分子文库，目的是识别干扰选择性结合 GRK2 激酶结构域的高亲和力 RNA 适体的化合物。这些化合物有可能与激酶结构域表面的新位点相互作用，从而作为 GRK2 的选择性抑制剂。