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Regulation of Opioid Sensitivity and Tolerance by Ubiquitin Ligase Signaling

通过泛素连接酶信号调节阿片类药物敏感性和耐受性

基本信息

批准号：
10490609
负责人：
Brock Grill
金额：
$ 62.96万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10490609
关键词：
26S proteasome Acute Affect Affinity Chromatography American Analgesics Animals Behavior Behavioral Behavioral Genetics Behavioral Model Binding Binding Proteins Biochemical Biochemistry Biological Assay Biology CRISPR/Cas technology Caenorhabditis elegans Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Dependence Development Disease Engineering Exhibits Fentanyl G-Protein-Coupled Receptors GTP-Binding Proteins Genes Genetic Genetic study Goals Human Impairment Lesion Mediating Medical Modeling Molecular Morphine Mutation Nematoda Nervous System Physiology Nervous system structure Opioid Opioid Analgesics Opioid Receptor Organism Orphan Orthologous Gene Outcome Pain Pain management Pathway interactions Peripheral Pharmaceutical Preparations Pharmacology Proteomics Receptor Signaling Regulation Research Rewards Rodent Role Shapes Signal Transduction Signaling Molecule System Testing Time Transgenic Organisms Ubiquitination Validation Withdrawal addiction base behavioral response behavioral tolerance chronic pain management desensitization drug action experimental study forward genetics genome sequencing high throughput screening high-throughput drug screening in vivo inhibitor innovation multicatalytic endopeptidase complex mutant novel opiate tolerance opioid exposure opioid use opioid use disorder prevent programs response screening side effect small molecule small molecule inhibitor targeted treatment ubiquitin ligase ubiquitin-protein ligase whole genome

项目摘要

SUMMARY Opioid drugs are the most widely used analgesics, but also abused substances. The adverse actions of these drugs, including peripheral side effects, dependence, tolerance and withdrawal, severely limit their utility for long term pain management. The -opioid receptor (MOR) is the primary target of opioid analgesia, addiction and withdrawal. Thus, efforts aimed at developing safer opioid treatments and managing problematic side effects, such as addiction and withdrawal, require a much deeper understanding of the genetic and molecular mechanisms that regulate MOR and the behavioral effects of opioid drugs. Our long-term goal is to use unbiased, large-scale forward genetics and proteomics, as well as targeted biochemistry and pharmacology to understand how ubiquitin ligase signaling affects opioid sensitivity and tolerance at a behavioral level. Towards this goal, we deploy a transgenic MOR (tgMOR) model, in which mammalian MOR is expressed in the nervous system of C. elegans. This imbues C. elegans with opioid- sensitive behaviors that we evaluate using computationally automated assays. Importantly, tgMOR animals exhibit the behavioral hallmarks of opioid responses in higher organisms including acute depressant effects, desensitization and tolerance. We previously used forward genetics and our tgMOR platform to unveil a novel, conserved anti-opioid system that functions from C. elegans through rodent behavioral models. This approach has now identified a ubiquitin ligase that regulates opioid sensitivity and behavioral tolerance to repeated opioid exposure. Our proposal represents an unprecedented opportunity to decipher how ubiquitin ligase signaling affects opioid drug responses on a behavioral genetic level. Our first aim will use two primary approaches to determine how this ubiquitin ligase shapes opioid behavioral responses. 1) We will use unbiased, large-scale forward suppressor genetics combined with whole- genome sequencing and CRISPR/Cas9 editing to identify mutations that rescue abnormal opioid responses in tgMOR ubiquitin ligase mutants. This will identify genes and genetic networks that are inhibited by ubiquitin ligase activity to influence opioid responses. 2) We will deploy unbiased, large-scale targeted proteomics with this ubiquitin ligase that will determine its interactome, binding proteins and putative substrates. Importantly, this aim features two approaches that are unbiased and large-scale but also complementary. In the second aim, we use in depth biochemical and structural studies to evaluate candidate substrates for this ubiquitin ligase. Using orthologous human ubiquitin ligases provides an important translational component to our proposal. We further aim to develop a high throughput screen (HTS) compatible assay for these human ubiquitin ligases. Finally, we use this HTS compatible assays in a pilot screen for small molecule inhibitors of these ubiquitin ligases. Our aims represent first of their kind studies on how ubiquitin ligase signaling affects opioid responses using an innovative new whole animal behavioral model.

摘要阿片类药物是使用最广泛的止痛药，也是滥用的物质。这些人的不良行为药物，包括外周副作用、依赖性、耐受性和戒断，严重限制了它们对长期疼痛管理。阿片受体(MOR)是阿片类药物镇痛、成瘾的主要靶点和戒烟。因此，旨在开发更安全的阿片类药物治疗和管理有问题的方面的努力诸如成瘾和戒断等影响需要对遗传和分子方面有更深入的了解调节MOR和阿片类药物行为效应的机制。我们的长期目标是使用公正的、大规模的正向遗传学和蛋白质组学，以及有针对性的生物化学和药理学以了解泛素连接酶信号如何影响阿片类药物敏感性和行为层面的容忍。为此，我们采用了一种转基因MOR(TgMOR)模型，在该模型中哺乳动物MOR在线虫的神经系统中表达。这给线虫注入了阿片类药物- 我们使用计算机自动分析来评估的敏感行为。重要的是，tgmor动物在高等生物体中表现出阿片类药物反应的行为特征，包括急性抑郁效应，脱敏和耐受。我们之前用正向遗传学和我们的tgMOR平台揭开了一部小说的面纱，保守的抗阿片系统，通过啮齿动物行为模型从线虫中发挥作用。这种方法现在已经确定了一种泛素连接酶，它调节阿片类药物的敏感性和对反复发作的行为耐受性阿片类药物暴露。我们的提议是破译泛素连接酶的一个前所未有的机会信号传递在行为遗传水平上影响阿片类药物的反应。我们的第一个目标将使用两种主要方法来确定这种泛素连接酶如何形成阿片类药物行为反应。1)我们将使用无偏见的、大规模的正向抑制基因结合整体- 基因组测序和CRISPR/Cas9编辑以确定挽救异常阿片类药物反应的突变 TgMOR泛素连接酶突变体。这将识别泛素抑制的基因和遗传网络影响阿片类药物反应的连接酶活性。2)我们将部署无偏见的大规模靶向蛋白质组学这种泛素连接酶将决定其相互作用体、结合蛋白和可能的底物。重要的是这一目标的特点是两种方法，它们是不偏不倚的、大规模的，但也是互补的。在第二个目的：我们使用深入的生化和结构研究来评估这种泛素的候选底物连接酶。使用同源的人类泛素连接酶为我们的求婚。我们的进一步目标是为这些人开发一种高通量筛选(HTS)兼容的检测方法泛素连接酶。最后，我们将这种与HTS兼容的分析用于小分子抑制剂的中试筛选这些泛素连接酶。我们的目标代表了他们对泛素连接酶信号如何影响的第一个研究使用创新的全动物行为模型的阿片类药物反应。