权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
10657793
负责人：
Brock Grill
金额：
$ 60.54万
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/10657793
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 Nervous System Physiology Opioid Opioid Analgesics Opioid Receptor Organism Orphan Orthologous Gene Outcome Pain Pain management Pathway interactions Peripheral Pharmaceutical Preparations Pharmacology Proteomics Regulation Research Rewards Rodent Role Shapes Signal Transduction Signaling Molecule System Testing Time Transgenic Organisms Ubiquitination Validation Withdrawal addiction 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.

总结阿片类药物是使用最广泛的镇痛药，也是被滥用的物质。这些不良行为药物，包括外周副作用、依赖性、耐受性和戒断，严重限制了它们用于长期疼痛管理阿片受体（莫尔）是阿片类药物镇痛、成瘾的主要靶点和撤退。因此，旨在开发更安全的阿片类药物治疗和管理有问题的副作用的努力，影响，如成瘾和戒断，需要更深入地了解遗传和分子机制，调节莫尔的机制和阿片类药物的行为效应。我们的长期目标是使用无偏见的，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的敏感性和行为层面的宽容。为了实现这一目标，我们部署了转基因莫尔（tg莫尔）模型，其中哺乳动物莫尔在C.优美的这是C。用阿片类药物治疗我们用计算机自动化分析来评估敏感行为。重要的是，tgMOR动物在高等生物体中表现出阿片样物质反应的行为特征，包括急性兴奋效应，脱敏和耐受。我们以前使用正向遗传学和tgMOR平台来揭示一种新的，保守的抗阿片系统，从C.通过啮齿动物的行为模型。这种方法现在已经确定了一种泛素连接酶，调节阿片类药物的敏感性和行为耐受性，阿片类药物暴露我们的建议代表了一个前所未有的机会，破译如何泛素连接酶信号传导在行为遗传水平上影响阿片类药物反应。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样物质行为反应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样物质反应的突变， tgMOR泛素连接酶突变体。这将确定基因和遗传网络是抑制泛素连接酶活性以影响阿片样物质反应。2)我们将部署无偏见的，大规模的靶向蛋白质组学，这种泛素连接酶将决定其相互作用组、结合蛋白和推定底物。重要的是，这一目标的特点是采取了两种无偏见和大规模但又互补的办法。在第二目的是，我们使用深入的生化和结构研究，以评估候选底物，这种泛素连接酶使用邻位的人泛素连接酶提供了一个重要的翻译组成部分，我们提议我们进一步的目标是开发一种高通量筛选（HTS）兼容的测定，用于这些人泛素连接酶。最后，我们使用这种HTS兼容的检测方法进行小分子抑制剂的中试筛选，这些泛素连接酶。我们的目标是首次对泛素连接酶信号如何影响阿片类药物的反应使用一个创新的新的整体动物行为模型。