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.

概括阿片类药物是使用最广泛的镇痛药，但也有滥用这些药物的不良作用。药物，包括外周副作用、依赖性、耐受性和戒断，严重限制了它们在治疗中的效用。长期疼痛管理。-阿片受体（MOR）是阿片类镇痛、成瘾的主要目标。因此，人们致力于开发更安全的阿片类药物治疗并管理有问题的方面。影响，例如成瘾和戒断，需要更深入地了解遗传和分子调节 MOR 和阿片类药物行为影响的机制。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为了实现这一目标，我们部署了转基因 MOR (tgMOR) 模型，其中哺乳动物 MOR 在秀丽隐杆线虫的神经系统中表达，这为秀丽隐杆线虫注入了阿片类药物。我们使用计算自动化分析评估 tgMOR 动物的敏感行为。表现出高等生物体中阿片类药物反应的行为特征，包括急性抑制作用，我们之前使用正向遗传学和我们的 tgMOR 平台推出了一种新颖的、保守的抗阿片类药物系统，通过啮齿动物行为模型从秀丽隐杆线虫中发挥作用。现在已经鉴定出一种泛素连接酶，可以调节阿片类药物敏感性和对重复性行为的耐受性我们的建议为破译泛素连接酶的机制提供了前所未有的机会。信号传导在行为基因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塑造阿片类药物 1）我们将使用无偏见的、大规模的前向抑制遗传学与整体- 基因组测序和 CRISPR/Cas9 编辑可识别可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被泛素抑制的基因和遗传网络。连接酶活性影响阿片类药物反应 2) 我们将部署公正的、大规模的靶向蛋白质组学。这种泛素连接酶将确定其相互作用组、结合蛋白和假定的底物。这一目标有两种方法，既公正、大规模，又互补。目的，我们利用深入的生化和结构研究来评估这种泛素的候选底物使用直系同源人类泛素连接酶为我们提供了重要的翻译组件。我们进一步的目标是为这些人开发一种高通量筛选（HTS）兼容的检测方法。最后，我们使用这种 HTS 兼容测定法来初步筛选泛素连接酶的小分子抑制剂。这些泛素连接酶的目标是关于泛素连接酶信号传导如何影响的首次研究。使用创新的整体动物行为模型进行阿片类药物反应。