Deciphering biased agonistic activation of mu-opioid receptor by novel optogenetic hydrogen peroxide sensor

新型光遗传学过氧化氢传感器破译μ阿片受体的偏向激动激活

基本信息

批准号：
10604662
负责人：
Justin Lee
金额：
$ 4.16万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10604662
关键词：
Acute Addictive Behavior Address Analgesics Animals Anxiety Arrestins Behavior Binding Proteins Brain Cells Chronic Clinic Codeine Complex Coupling Dependence Detection Directed Molecular Evolution Disease Dose Drug Addiction Drug Design Drug Exposure Drug Tolerance Drug Withdrawal Symptoms Embryo Emotions Engineering Enhancers Enterobacteria phage P1 Cre recombinase Epidemic Equipment Exhibits Face Fentanyl Funding Future G Protein-Coupled Receptor Signaling GTP-Binding Proteins Goals Green Fluorescent Proteins Health Homologous Gene Human Hydrogen Peroxide In Vitro Injections Kinetics Ligands Link MAPK8 gene Machine Learning Measures Mediating Molecular Molecular Target Monitor Morphine Mus NADPH Oxidase National Institute of Drug Abuse Neurobiology Neurodegenerative Disorders Neuronal Plasticity Neurons Opioid Opioid Analgesics Opioid Receptor Opioid agonist Output Pain Pain management Pathologic Pathway interactions Performance Persons Pharmaceutical Preparations Pharmacology Physiological Adaptation Problem Solving Production Property Protein Activation Pathway Protein Engineering Proteins Public Health Reaction Reaction Time Reactive Oxygen Species Receptor Activation Reporter Research Rodent Model Signal Pathway Signal Transduction Site Slice Source Specificity Stress Structure System Technology Time Tissues Toxic effect United States Dept. of Health and Human Services Universities Validation Ventilatory Depression Ventral Tegmental Area Virus Washington addiction animal tissue base brain tissue cell type clinically relevant desensitization design disease phenotype drug development drug withdrawal fluorescence imaging high throughput screening improved in vivo innovation insight kappa opioid receptors kidney cell motivated behavior mu opioid receptors neurotransmission novel opioid epidemic opioid exposure opioid overdose opioid use optogenetics overdose death peroxiredoxin prevent prototype quantum receptor response sensor side effect spatiotemporal targeted imaging tool

项目摘要

ABSTRACT: In 2017 the U.S. Department of Health and Human Services declared the ongoing opioid epidemic a public health crisis and more than 100,000 people died due to opioid overdose in 2021. A critical part of the solution is to understand the fundamental reaction and adaptation of brain circuits to stimulation by opioids. For example, the desensitization of opioid receptors is a critical problem in pain management because it requires increasing doses of analgesic compounds, which could contribute to developing a drug addiction. Recently, it has been shown that the activation of mu and kappa opioid receptors in neurons causes the production of reactive oxygen species (ROS) through a pathway involving NADPH oxidase and c-Jun N-terminal kinase. Therefore, this distinct response, downstream from the receptor, could be utilized to detect specific opioid receptor activation and modulation. However, we currently lack sensitive fluorescent sensors, which would allow us to directly monitor pathways downstream from mu-opioid receptor (MOR) activation in real-time. Current limitations of contemporary sensors are slow response time, low specificity, low signal output, low brightness, and toxicity. My goal is to develop a genetically encoded sensor protein that detects ROS levels at endogenous levels with response time and signal amplitudes that will enable monitoring of neuronal systems upon MOR activation in brain tissue. I have developed a novel fluorescent ROS sensor with significantly improved signaling amplitude, sensitivity, and response kinetics compared to previous sensors. I used a newly identified insertion site on OxyR, a bacterial hydrogen peroxide binding protein, that putatively improved allosteric coupling to the fluorescent reporter domain. We will increase the fidelity of this tool with new green fluorescent protein (AausFP1) that exhibits exceptional quantum yield and brightness. I will optimize a new ROS sensor at an unprecedented rate through a multifaceted approach that combines rational, computational, and evolutionary protein engineering. My objective is to express this novel tool in MOR positive neurons and to link ROS signals to MOR activity pharmacologically. I hypothesize that ROS signals in MOR neurons will increase under morphine but not fentanyl through a pathway including c-Jun N-terminal Kinase, Peroxiredoxin 6 and NADPH oxidase. Furthermore, I hypothesize that we will observe a decrease in ROS transients under repeated drug exposure reflecting the desensitization of MORs. At the end, I will have a novel and highly specific sensor for monitoring opioid receptor activity and adaptivity. My proposal is significant because, for the first time, we will be able to monitor the adaptation of this clinically relevant signaling pathway to opioid exposure. My approach is innovative because it combines novel protein engineering and monitoring of opioid-triggered signals to dissect a difficult-to-access neuronal signaling pathway. Furthermore, this approach could be broadly applied in future studies to monitor the activity of opioid receptors during drug exposure and link the subsequent changes in neuronal signaling and plasticity to motivated behaviors, or analgesic tolerance.

摘要：2017 年，美国卫生与公众服务部宣布阿片类药物流行病持续存在 2021 年，一场公共卫生危机，超过 10 万人因阿片类药物过量死亡。解决方案是了解大脑回路对阿片类药物刺激的基本反应和适应。为了例如，阿片受体的脱敏是疼痛管理中的一个关键问题，因为它需要增加镇痛化合物的剂量，这可能会导致药物成瘾。最近，它研究表明，神经元中 mu 和 kappa 阿片受体的激活会导致产生通过涉及 NADPH 氧化酶和 c-Jun N 末端激酶的途径释放活性氧 (ROS)。因此，受体下游的这种独特反应可用于检测特定的阿片类药物受体激活和调节。然而，我们目前缺乏灵敏的荧光传感器，这使得我们可以直接实时监测 mu-阿片受体 (MOR) 激活的下游途径。当前的现代传感器的局限性是响应时间慢、特异性低、信号输出低、亮度低、和毒性。我的目标是开发一种基因编码的传感器蛋白，可以检测 ROS 水平具有响应时间和信号幅度的内源水平，可以监测神经元脑组织中 MOR 激活后的系统。我开发了一种新型荧光 ROS 传感器与以前的传感器相比，显着提高了信号幅度、灵敏度和响应动力学。我使用了 OxyR（一种细菌过氧化氢结合蛋白）上新发现的插入位点，推测改善与荧光报告结构域的变构耦合。我们将通过新的方法来提高该工具的保真度绿色荧光蛋白 (AausFP1)，具有出色的量子产率和亮度。我会优化一个新的ROS传感器以前所未有的速度通过多方面的方法结合了理性、计算和进化蛋白质工程。我的目标是以 MOR 积极的方式表达这个新颖的工具神经元并在药理学上将 ROS 信号与 MOR 活性联系起来。我假设 ROS 在 MOR 中发出信号神经元在吗啡作用下会通过包括 c-Jun N 末端激酶在内的途径增加，但在芬太尼作用下不会增加，过氧化还原蛋白 6 和 NADPH 氧化酶。此外，我假设我们会观察到 ROS 的减少重复药物暴露下的瞬变反映了 MOR 的脱敏。最后我会写一本小说用于监测阿片受体活性和适应性的高度特异性传感器。我的建议意义重大因为，我们将第一次能够监测这一临床相关信号通路的适应情况阿片类药物暴露。我的方法是创新的，因为它结合了新颖的蛋白质工程和监测阿片类药物触发的信号来剖析难以访问的神经元信号通路。此外，这种方法可以广泛应用于未来的研究中，以监测药物暴露期间阿片受体的活性，将神经元信号和可塑性的后续变化与动机行为或镇痛耐受性联系起来。