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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，阿片类药物正在流行，公共卫生危机，2021年有超过10万人死于阿片类药物过量。的关键部分解决办法是了解大脑回路对阿片类药物刺激的基本反应和适应。为例如，阿片受体的脱敏是疼痛管理中的一个关键问题，因为它需要增加止痛化合物的剂量，这可能有助于发展药物成瘾。近日已经表明神经元中μ和κ阿片样物质受体的激活引起活性氧（ROS）通过涉及NADPH氧化酶和c-Jun N-末端激酶的途径。因此，这种独特的反应，下游的受体，可以用来检测特定的阿片类药物受体激活和调节。然而，我们目前缺乏敏感的荧光传感器，这将允许我们直接监测μ阿片受体（莫尔）激活的下游通路。电流当前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，内源性水平与响应时间和信号幅度，这将使监测神经元脑组织中的莫尔激活后，我开发了一种新型的荧光ROS传感器，与以前的传感器相比，显著改善了信号幅度、灵敏度和响应动力学。我使用了一个新发现的OxyR插入位点，OxyR是一种细菌过氧化氢结合蛋白，与荧光报告结构域的改善的变构偶联。我们将使用新的绿色荧光蛋白（AausFP 1），具有出色的量子产率和亮度。我将优化一个新的ROS传感器以前所未有的速度通过多方面的方法，计算和进化蛋白质工程。我的目标是用莫尔积极的方式来表达这个新颖的工具并将ROS信号与莫尔活性联系起来。我假设在莫尔中ROS信号神经元将在吗啡而不是芬太尼下通过包括c-Jun N-末端激酶的途径增加， Peroxiredoxin 6和NADPH氧化酶。此外，我假设我们将观察到ROS的减少，反映MORs脱敏的重复药物暴露下的瞬变。最后，我会有一本小说以及用于监测阿片受体活性和适应性的高度特异性传感器。我的提议意义重大因为，这是第一次，我们将能够监测这种临床相关信号通路的适应性，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一个难以进入的神经元信号通路。此外，这种方法可以广泛应用于未来的研究，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。