Identifying novel molecular targets, signaling pathways and mechanisms underlying fentanyl overdose-induced severe respiratory depression and lethality in rats using TMT phosphoproteomics/proteomics

使用 TMT 磷酸蛋白质组学/蛋白质组学识别芬太尼过量引起大鼠严重呼吸抑制和致死的新分子靶标、信号通路和机制

• 批准号: 10831163
• 负责人: YING-XIAN PAN
• 金额: $ 47.1万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10831163
• 关键词: Alfentanil; Amygdaloid structure; Analgesics; Apnea; Brain Stem; Brain region; Breathing; Breeding; Carotid Body; Cells; Cessation of life; Complex; Data; Dose; Exons; Fentanyl; GPCR Signaling Pathway; Gene Targeting; Goals; Heroin; In Vitro; Intravenous; Ion Channel; Length; Lethal Dose 50; LoxP-flanked allele; Lung; Measures; Mediating; Messenger RNA; Modeling; Molecular; Molecular Target; Morphine; Muscle Rigidity; Neurons; Nucleus solitarius; Opioid; Opium; Pathologic Processes; Peripheral; Pharmacology; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological Processes; Play; Post-Translational Protein Processing; Prefrontal Cortex; Protein Kinase; Protein Kinase Protein Phosphorylation; Protein phosphatase; Proteins; Proteomics; Rattus; Regulation; Respiratory physiology; Role; Shapes; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Specific qualifier value; Sprague-Dawley Rats; System; Testing; Time; Tissues; Toxic effect; United States; Ventilatory Depression; analog; candidate selection; carfentanil; combat; falls; fentanyl overdose; in vivo; lipophilicity; locus ceruleus structure; midbrain central gray substance; mu opioid receptors; novel; novel therapeutic intervention; opioid mortality; overdose death; parabrachial nucleus; phosphoproteomics; preBotzinger complex; protein expression; respiratory

Project Summary/Abstract Fentanyl is a potent synthetic, lipophilic phenylpiperidine analgesic that primarily acts through the Oprm1 Exon 1 (E1)-associated full-length 7TM mu opioid receptors (MORs). Fentanyl has its unique pharmacology that distinguishes from opium-derived mu opioids such as morphine and heroin, which includes its faster onset and higher potency. Our preliminary studies showed that a lethal dose of fentanyl (10 mg/kg, i.v.) had no effect on persistent apnea, muscle rigidity and lethality, but induced modest respiratory depression in our Oprm1 E1-KO (rE1d/d) rats, suggesting that fentanyl overdose-induced severe respiratory depression (FOISRD), persistent apnea, muscle rigidity and lethality are primarily mediated through MORs with additional non-MOR mechanisms, particularly of the respiratory depression, in our rat models. Respiratory regulation in mammalians is very complex and dynamic and involves coordination among multiple brain regions and several peripheral tissues such as the lung and carotid body at the molecular, cellular, and network levels. MORs are widely expressed at these sites. Increasing evidence has indicated that MORs play an essential role in mediating opioid-induced respiratory depression (OIRD). However, the molecular mechanisms and related signaling pathways underlying FOISRD and other toxicities via MORs or non-MOR systems remain largely unknown. Reversible protein phosphorylation by protein kinases and phosphatases is one of the most important post-translational modifications that plays a fundamental role in regulating almost all physiological and pathological processes. Mu opioid-induced phosphorylation occurs very rapidly, commonly within seconds or minutes, dynamically shaping the signaling. However, we know little about in vivo phosphorylation induced by mu opioids, particularly by fentanyl overdose. Our preliminary data using tandem mass tags-based phosphoproteomics/proteomics (TMT PP/P-omics) showed that fentanyl overdose can rapidly modify phosphorylation status of many signaling molecules, some of which likely contribute to FOISRD, persistent apnea, muscle rigidity and death. These data strongly support our overarching hypothesis that fentanyl overdose induces differential phosphorylation of crucial signaling molecules in multiple brain regions and peripheral tissues via MORs or non-MOR system and subsequently alters their related signaling pathways, leading to severe respiratory depression, persistent apnea, muscle rigidity and lethality. To test the hypothesis, we proposed the following Specific Aims: (1) Examine fentanyl overdose-induced phosphoproteins/proteins' changes in multiple brain regions and peripheral tissues of our rat Oprm1 gene targeting models using TMT PP/P-omics; (2) Identify molecular targets and signaling pathways that are responsible for FOISRD from selected candidate phosphoproteins from TMT PP/P-omics study. The proposed studies well fall into the goal of this specific RFA and promise to identify novel molecular mechanisms underlying FOISRD, persistent apnea, muscle rigidity and lethality and may identify the potential molecular targets for developing novel therapeutic strategies to combat FOISRD and fentanyl overdose death.

项目概要/摘要 芬太尼是一种有效的合成亲脂性苯基哌啶镇痛药，主要通过 Oprm1 外显子发挥作用 1 (E1) 相关全长 7TM mu 阿片受体 (MOR)。芬太尼有其独特的药理作用 与吗啡和海洛因等鸦片衍生的 mu 阿片类药物不同，其起效更快，且 更高的效力。我们的初步研究表明，致死剂量的芬太尼（10 mg/kg，静脉注射）对 持续性呼吸暂停、肌肉僵硬和致命性，但在我们的 Oprm1 E1-KO 中引起适度的呼吸抑制 (rE1d/d) 大鼠，表明芬太尼过量引起严重呼吸抑制 (FOISRD)，持续存在 呼吸暂停、肌肉僵硬和致死率主要通过 MOR 以及其他非 MOR 机制介导， 特别是在我们的大鼠模型中，呼吸抑制。哺乳动物的呼吸调节非常 复杂且动态，涉及多个大脑区域和多个周围组织之间的协调 例如分子、细胞和网络水平上的肺和颈动脉体。 MOR 广泛表达于 这些网站。越来越多的证据表明，MOR 在介导阿片类药物诱发的应激反应中发挥着重要作用。 呼吸抑制（OIRD）。然而，其分子机制和相关信号通路 FOISRD 和 MOR 或非 MOR 系统产生的其他毒性仍然很大程度上未知。可逆蛋白质 蛋白激酶和磷酸酶的磷酸化是最重要的翻译后过程之一 在调节几乎所有生理和病理过程中发挥基础作用的修饰。亩 阿片类药物诱导的磷酸化发生得非常快，通常在几秒或几分钟内，动态塑造 信号。然而，我们对 mu 阿片类药物诱导的体内磷酸化知之甚少，特别是 芬太尼过量。我们使用基于串联质量标签的磷酸蛋白质组学/蛋白质组学 (TMT PP/P-omics）表明芬太尼过量可以快速改变许多信号传导的磷酸化状态 分子，其中一些可能导致 FOISRD、持续性呼吸暂停、肌肉僵硬和死亡。这些数据 强烈支持我们的总体假设，即芬太尼过量会导致关键的磷酸化差异 通过 MOR 或非 MOR 系统在多个大脑区域和周围组织中传递信号分子 随后改变其​​相关的信号传导途径，导致严重的呼吸抑制、持续性呼吸暂停、 肌肉僵硬和致命性。为了检验假设，我们提出了以下具体目标：（1）检查 芬太尼过量引起脑部多个区域和周围组织磷蛋白/蛋白质的变化 使用 TMT PP/P-omics 建立我们的大鼠 Oprm1 基因靶向模型； (2) 识别分子靶点和信号传导 从 TMT PP/P-omics 选定的候选磷蛋白中找出导致 FOISRD 的途径 学习。拟议的研究完全符合该特定 RFA 的目标，并有望鉴定出新的分子 FOISRD、持续性呼吸暂停、肌肉僵硬和致命性的潜在机制，并可能确定潜在的 开发新的治疗策略以对抗 FOISRD 和芬太尼过量死亡的分子靶标。