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.

项目总结/摘要 芬太尼是一种有效的合成亲脂性苯基哌啶镇痛剂，主要通过Oprm 1外显子发挥作用 1（E1）相关全长7 TM μ阿片受体（MORs）。芬太尼有其独特的药理作用， 区别于阿片衍生的μ阿片类药物，如吗啡和海洛因，包括其更快的起效， 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，i. v.）不影响 持续性呼吸暂停、肌肉僵硬和致死性，但在我们的Oprm 1 E1-KO中诱导中度呼吸抑制 (rE1d/d）大鼠，表明芬太尼过量诱导的严重呼吸抑制（FOISRD），持续 呼吸暂停、肌肉僵硬和致死性主要通过MOR与另外的非MOR机制介导， 特别是在我们的老鼠模型中的呼吸抑制。呼吸调节在保加利亚人是非常 复杂和动态的，涉及多个脑区和几个外周组织之间的协调 例如肺和颈动脉体在分子、细胞和网络水平上。MORs广泛表达于 这些网站。越来越多的证据表明MORs在介导阿片类药物诱导的神经元凋亡中起重要作用。 呼吸抑制（OIRD）。然而，潜在的分子机制和相关的信号通路， FOISRD和其他通过MOR或非MOR系统的毒性在很大程度上仍然未知。可逆蛋白 蛋白激酶和磷酸酶的磷酸化是最重要的翻译后 这些修饰在调节几乎所有生理和病理过程中起着重要作用。亩 阿片样物质诱导的磷酸化发生非常迅速，通常在数秒或数分钟内， 信号。然而，我们对μ阿片类物质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化蛋白质组学/蛋白质组学（TMT） PP/P-omics）表明，芬太尼过量可迅速改变许多信号转导的磷酸化状态， 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，芬太尼过量诱导关键蛋白的差异磷酸化， 通过MOR或非MOR系统在多个脑区域和外周组织中的信号传导分子， 随后改变其相关的信号通路，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验证这一假设，我们提出了以下具体目标：（1）检查 芬太尼过量致多个脑区和外周组织磷蛋白/蛋白质改变 利用TMT PP/P-omics构建大鼠Oprm 1基因打靶模型;（2）分子靶点和信号转导 负责来自TMT PP/P-omics的选定候选磷蛋白的FOISRD的途径 study.所提出的研究很好地符合这种特定RFA的目标，并有望鉴定新的分子。 FOISRD的潜在机制，持续性呼吸暂停，肌肉僵硬和致死性，并可能确定潜在的 分子靶点开发新的治疗策略，以打击FOISRD和芬太尼过量死亡。