Precision pharmacology of the opioids

项目摘要/摘要 随着时间的推移，神经元中基因表达程序的分子变化导致了物质滥用行为。 吗啡和海洛因是高度成瘾的阿片类药物，主要刺激u阿片受体，导致短时间- 术语欣快感。与天然阿片肽不同，吗啡和海洛因的成瘾性质可能是由于它们的 细胞通透性，从而直接影响导致成瘾的基因表达程序。然而， 由于缺乏技术来了解这些精确的分子机制，通常是具有挑战性的 精确绘制小分子的全球分子靶点和路径。我们已经开发出一种 跨学科精确药理学策略，绘制小分子在体内的直接和间接影响 细胞蛋白质组，我们建议将其应用于这些成瘾的阿片类药物。我们的方法借鉴了以下领域 化学生物学、质谱学和数据科学，使人们能够洞察各种分子 相互作用，相互作用部位的结构生物学，以及在一个 单次实验。该策略包括：(1)用感兴趣的小分子处理整个细胞，(2) 分离得到的全球分子结合位点和(3)基于信任质谱学的指认。 我们最近应用这个平台研究了三种非类固醇抗炎药(NSAIDs)。我们的结果 揭示了与非类固醇抗炎药相互作用的几个参与基因表达的蛋白质复合体，包括一个 直接与核小体结合。这些结果指向了目前广泛存在的分子机制网络 通过精确药理学可以观察到的。在这项提案中，我们将把我们的技术应用于吗啡和海洛因，以 确定它们对导致药物滥用的基因表达的直接影响。我们将首先开发一套 “点击阿片类药物”和“照片点击阿片类药物”是追踪生物学中阿片类药物机制的普遍有用的探针。 我们将专门应用这些探针来表征阿片类药物驱动的神经元遗传和表观遗传调控。 细胞首先在组织培养，随后以小鼠为成瘾模型。一张阿片类药物的全球地图 这些数据将揭示阿片类药物与核小体通过结合、共价修饰、 或介导乙酰化标记，以及对驱动基因的上游转录程序的间接影响 表情发生了变化。通过描述阿片类药物更广泛的相互作用，我们准备揭露 导致成瘾的分子机制，确定新的治疗和诊断的新靶点，以及 开启了小分子生物调控的新范式。概述的研究在概念上是一种新奇。 由于具有广阔的潜力，开展适合Avenir奖的行动机制研究的方法 对于本研究的影响和早期阶段。

PROJECT SUMMARY/ABSTRACT Substance abuse behaviors result from molecular changes to gene expression programs in neurons over time. Morphine and heroin are highly addictive opioids that primarily stimulate the mu opioid receptor resulting in short- term euphoria. The addictive nature of morphine and heroin, unlike natural opioid peptides, may result from their cell permeability and thus direct influence on gene expression programs leading to addiction. However, understanding these precise molecular mechanisms is typically challenging due to the dearth of technologies to precisely map global molecular targets and pathways of a small molecule. We have developed an interdisciplinary precision pharmacology strategy to map the direct and indirect effects of a small molecule in the cellular proteome that we propose to apply to these addictive opioids. Our approach draws on the fields of chemical biology, mass spectrometry and data science to enable insight to the full range of molecular interactions, the structural biology underlying the interaction site, and changes to downstream pathways in a single experiment. The strategy involves: (1) treatment of whole cells with the small molecule of interest, (2) isolation of the resulting global molecular binding sites and (3) confident mass spectrometry-based assignment. We recently applied this platform to study three non-steroidal anti-inflammatory drugs (NSAIDs). Our results revealed several protein complexes involved in gene expression that the NSAIDs interact with, including a directly with the nucleosome. These results point to the vast web of molecular mechanisms that is now observable by precision pharmacology. In this proposal, we will apply our technology to morphine and heroin to determine their direct influence on gene expression leading to substance abuse. We will first develop a set of “click opioids” and “photo-click opioids” as generally useful probes for tracking opioid mechanisms in biology. We will specifically apply these probes to characterize opioid-driven genetic and epigenetic regulation in neuronal cells first in tissue culture and subsequently to mouse models of addiction. With a global map of the opioid interactome, these data will reveal direct opioid interactions with nucleosomes by binding, covalent modification, or mediating acetylation marks, and the indirect influence on upstream transcriptional programs that drive gene expression changes. By characterizing the broader interactions of the opioids, we are poised to expose molecular mechanisms leading to addiction, identify novel targets for new therapeutics and diagnostics, and open new paradigms in biological regulation by small molecules. The outlined research is a conceptually novel approach to perform mechanism of action studies that is suitable for the Avenir Award due to the broad potential for impact and early stage of this research.