Precision pharmacology of the opioids

项目概要/摘要 物质滥用行为是神经元中基因表达程序随时间发生分子变化的结果。 吗啡和海洛因是高度成瘾的阿片类药物，主要刺激μ阿片受体，导致短暂的- 术语欣快症。与天然阿片肽不同，吗啡和海洛因的成瘾性可能是由于它们的 细胞渗透性，从而直接影响基因表达程序，导致成瘾。然而，在这方面， 理解这些精确的分子机制通常具有挑战性，因为缺乏技术， 精确绘制小分子的全局分子靶点和途径。我们已经开发了一个 跨学科的精确药理学策略，以映射小分子在 细胞蛋白质组，我们建议将其应用于这些成瘾性阿片类药物。我们的方法借鉴了以下领域 化学生物学、质谱分析和数据科学，使人们能够深入了解各种分子 相互作用，相互作用位点的结构生物学，以及相互作用中下游途径的变化。 单一实验该策略包括：（1）用感兴趣的小分子处理全细胞，（2） 分离得到的全局分子结合位点和（3）基于可靠质谱的分配。 我们最近应用该平台研究了三种非甾体抗炎药（NSAID）。我们的结果 揭示了几种参与基因表达的蛋白质复合物，这些蛋白质复合物与NSAID相互作用，包括 直接与核小体接触这些结果指向了现在已经形成的巨大的分子机制网络。 通过精密药理学观察。在这个提案中，我们将把我们的技术应用于吗啡和海洛因， 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 “点击阿片样物质”和“光点击阿片样物质”作为用于在生物学中追踪阿片样物质机制的通常有用的探针。 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细胞中的遗传和表观遗传调控。 首先在组织培养中培养细胞，随后用于成瘾小鼠模型。全球阿片类药物分布图 相互作用体，这些数据将揭示直接阿片类药物相互作用与核小体的结合，共价修饰， 或介导乙酰化标记，以及间接影响上游转录程序， 表情变化。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，确定新治疗和诊断的新靶点， 开启了小分子生物调控的新范例。概述的研究是一个概念新颖 由于具有广泛的潜力，适合Avenir奖的作用机制研究方法 for impact影响and early早期stage阶段of this research研究.

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.