Cross platform analysis of drug targets and toxicity of bath salts

项目摘要/摘要 美国正处于药物滥用和精神健康流行病之中。在过去的十年里， 滥用精神刺激剂(有时是致幻剂)卡西酮，通常被称为“浴盐”，以及 像flakka这样的变体在美国年轻人中越来越受欢迎。尽管有大量的 研究其中一些制剂的成瘾和不良影响的潜在机制，如 3，4-亚甲基二氧基甲基苯丙胺(MDMA)，各种各样的变种可以在互联网上出售。 此外，这些只是可以以公斤为单位生产的可想到的结构的一小部分 在线销售规模。更全面地了解与以下药物相关的药物靶点和毒性 这些药物是治疗药物滥用障碍和间歇性毒性的重要一步。 我们工作的总体目标是统一涉及神经行为的整个系统层面的分析 在分子水平上研究结合伴侣对精神活性物质的影响。这件事的重点是 建议实施一种新的体内光亲和标记(PAL)方法来跨平台分析 精神活性物质，重点是与亚甲基二氧基吡喃酮和α相关的结构- 吡咯烷基丙酮(α-PVP)。在目标1中，我们将合成一组精心设计的探针，以 已编入AIM 2分析。这包括PAL探针的立体选择性合成、构效关系 目标2C中用于LC-MS研究的(SAR)对照和稳定同位素标记标准。合成的文库将 测定1)受体结合谱：2)对大鼠突触体单胺摄取和释放的影响； 3)多细胞系HepG2(肝脏)、SHSY-5Y(神经细胞)、AC16(心肌细胞)和PC-12的毒性 (神经元[大鼠])。在目标2C中，活性化合物(和选定的非活性对照)将在成年斑马鱼中进行研究 在活体PAL同时进行的新型坦克测试的行为范式。每种药物的浓度 将通过LC-MS/MS进行测量，以将目标组织(脑)药物暴露与行为反应相关联。 选定的化合物将在AIM 2检测中重新检查，使用PAL进行结合的并排比较 AIM 2实验系统的相互作用组与体内结合的相互作用组。AIM 3中的化学生物学 将利用点击化学通过SDS PAGE凝胶的荧光成像来可视化蛋白质标记。蛋白质组学将 亲和纯化后确定蛋白质鉴定，以确定每个探针的结合相互作用体。一个 将进行各种竞赛实验来表征背景PAL并识别真实的PAL 目标。生物信息学将用于网络分析，以提供结合的公正比较 每个实验系统的互动，以提出潜在的治疗靶点来治疗毒性和发作 这些特工的杀伤力。

Project Summary/Abstract The United States is in the midst of a substance abuse and mental health epidemic. Over the past decade, abuse of psychostimulant (and sometimes hallucinogenic) cathinones, often referred to as “bath salts”, and variants such as ‘flakka’, has become increasing popular in America’s youth. Although there has been substantial research into the mechanisms underlying the addictive and adverse effects of some of these agents, such as 3,4-methylenedioxymethamphetamine (MDMA), a wide variety of variants are available for sale on the internet. Furthermore, these represent only a small fraction of conceivable structures which can be produced on kilograms scale for sale online. Gaining a more complete understanding of the drug targets and toxicity associated with these agents is an important step to treat substance abuse disorders and episodic toxicity. The over-arching goal of our work is to unite whole system level analysis involving the neurobehavioral effects of psychoactive agents with the study of binding partners at the molecular level. The focus of this proposal is to implement a novel in vivo photoaffinity labeling (PAL) approach for cross platform analysis of psychoactive agents, with an emphasis on structures related methylenedioxypyrovalerone (MDPV) and α- pyrrolidinovalerophenone (α-PVP). In Aim 1 we will synthesize a carefully designed set of probes to be cataloged in Aim 2 assays. This includes stereoselective synthesis of PAL probes, structure activity relationship (SAR) controls, and stable isotope labeled standards for LC-MS studies in Aim 2C. The synthesized library will be assayed for 1) receptor binding profile; 2) effect on monoamine uptake and release in rat synaptosomes; and 3) toxicity in multiple cell lines HepG2 (liver), SHSY-5Y (neuronal), AC16 (cardiac myocyte) and PC-12 (neuronal [rat]). In Aim 2C, active compounds (and select inactive controls) will be studied in an adult zebrafish behavioral paradigm utilizing the novel tank test with concurrent in vivo PAL. The concentration of each drug will be measured by LC-MS/MS to correlate target tissue (brain) drug exposure with behavioral responses. Select compounds will be re-examined in the Aim 2 assays using PAL for side-by-side comparison of binding interactome of Aim 2 experimental systems versus the in vivo binding interactome. Chemical biology in Aim 3 will utilize click chemistry to visualize protein labeling via fluoroimaging of SDS PAGE gels. Proteomics will determine protein identification after affinity purification to define the binding interactome of each probe. A variety of competition experiments will be performed to characterize background PAL and recognize bona fide targets. Bioinformatics will be used for network analysis to provide an unbiased comparison of the binding interactomes for each experimental system to propose potential therapeutic targets to treat toxicity and episodic lethality of these agents.