Cross platform analysis of drug targets and toxicity of bath salts

药物靶点和浴盐毒性的跨平台分析

基本信息

批准号：
10704604
负责人：
Isaac T Schiefer
金额：
$ 55.65万
依托单位：
UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10704604
关键词：
Adult Adverse effects Affinity Affinity Chromatography Alkynes Azides Behavioral Behavioral Paradigm Binding Binding Proteins Bioinformatics Biological Assay Biology Biotin Brain Cardiac Myocytes Cell Line Chemicals Chemistry Databases Development Disease Drug Exposure Drug Targeting Drug toxicity Epidemic Funding Future Gel Generations Goals Grant Heart HepG2 Hepatic In Vitro Internet Investments Kilogram Label Lead Learning Libraries Liver Measures Mediating Mental Health Modeling Molecular Molecular Target National Institute of Drug Abuse Neurons Neurotransmitters PC12 Cells Pathway Analysis Pathway interactions Pharmaceutical Preparations Phenethylamines Phenotype Photoaffinity Labels Proteins Proteomics Psychotropic Drugs Rattus Research Route Sales Side Site Societies Stable Isotope Labeling Structure Structure-Activity Relationship Substance abuse problem Synaptosomes System Testing Therapeutic Time Tissues Toxic effect United States Variant Visit Visualization Work Youth Zebrafish analog bath salts behavioral response biological systems cathinone cell immortalization design ecstasy efficacy evaluation experimental study fluorophore in vivo innovation insight knock-down monoamine neurobehavioral new therapeutic target novel receptor receptor binding small molecule stimulant abuse therapeutic target uptake

项目摘要

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.

项目摘要/摘要美国正处于药物滥用和心理健康流行病之中。在过去的十年中，滥用心理刺激剂（有时甚至是致幻剂）的载体，通常称为“浴沙拉”和诸如“ Flakka”之类的变体在美国青年时代变得越来越受欢迎。虽然有很大的研究其中一些药物的添加和不利影响的基础机制，例如 3,4-甲基二甲基甲基苯丙胺（MDMA），可以在互联网上出售多种变体。此外，这些仅代表一小部分可想象的结构，可以在公斤上产生在线出售范围。对与与之相关的药物靶标和毒性有更全面的了解这些药物是治疗药物滥用障碍和情节毒性的重要步骤。我们工作的整个目标是使整个系统级分析涉及神经行为通过研究分子水平的结合伴侣的研究的影响。重点建议是实施一种新颖的体内光相位标签（PAL）方法，以进行跨平台分析精神活性剂，重点是与结构相关的甲基二邻苯甲酮（MDPV）和α- 吡咯烷蛋白甲酚（α-PVP）。在AIM 1中，我们将综合一组精心设计的问题在AIM 2分析中分类。这包括对PAL问题的立体选择性合成，结构活动关系（SAR）在AIM 2C中进行LC-MS研究的对照和稳定的同位素标记。合成的库将为1）受体结合曲线分配； 2）对大鼠突触体中单胺摄取和释放的影响； 3）在多个细胞系HEPG2（肝），SHSY-5Y（神经元），AC16（心肌细胞）和PC-12中的毒性（神经元[大鼠]）。在AIM 2C中，将在成年斑马鱼中研究活性化合物（和选择的无活性控制）采用新型储罐测试的行为范式以及同时的体内PAL。每种药物的浓度将通过LC-MS/MS测量，以将靶组织（脑）药物与行为反应相关联。选择化合物将在AIM 2分析中使用PAL重新检查，以并排比较绑定 AIM 2实验系统与体内结合相互作用组的相互作用组。 AIM中的化学生物学3 是否会通过SDS Page Gels的荧光影像学来使用点击化学来可视化蛋白质标记。蛋白质组学会确定亲和力纯化后的蛋白质鉴定，以定义每个探针的结合相互作用。一个将进行各种竞争实验以表征背景PAL并认识真正的善意目标。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相互作用体提出了潜在的治疗靶标，以治疗毒性和发作这些代理人的致命性。