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

• 批准号: 10444755
• 负责人: Isaac T Schiefer
• 金额: $ 48.01万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 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/10444755
• 关键词: Adult; Adverse effects; Affinity; Affinity Chromatography; Alkynes; Americas; Behavioral; Behavioral Paradigm; Binding; Binding Proteins; Bioinformatics; Biological Assay; Biology; Biotin; Brain; Cardiac Myocytes; Cell Line; Chemicals; Chemistry; Databases; Development; Disease; Drug Compounding; Drug Exposure; Drug Targeting; Drug toxicity; Epidemic; Foundations; Funding; Future; Gel; Generations; Goals; Grant; Heart; HepG2; Hepatic; In Vitro; Internet; Investments; Kilogram; Label; Lead; 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; 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

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.

项目概要/摘要 在过去的十年里，美国正处于药物滥用和心理健康流行之中。 滥用精神兴奋剂（有时致幻）卡西酮，通常被称为“浴盐”，以及 诸如“flakka”之类的变体在美国年轻人中越来越流行，尽管数量很大。 研究其中一些药物成瘾和不良反应的机制，例如 3,4-亚甲二氧基甲基苯丙胺 (MDMA) 的多种变体均可在互联网上出售。 此外，这些仅代表可以以公斤为单位生产的可想象结构的一小部分 更全面地了解药物靶标和相关毒性。 这些药物是治疗药物滥用疾病和偶发性毒性的重要一步。 我们工作的首要目标是将涉及神经行为的整个系统级分析结合起来 精神活性药物的影响与分子水平上结合伙伴的研究是本研究的重点。 建议实施一种新颖的体内光亲和标记（PAL）方法，用于跨平台分析 精神活性药物，重点是亚甲二氧基吡咯戊酮 (MDPV) 和 α- 相关结构 在目标 1 中，我们将合成一组精心设计的探针。 在 Aim 2 分析中进行分类，这包括 PAL 探针的立体选择性合成、结构活性关系。 (SAR) 对照，以及用于 Aim 2C 中 LC-MS 研究的稳定同位素标记标准品。 测定 1) 受体结合谱；2) 对大鼠突触体中单胺摄取和释放的影响； 3) 多种细胞系 HepG2（肝脏）、SHSY-5Y（神经元）、AC16（心肌细胞）和 PC-12 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（和选择的非活性对照）。 行为范例利用新颖的罐测试与同时体内 PAL 的每种药物的浓度。 将通过 LC-MS/MS 进行测量，以将目标组织（大脑）药物暴露与行为反应相关联。 选择的化合物将在 Aim 2 中使用 PAL 进行重新检查，以进行结合分析的并行比较 Aim 2 实验系统的相互作用组与 Aim 3 中的体内结合化学生物学。 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。 亲和纯化后确定蛋白质鉴定，以确定每个探针 A 的结合相互作用组。 将进行各种竞争实验来表征背景 PAL 并识别真实性 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组，以提出治疗毒性和偶发性的潜在治疗靶点 这些药剂的杀伤力。