CYTOCHROME P450 2D6 VARIANTS IN NEUROTOXICITY

细胞色素 P450 2D6 变体的神经毒性

• 批准号: 2892022
• 负责人: TIMOTHY L MACDONALD
• 金额: $ 19.77万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2892022
• 关键词: PC12 cells; Sf9 cell line; complementary DNA; cytochrome P450; drug metabolism; enzyme inhibitors; free radical oxygen; genetic polymorphism; isoquinolines; isozymes; neurotoxicology; protein structure function; pyridine; pyridoindole; transfection

DESCRIPTION: (Adapted from Applicant's Abstract): The goal of this research is to elucidate the role that cytochrome P450 2D6 variants may play in neurotoxicity and neurodegenerative diseases. Polymorphism in cytochrome P450 2D6 (P450 2D6), an enzyme that has been identified in human brain and known to play a role in the metabolism of both foreign and endogenous pro-neurotoxins has been proposed to have an association with Parkinson's disease. It is our goal to move the understanding of the relationship between P450 2D6 polymorphism and Parkinson's disease to the molecular level and, ultimately, to provide a mechanistic basis for the complex interplay of genetic and environmental factors in the etiology of this disease. Although this application focuses on the evaluation of a single P450 2D6 mutant, which exhibits an Arg296 to Sys296 mutation and has the highest risk factor yet identified for the disease (=5.56), the protocol developed will be generally applicable to the investigation of the roles of any P450 variant(s) in neurotoxicity. Specifically, we propose to produce P450 2D6 and its variant in sufficient quantity for in vitro investigations by over expression in COS and Sf9 cells. We will define the in vitro structure-function relationships between the wild type and mutant P450 2D6 enzymes with regard to: (1) substrate metabolism and/or enzyme inhibition; and (2) the generation of reactive intermediates or reactive oxygen species or a family of structurally related tetrahydro-isoquinoline, b-carboline, and pyridine compounds, that are established or proposed to contribute to the etiology of Parkinson's disease. Finally, we propose to transfect a catecholaminergic cell line, rat pheochromocytoma PC12 cells, with the wild type and mutant enzymes. Differential sensitivities between the transfectants with regard to markers for neuronal stress and toxicity upon treatment with the potential pro-neurotoxins identified through our in vitro studies may illustrate the cellular consequences of such metabolic differences. With validation, these and analogously transfected neuronal cell lines may enable the development of assays for assessing the neurotoxicity of endogenous and foreign compounds that are responsible to cerebral metabolism by P450 2D6.

描述：（改编自申请人的摘要）：本发明的目标是 研究旨在阐明细胞色素P450 2D 6变体可能发挥的作用。 神经毒性和神经退行性疾病。 细胞色素多态性 P450 2D 6（P450 2D 6）是一种在人脑中发现的酶， 已知在外源性和内源性代谢中发挥作用 前神经毒素被认为与帕金森氏症有关 疾病 我们的目标是推动对这种关系的理解 P450 2D 6多态性与帕金森病在分子水平上的关系 并最终为以下复杂的相互作用提供一个机械基础： 遗传和环境因素在这种疾病的病因。 虽然 本申请集中于评价单个P450 2D 6突变体， 其表现出Arg 296到Sys 296的突变，并且具有最高的风险因子。 尚未确定的疾病（=5.56），制定的协议将是 一般适用于调查任何P450的作用 神经毒性的变异。 具体来说，我们建议生产P450 2D 6 及其变体，其量足以进行体外研究 在COS和Sf 9细胞中表达。 我们将定义体外 野生型和突变型P450 2D 6之间的结构-功能关系 （1）底物代谢和/或酶抑制; 和（2）反应性中间体或活性氧物质的产生 或结构上相关的四氢异喹啉，β-咔啉， 和吡啶化合物，已建立或建议有助于 帕金森病的病因 最后，我们建议选择一个 儿茶酚胺能细胞系，大鼠嗜铬细胞瘤PC 12细胞，与野生型 类型和突变酶。 之间的差异敏感性 关于神经元应激的标志物和对神经元的毒性， 治疗与潜在的前神经毒素确定通过我们的体外 研究可以说明这种代谢的细胞后果， 差异 通过验证，这些和类似转染的神经元 细胞系可以使得能够开发用于评估 内源性和外源性化合物的神经毒性， 脑代谢P450 2D 6。