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）：本次会议的目标 研究旨在阐明细胞色素 P450 2D6 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 2D6 (P450 2D6)，一种已在人脑中发现的酶， 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病有关 疾病。 我们的目标是增进对这种关系的理解 P450 2D6 多态性与帕金森病在分子水平上的关系 并最终为复杂的相互作用提供机械基础 本病的病因有遗传和环境因素。 虽然 该应用侧重于评估单个 P450 2D6 突变体， 表现出 Arg296 到 Sys296 突变，且风险因素最高 尚未确定该疾病（= 5.56），制定的方案将是 一般适用于调查任何P450的角色 神经毒性的变体。 具体来说，我们建议生产P450 2D6 及其变体的数量足以进行体外研究 在 COS 和 Sf9 细胞中表达。 我们将定义体外 野生型和突变型 P450 2D6 之间的结构-功能关系 与以下方面有关的酶：(1)底物代谢和/或酶抑制； (2)反应中间体或活性氧的产生 或结构相关的四氢异喹啉、b-咔啉家族， 和吡啶化合物，已建立或提议有助于 帕金森病的病因学。 最后，我们建议转染 儿茶酚胺能细胞系，大鼠嗜铬细胞瘤PC12细胞，与野生 型和突变型酶。 之间的灵敏度差异 转染子的神经元应激和毒性标记物 使用通过我们的体外鉴定的潜在前神经毒素进行治疗 研究可能会说明这种代谢的细胞后果 差异。 经过验证，这些和类似转染的神经元 细胞系可能有助于开发用于评估 内源性和外源性化合物的神经毒性 P450 2D6 的脑代谢。