GSH MEDIATED MECHANISMS IN NITROSOUREA TOXIC EFFECTS

GSH 介导的亚硝脲毒性作用机制

• 批准号: 3298619
• 负责人: DONALD C DE LUCA
• 金额: $ 9.99万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298619
• 关键词: affinity chromatography; carmustine; chemical structure function; chemical synthesis; drug adverse effect; electrofocusing; enzyme inhibitors; enzyme mechanism; enzyme structure; erythrocytes; glutathione; glutathione reductase; glutathione transferase; high performance liquid chromatography; human tissue; hydrolysis; mass spectrometry; molecular biology; nitrosourea

One of the consequences of the therapeutic use of BCNU is the inactivation of GSSG reductase. There is mounting evidence that the inactivation of this key antioxidant enzyme, leading to the cellular accumulation of toxic levels of GSSG, is a major factor in pulmonary and cerebral toxicities of BCNU. This project explores the molecular consequences of a BCNU-GSH derived inactivator of GSSG reductase. Preliminary studies revealed that an active site-directed inactivator of GSSG reductase, designated GS-I, was formed by the reaction of a BCNU metabolite with GSH. GS-I will be chemically synthesized and its structure proven by FAB mass spectrometry and 500 MHz NMR analyses. The kinetics and stoichiometry of the inactivation of purified human erythrocyte GSSG reductase by GS-I will be studied. Intact erythrocytes will be used to determine if exogenous GS-I inactivates erythrocytic GSSG reductase. It will also be determined if GS-I is synthesized and secreted by erythrocytes. Studies are designed to establish whether GSH transferase isoenzymes from human liver and erythrocytes catalyze the synthesis of GS-I from BCNU or limit the formation of GS-I by catalyzing the conversion of BCNU to other products, and whether such products are inactivators of GSSG reductase. The proposed structure for GS-I strongly suggests that it may serve as an active site-directed inactivator of other GSH- and GSSG-linked enzymes, which may then be additional specific molecular targets for BCNU toxic effects. The intrinsic susceptibilities to GS-I of purified gamma- glutamyltranspeptidase, GSH transferase isoenzymes, and glyoxalases I and II will be examined. Whether GS-I is a substrate for hydrolysis by glyoxalase II and/or by gamma-glutamyltrans- peptidase will also be investigated. This project is expected to (a) reconcile the in vitro and in vivo observations of others concerning the basis of BCNU-induced GSSG reductase deficiency, (b) increase understanding of nitrosourea activation and inactivation via GSH-dependent pathways, (c) reveal additional specific molecular targets of BCNU toxicities, and (d) provide information that will aid in predicting toxic effects of new nitrosoureas while these drugs are still at the design stage.

BCNU治疗用药的后果之一是 GSSG还原酶失活。越来越多的证据表明 这一关键抗氧化酶的失活，导致 GSSG的毒性水平在细胞内积累，是导致 BCNU的肺毒性和脑毒性。这个项目探索 BCNU-GSH衍生灭活剂的分子后果 GSSG还原酶。 初步研究表明，一种活跃的定点定向 GSSG还原酶失活剂，命名为GS-I BCNU代谢物与GSH的反应。GS-I会是 化学合成及其FAB MS确证其结构 光谱分析和500 MHz核磁共振分析。动力学和 纯化人红细胞失活的化学计量 对GS-I产生的GSSG还原酶进行了研究。完整的红细胞将 用于确定外源性GS-I是否使红细胞失活 GSSG还原酶。还将确定是否合成了GS-I 并由红细胞分泌。研究的目的是建立 人肝谷胱甘肽转移酶同工酶和 红细胞催化BCNU或LIMIT合成GS-I 催化BCNU转化生成GS-I 其他产品，以及这些产品是否是 GSSG还原酶。GS-I的拟议结构强烈 提示它可能是一种活性位点定向失活剂。 其他GSH和GSSG连接的酶，然后可能是 针对BCNU毒性效应的其他特定分子靶点。 纯化的伽马射线对GS-I的本征敏感性 谷氨酰转肽酶、谷胱甘肽转移酶同工酶和 乙二醛酶I和II将被检查。GS-I是否为底物 用于乙草酸酶II和/或伽马-谷氨酰反式- 还将对多肽酶进行调查。 该项目有望(A)协调体外和体内的 关于BCNU诱导的基础的其他人的观察 GSSG还原酶缺乏症，(B)增加对 谷胱甘肽依赖的亚硝脲激活和失活 途径，(C)揭示了其他特定的分子靶点 BCNU毒性，以及(D)提供有助于 预测新亚硝脲的毒性作用，同时这些药物 目前仍处于设计阶段。