GSH MEDIATED MECHANISMS IN NITROSOUREA TOXIC EFFECTS

GSH 介导的亚硝脲毒性作用机制

• 批准号: 3298618
• 负责人: DONALD C DE LUCA
• 金额: $ 10.7万
• 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/3298618
• 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的治疗用途的后果之一是， GSSG还原酶失活。 有越来越多的证据表明 这种关键的抗氧化酶的失活，导致 GSSG的毒性水平的细胞积累是一个主要因素， BCNU的肺和脑毒性。 该项目探索 BCNU-GSH衍生灭活剂的分子后果 GSSG reductase还原酶 初步研究表明，一个活跃的定点 GSSG还原酶的灭活剂，命名为GS-I，通过以下步骤形成： BCNU代谢物与GSH的反应。 GS-我会 化学合成并通过FAB质量证明其结构 光谱和500 MHz NMR分析。 的动力学和 纯化人红细胞灭活的化学计量学 将研究GS-I的GSSG还原酶。 完整的红细胞将 用于确定外源性GS-I是否使红细胞 GSSG还原酶。 还将确定是否合成了GS-I 并由红细胞分泌。 研究旨在建立 是否来自人肝脏的GSH转移酶同工酶和 红细胞催化BCNU或限制GS-I的合成 通过催化BCNU转化为 其他产品，以及这些产品是否是 GSSG还原酶。GS-I的拟议结构 表明它可以作为一种活性的定点灭活剂 其他GSH和GSSG连接的酶，然后可能是 BCNU毒性作用的其他特异性分子靶点。 纯化的γ-葡聚糖对GS-I的固有亲和性， 谷氨酰转肽酶，GSH转移酶同工酶，和 将检测糖苷酶I和II。 GS-I是否为底物 用于通过谷胱甘肽酶II和/或通过γ-谷氨酰反式- 还将研究肽酶。 该项目预计将（a）协调体外和体内 其他人关于BCNU诱导的基础的意见 GSSG还原酶缺乏，（B）增加对 亚硝基脲激活和失活通过GSH依赖 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 预测新的亚硝基脲的毒性作用，而这些药物是 还在设计阶段。