GLUTATHIONE METABOLISM IN TRYPANOSOMATIDS

锥虫中的谷胱甘肽代谢

• 批准号: 3131546
• 负责人: ALAN H FAIRLAMB
• 金额: $ 7.26万
• 依托单位: UNIVERSITY OF LONDON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3131546
• 关键词: Trypanosoma cruzi; affinity chromatography; antiprotozoal agents; autoradiography; crystallization; drug design /synthesis /production; endonuclease; enzyme biosynthesis; enzyme inhibitors; enzyme mechanism; gel filtration chromatography; glutathione; glutathione analog; high performance liquid chromatography; ion exchange chromatography; laboratory mouse; leishmaniasis; ligase; molecular cloning; nucleic acid sequence; oxidoreductase; parasitic diseases; peroxidases; protein biosynthesis; protein metabolism; radionuclides; scintillation counter; spermidine; trypanosomiasis

The ultimate goal of this research is to identify biochemical differences between mammalian host and parasite that can be exploited for the development of new and better chemotherapeutic compounds against diseases caused by Trypanosoma cruzi (Chagas' disease), Leishmania spp. (cutaneous, mucocutaneous and visceral Leishmaniasis) and T.rhodesiense or T.gambiense (human African trypanosomiasis). The general approach relies on the identification of novel and unique pathways, essential for the survival of the parasite, but not the human host. By characterizing such biochemical pathways and, in particular, gaining an understanding at the molecular level of the chemical mechanisms of key enzymatic reactions, it is hoped to provide a rational basis for the design of new inhibitors. The present application is a continuation of a previous grant, which led to the discovery of the novel dithiol spermidine-peptide, trypanothione (bis(glutathionyl)-spermidine). Three unique and key target enzymes have been identified so far: trypanothione reductase, trypanothione synthetase and trypanothione peroxidase. During the next grant period, it is intended to focus on T.cruzi (using C.fascicufata as a model, where appropriate) in order to achieve the following specific aims: 1. To purify and characterize trypanothione synthetase from C.fasciculata, the enzyme required for biosynthesis of trypyanothione and glutathionylspermidine. To design and test analogs of glutathione, spermidine and glutathionylspermidine as inhibitors. 2. To clone and sequence the genes encoding enzymes related to trypanothione metabolism, particularly with a view to expressing and producing T.cruzi trypanothione reductase and trypanothione synthetase in large amounts for drug development and crystallographic studies. 3. To further elucidate the roles of glutathione, glutathionylspermidine and trypanothione in the metabolism of these parasites, with particular emphasis given to: a) how glutathionylspermidine modulates intracellular levels of free spermidine. b) purification and characterization of trypanothione-dependent peroxidase activity. c) a comprehensive search for other glutathione- or trypanothione-dependent enzymes.

本研究的最终目标是确定生化 哺乳动物宿主和寄生虫之间的差异 为开发新的、更好的产品而开发 针对由以下原因引起的疾病的化疗化合物 克氏锥虫（恰加斯病）、利什曼原虫属。 （皮肤、 粘膜皮肤和内脏利什曼病）和 T.rhodesense 或 T.gambiense（非洲人类锥虫病）。 将军 方法依赖于新颖且独特的识别 途径，对于寄生虫的生存至关重要，但不是 人类宿主。 通过表征此类生化途径，并在 特别是在分子水平上获得了解 关键酶促反应的化学机制，希望能够 为新型抑制剂的设计提供合理依据。 本申请是先前资助的延续， 这导致了新型二硫醇亚精胺肽的发现， 锥硫酮（双（谷胱甘肽）-亚精胺）。 三个独特且 迄今为止已确定关键靶酶：锥硫酮 还原酶、锥硫酮合成酶和锥硫酮 过氧化物酶。 在下一个资助期内，计划重点关注 T.cruzi （在适当的情况下，使用 C.fascicufata 作为模型） 实现以下具体目标： 1. 纯化并表征锥硫酮合成酶 C.fasciculata，生物合成所需的酶 锥硫酮和谷胱甘肽亚精胺。 设计和测试 谷胱甘肽、亚精胺和谷胱甘肽亚精胺的类似物 抑制剂。 2. 克隆并测序编码相关酶的基因 锥硫酮代谢，特别是为了表达 并产生T.cruzi锥硫酮还原酶和锥硫酮 大量合成酶用于药物开发和 晶体学研究。 3. 为了进一步阐明谷胱甘肽的作用， 谷胱甘肽亚精胺和锥硫酮在代谢中的作用 这些寄生虫，特别强调： a) 谷胱甘肽亚精胺如何调节细胞内的水平 游离亚精胺。 b) 锥硫酮依赖性的纯化和表征 过氧化物酶活性。 c) 全面搜索其他谷胱甘肽或 锥硫酮依赖性酶。