PURINE METABOLISM IN SCHISTOSOMA MANSONI

曼索尼血吸虫的嘌呤代谢

• 批准号: 2062433
• 负责人: Ching Chung WANG
• 金额: $ 16.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2062433
• 关键词: Schistosoma mansoni; X ray crystallography; affinity labeling; anthelmintics; chemical structure function; circular dichroism; computer graphics /printing; drug design /synthesis /production; enzyme inhibitors; enzyme structure; gel electrophoresis; guanine; high performance liquid chromatography; hypoxanthine phosphoribosyltransferase; microorganism culture; molecular cloning; parasitic disease chemotherapy; purine /pyrimidine metabolism; schistosomiasis; site directed mutagenesis

The overall purpose of this research project is to employ biochemical, molecular biological and biophysical means to discover an effective and nontoxic antischistosomal agent. The specific approach is to try to design a specific, potent inhibitor of the hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) in Schistosoma mansoni through a thorough understanding of the structures and properties of S. mansoni and human HGPRTases as well as the discrepancies between the two enzymes. The reasons justifying this particular plan of study are based on the knowledge that schistosomes are incapable of de novo synthesis of purine nucleotides, and have to rely on the function of HGPRTase as the primary means of fumishing guanine nucleotides. Full-length cDNAs encoding the S. mansoni and human enzymes have been cloned and expressed in transformed Escherichia coli to produce native enzymes in large quantities (15 to 25 mg of purified enzyme per liter of bacterial pulture). Both enzymes have been crystallized. Preliminary X-ray diffraction patterns of a 2.9 Angstrom resolution have been recorded from the crystals of S. mansoni HGPRTase. For the next granting period, we plan to focus on resolution of detailed 3-dimensional structures of both S. mansoni and human HGPRTase by X-ray crystallography. By all the initial indications we have collected thus far, we have every confidence to believe that this objective will be fully accomplished. We shall then use computer graphic programs to search for the appropriate chemical structure of a specific inhibitor of S. mansoni HGPRTase, and design site-directed mfitagenesis to generate mutant enzymes for additional structural and kinetic analysis aimed at further in-depth understandings of the distinctive properties between the host and parasite enzymes. Meanwhile, chemical rnodifications of the two enzymes by a photoaffinity label 8-azidohypoxanthine and 2', 3'-dialdehyde derivatives of IMP, GMP and PRPP will be performed to identify specific amino acid residues in the active pockets involved with substrate-bindings. Iodoacetate labelings of the enzymes in the presence of PRPP may also identify the potential cysteine residue(s) responsible for PRPP binding to HGPRTase. Circular dichroism spectral analysis will provide information on conformational changes of the two proteins with changing environments, and may explain the remarkable stabilides of the two enzymes at elevated tempemtures (80 degrees C). Finally, the fine collection of hypoxanthine and guanine analogs at the Wellcome Research Laboratories will be tested on the transformed E. coli whose survival depends on a functioning S. mansoni HGPRTase in order to discover a specific inhibitor of this enzyme. Thus, we are approaching the final stage of a long struggle toward establishing a model for biochemical approaches to antiparasitic chemotherapy (or any chemotherapy). There is every reason for us to feel optimistic about the eventual outcome.

本研究项目的总体目的是利用 生物化学、分子生物学和生物物理手段来发现 一种有效且无毒的抗血吸虫病药物。具体的 方法是尝试设计一种特定的、有效的抑制 次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HGPRTase) 通过对曼氏血吸虫的透彻了解 曼氏沙门氏菌和人HGPRTase的结构和性质 因为这两种酶之间的差异。原因何在 证明这一特定学习计划的合理性是基于 血吸虫不能从头合成嘌呤 核苷酸，必须依赖于HGPRTase作为 烟熏鸟嘌呤核苷酸的主要方法。全长cDNA 编码曼氏链球菌和人类酶的基因已经被克隆并 在转化的大肠杆菌中表达以产生天然酶 大量(每升15至25毫克纯化酶 细菌培养)。这两种酶都已结晶。 2.9埃分辨率的初步X射线衍射图 都是从曼氏链球菌HGPRTase的晶体中记录的。为 在下一个授权期内，我们计划重点解决详细的 曼氏血吸虫和人HGPRTase的三维结构 X射线结晶学。从我们所掌握的所有初步迹象来看 到目前为止，我们完全有信心相信这一点 目标将全面实现。然后我们将使用计算机 用于搜索适当化学结构的图形化程序 一种曼氏葡萄球菌HGPRTase的特异性抑制剂，并设计 定点错配发生产生额外的突变酶 旨在进一步深入的结构和动力学分析 对主办方和主办方之间不同性质的理解 寄生虫酶。同时，对两者的化学修饰进行了研究 酶通过光亲和标记8-叠氮次黄嘌呤和2‘， IMP、GMP和PRPP的3‘-二醛衍生物将被用于 确定参与活动的口袋中的特定氨基酸残基 与底物结合。三种酶的碘乙酸酯标记 PRPP的存在也可能识别潜在的半胱氨酸 残基(S)负责PRPP与HGPRTase的结合。循环式 二向色性光谱分析将提供有关 两种蛋白质的构象随时间的变化 环境，并可能解释了这两种环境的显著稳定剂 酶在高温下(80摄氏度)。最后，罚款 在Wellcome收藏次黄嘌呤和鸟嘌呤类似物 研究实验室将对转化后的大肠杆菌进行测试 它的生存依赖于功能正常的曼氏链球菌HGPRTase 以发现这种酶的一种特定的抑制剂。因此，我们是 为建立一个国家而进行的长期斗争接近最后阶段 抗寄生虫化疗的生化方法模型(或 任何化疗)。我们完全有理由感到乐观 关于最终的结果。