PURINE METABOLISM IN SCHISTOSOMA MANSONI

曼索尼血吸虫的嘌呤代谢

• 批准号: 3136695
• 负责人: Ching Chung WANG
• 金额: $ 15.45万
• 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/3136695
• 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学手段， 一种有效且无毒的抗组胺剂。 具体 方法是尝试设计一种特异的，有效的抑制剂， 次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT酶）， 通过对曼氏血吸虫的深入了解， 的结构和性质。mansoni和人HGPRT酶 两种酶之间的差异。 的原因 证明这个特殊的研究计划是基于知识 染色体不能从头合成嘌呤 核苷酸，并且必须依赖于HGPRTase的功能， 鸟嘌呤核苷酸烟化的主要手段。 全长cdna 对S.已经克隆了mansoni和人类酶， 在转化的大肠杆菌中表达以产生天然酶 以大量（每升15至25毫克纯化酶， 细菌培养物）。 两种酶都已结晶。 2.9埃分辨率的初步X射线衍射图 从S. mansoni HGPRT酶。 为 下一个授予期，我们计划集中解决详细的 3-两种S. mansoni和人HGPRT酶， X射线晶体学 根据我们掌握的所有初步迹象 到目前为止，我们有信心相信， 目标将完全实现。 我们将使用计算机 图形程序来搜索合适的化学结构 一种特异性的S. mansoni HGPRT酶，和设计 定点诱变以产生突变酶， 结构和动力学分析，旨在进一步深入 理解宿主与宿主之间的独特属性， 寄生虫酶 与此同时， 酶通过光亲和标记8-叠氮基次黄嘌呤和2 ′， 将进行IMP、GMP和PRPP的3'-二醛衍生物， 鉴定所涉及的活性口袋中的特定氨基酸残基 与衬底绑定。 碘乙酸标记的酶， PRPP的存在也可以鉴定潜在的半胱氨酸 负责PRPP与HGPRT酶结合的残基。 圆形 二向色光谱分析将提供信息， 这两种蛋白质的构象变化， 环境，并可能解释显着的稳定性，这两个 酶在高温（80摄氏度）。 最后，罚款 次黄嘌呤和鸟嘌呤类似物的收集在惠康 研究实验室将在转化的E.杆菌 他们的生存依赖于一个功能正常的S mansoni HGPRT酶的顺序 来发现这种酶的特异性抑制剂 因此，我们 接近建立长期斗争的最后阶段 抗寄生虫化疗的生物化学方法模型（或 任何化疗）。 我们完全有理由感到乐观 关于最终的结果