MECHANISM-BASED DRUG SELECTION AND DESIGN: NUCLEOTIDE SA

基于机制的药物选择和设计：核苷酸 SA

• 批准号: 6374328
• 负责人: BUDDY ULLMAN
• 金额: $ 70.79万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374328
• 关键词: Escherichia coli; Toxoplasma gondii; X ray crystallography; Xenopus oocyte; acid anhydride hydrolase; adenosine; adenosine kinase; antiprotozoal agents; drug design /synthesis /production; enzyme mechanism; enzyme structure; gene mutation; hypoxanthine phosphoribosyltransferase; membrane transport proteins; nucleotide metabolism; pentosyltransferase; recombinant proteins; uracil

DESCRIPTION (adapted from the application): This R01 application was submitted in response to PA AI-98-100, "National Cooperative Drug Discovery Groups-Opportunistic Infections" (NCDDG-OI) by a group of investigators from the Oregon Health Sciences University, the University of Pennsylvania, and the Yale University School of Medicine. It represents the continuation of a long-term formal collaboration among the investigators of these three institutions, supported since 1991 with a grant under the auspices of the National Cooperative Drug Discovery Group (NCDDG). The major scientific objective of the proposed studies is to integrate genetic, biochemical, and structural studies on key transporters and enzymes of the pyrimidine and purine salvage pathways in Toxoplasma gondii and related apicomplexan parasites, including Cryptosporidium, Sarcocystis, and Plasmodium. The long-term goal will be focused on developing better and more efficacious antiparasitic drugs for these parasites - particularly opportunistic pathogens associated with AIDS. Interference with pyrimidine synthesis has traditionally provided the most effective tool for management of clinical toxoplasmosis. However, despite the fact that (i) all of these pathogens are purine auxotrophs, (ii) the existing precedent of subversive purines as effective treatment for other parasitic diseases, and (iii) that the availability of antiparasitic lead compounds that target either purine or pyrimidine salvage pathways, the latter two pathways have not been extensively explored as targets for chemotherapeutic treatment of either T. gondii or other apicomplexans. Reagents previously developed through this research collaboration include: (1) a genetic map of nucleotide salvage pathways in Toxoplasma; (2) molecular clones encoding T. gondii UPRT, HGXPRT, AK, NTPase, and the parasite's major adenosine transporter; (3) milligram quantities of each of the above soluble enzymes purified to homogeneity, and heterologous systems for transporter expression; (4) high-resolution crystal structures for UPRT, HGXPRT, and AK; and (5) transgenic parasites harboring mutations in (or altered expression of) each of the above genes. These reagents are expected to permit structure-based discovery of new drug classes that target proteins necessary for parasite survival. Currently available molecular, biochemical, and cellular reagents and data that have become available through these studies include: (i) Molecular clones. Full length genomic and cDNA sequences for T. gondii uracil phosphoribosyl transferase (UPRT), nucleoside triphosphate hydrolyze (NTPase), hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRT), adenosine kinase (AK), and the parasite's major adenosine transporter (AT). (ii) Recombinant proteins. E. coli that overexpresses T. gondii UPRT, HGXPRT, AK, or NTPase and effectively provides unlimited quantities of these proteins purified to homogeneity. Functional expression of recombinant AT in a Xenopus oocyte assay system. (iii) Crystal structures. High-resolution three-dimensional crystal structures for the UPRT, HGXPRT, and AK proteins determined by x-ray crystallography. (iv) Transgenic parasites. UPRT-, HGXPRT-, and AK-knockout transgenics created by homologous gene replacement in otherwise syngeneic wild type parasites. AT and xanthine transporter (XT) transgenics isolated by imertional mutagenesis. NTPase-deficient transgenics in which the endogenous enzyme is down-regulated by antisense expression. Specific aims for the proposed studies include: (1) performing a detailed biochemical and structural characterization of T. gondii UPRT and AK enzymes. The resolution of the T. gondii UPRT and AK enzymes will be extended and enzyme-substrate and enzyme-product structures determined in order to provide a full understanding of their catalytic mechanisms, and to facilitate the discovery of novel inhibitors through computational methods (see Specific Aim 2). High-resolution crystal structures of a series of site-directed mutant UPRT and AK proteins will also be carried out to assess the roles of key residues in substrate specificity and catalysis. Mutant enzymes will be purified from E. coli for kinetic appraisal, and crystal structures will be determined by molecular modification or molecular replacement to ascertain structural changes. The phenotypic consequences of mutations of interest will be tested in intact parasites by replacement of either the wild type UPRT or AK allele with an appropriate targeting construct; (2) developing screens for identifying and evaluating novel classes of potential antiparasitic drugs that target either UPRT or AK. Small-molecule structural databases will be screened computationally using the crystallographically determined high-resolution apo- and substrate- and product-bound UPRT and AK structures to identify novel compounds that may interact with the active sites of either enzyme. Compounds that are computationally predicted to target the active site of the T. gondii UPRT or AK enzymes will be evaluated as potential lead compounds against the purified UPRT or AK enzymes; E. coli expressing T. gondii UPRT or AK cDNAs; and wild type, UPRT- or AK-T. gondii parasites in culture. The crystal structures will be solved for UPRT or AK co-crystallized with promising lead compounds; (3) functionally characterize, localize, and genetically dissect the T. gondii adenosine transporter (AT). AT ligand specificity and kinetic parameters will be determined in detail by functional expression of the AT cDNA in Xenopus laevis oocytes and/or nucleoside transport (NT)-deficient Leishmania donovani. The applicants also plan to use electrophysiologic approaches in the Xenopus expression system to ascertain whether AT is a proton- or Na(+)-coupled symporter that actively concentrates adenosine. Antibodies against AT will be used to determine the subcellular location of AT by immunofluorescence and immunoelectron microscopy. Finally, forward genetic approaches will be implemented to initiate a structure-function analysis of key amino acids of AT that participate in ligand recognition or that govern substrate specificity; (4) isolation of XT cDNA and characterizing the properties of T. gondii XT. The XT gene will be isolated from insertional mutants by marker rescue and used to obtain full-length cDNA clones. Functional properties of XT will be evaluated after heterologous expression of XT cDNA in Xenopus oocytes, and the transporter will be immunolocalized after generating monospecific antibodies; and (5) to crystallize and solve the x-ray structure of the T. gondii NTPase and determine how enzymatic activity is regulated. The NTPase cDNA has been overexpressed in E. coli providing ample and replenishable quantities of monomeric recombinant protein for initial crystallization trials. In parallel, the production, purification, and crystallization of enzymatically active oligomeric NTPase will be pursued. Ultimately, these crystallization experiments will lead to the x-ray structure determination of the NTPase by multiple isomorphous replacement. Concurrent experiments will determine how NTPase functions in AK HGXPRT-, and AT parasites, identify the protein(s) which regulate NTPase enzymatic activity, and evaluate the impact of abrogating NTPase expression on parasite viability.

描述（改编自应用程序）：此 R01 应用程序是 提交回应 PA AI-98-100，“国家合作药物发现 群体机会性感染”（NCDDG-OI）由一组研究人员 俄勒冈健康科学大学、宾夕法尼亚大学和 耶鲁大学医学院。 它代表着一个 这三个研究人员之间的长期正式合作 自 1991 年以来，在 国家药物发现合作小组（NCDDG）。 主要科学 拟议研究的目标是将遗传、生化和 嘧啶和关键转运蛋白和酶的结构研究 弓形虫和相关顶复门中的嘌呤补救途径 寄生虫，包括隐孢子虫、肉孢子虫和疟原虫。 这 长期目标将集中于发展更好、更有效 针对这些寄生虫（特别是机会性病原体）的抗寄生虫药物 与艾滋病有关。 干扰嘧啶合成 传统上为临床管理提供了最有效的工具 弓形体病。 然而，尽管 (i) 所有这些病原体都是 嘌呤营养缺陷型，（ii）颠覆性嘌呤的现有先例 对其他寄生虫病的有效治疗，以及 (iii) 靶向嘌呤或的抗寄生虫先导化合物的可用性 嘧啶补救途径，后两条途径尚未得到广泛研究 探索作为弓形虫化疗治疗的靶标 其他顶端复合物。 先前通过本研究开发的试剂 合作包括：（1）核苷酸挽救途径的遗传图谱 弓形虫； (2)编码弓形虫UPRT、HGXPRT、AK、NTPase的分子克隆， 以及寄生虫的主要腺苷转运蛋白； (3) 毫克量 上述每种可溶性酶均纯化至同质和异源 转运蛋白表达系统； (4) 高分辨率晶体结构 UPRT、HGXPRT 和 AK； (5) 携带突变的转基因寄生虫（或 改变了上述每个基因的表达。 这些试剂预计 允许基于结构的靶向蛋白质的新药物类别的发现 寄生虫生存所必需的。 目前可用的分子、生化和细胞试剂和数据 通过这些研究获得的成果包括： (i) 分子克隆。 弓形虫全长基因组和 cDNA 序列 尿嘧啶磷酸核糖转移酶 (UPRT)、三磷酸核苷水解 (NTPase)、次黄嘌呤-鸟嘌呤-黄嘌呤磷酸核糖转移酶 (HGXPRT)、 腺苷激酶 (AK) 和寄生虫的主要腺苷转运蛋白 (AT)。 (ii) 重组蛋白。 过度表达弓形虫 UPRT 的大肠杆菌， HGXPRT、AK 或 NTPase，并有效地提供无限数量的这些 蛋白质纯化至均质。 重组AT的功能表达 非洲爪蟾卵母细胞测定系统。 (iii) 晶体结构。 高分辨率三维晶体 X 射线确定的 UPRT、HGXPRT 和 AK 蛋白的结构 晶体学。 (iv) 转基因寄生虫。 UPRT、HGXPRT 和 AK 敲除转基因 通过同源野生型中的同源基因替换而产生 寄生虫。 AT 和黄嘌呤转运蛋白 (XT) 转基因分离 惯性诱变。 NTPase 缺陷型转基因，其中内源性 酶被反义表达下调。 拟议研究的具体目标包括： (1) 进行详细的生化和结构表征 弓形虫 UPRT 和 AK 酶。 弓形虫 UPRT 和 AK 的分辨率 酶将被扩展，酶-底物和酶-产物结构 确定以便充分了解其催化作用 机制，并通过以下方式促进新型抑制剂的发现 计算方法（参见具体目标 2）。 高分辨率晶体 一系列定点突变 UPRT 和 AK 蛋白的结构也将 进行评估关键残基在底物特异性中的作用 和催化作用。 将从大肠杆菌中纯化突变酶用于动力学 鉴定，并通过分子修饰确定晶体结构 或分子替换以确定结构变化。 表型 感兴趣的突变的后果将在完整的寄生虫中进行测试 用适当的等位基因替换野生型 UPRT 或 AK 等位基因 靶向构建； (2) 开发用于识别和评估新类别的屏幕 靶向 UPRT 或 AK 的潜在抗寄生虫药物。 小分子 结构数据库将使用计算筛选 晶体学测定的高分辨率 apo- 和底物- 和 产物结合的 UPRT 和 AK 结构，以鉴定可能的新型化合物 与任一酶的活性位点相互作用。 化合物是 经计算预测可靶向弓形虫 UPRT 的活性位点或 AK 酶将作为针对纯化的潜在先导化合物进行评估 UPRT 或 AK 酶；表达弓形虫 UPRT 或 AK cDNA 的大肠杆菌；和狂野的 类型，UPRT-或AK-T。文化中的弓形虫寄生虫。 晶体结构将 解决 UPRT 或 AK 与有前途的先导化合物共结晶的问题； (3) 对弓形虫进行功能表征、定位和基因解剖 腺苷转运蛋白（AT）。 AT配体特异性和动力学参数将 通过非洲爪蟾 AT cDNA 的功能表达来详细确定 莱维斯卵母细胞和/或核苷转运 (NT) 缺陷的杜氏利什曼原虫。 申请人还计划在非洲爪蟾中使用电生理学方法 表达系统以确定 AT 是质子偶联还是 Na(+) 偶联 主动浓缩腺苷的同向转运蛋白。 针对 AT 的抗体将 用于通过免疫荧光法确定 AT 的亚细胞位置 免疫电子显微镜。 最后，正向遗传方法将 用于启动 AT 关键氨基酸的结构功能分析 参与配体识别或控制底物特异性； (4) XT cDNA的分离并表征弓形虫XT的特性。 XT 基因将通过标记拯救从插入突变体中分离出来 用于获得全长 cDNA 克隆。 XT 的功能特性为 在爪蟾卵母细胞中异源表达 XT cDNA 后进行评估，并且 转运蛋白产生单特异性抗体后将被免疫定位； 和 (5) 结晶并解析弓形虫NTPase的X射线结构 确定酶活性的调节方式。 NTPase cDNA 已被 在大肠杆菌中过度表达，提供充足且可补充的数量 用于初始结晶试验的单体重组蛋白。 在 平行地，酶法生产、纯化和结晶 将追求活性寡聚 NTPase。 最终这些结晶 实验将导致 NTPase 的 X 射线结构测定 多重同构置换。 并行实验将确定如何 NTPase 在 AK HGXPRT- 和 AT 寄生虫中发挥作用，识别蛋白质 调节 NTPase 酶活性，并评估其影响 消除 NTPase 表达对寄生虫活力的影响。