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: 6170897
• 负责人: BUDDY ULLMAN
• 金额: $ 68.81万
• 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/6170897
• 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（根据应用程序改编）：此R01应用程序是 提交了对PA AI-98-100的回应，“国家合作毒品发现 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。 它代表了一个延续 这三个调查人员之间的长期正式合作 自1991年以来支持的机构在主持下获得了赠款 国家合作药物发现小组（NCDDG）。 主要科学 拟议的研究的目的是整合遗传，生化和 关于嘧啶和关键转运蛋白和酶的结构研究 弓形虫和相关的apicomplexan中的嘌呤挽救途径 寄生虫，包括隐孢子虫，肌囊肿和疟原虫。 这 长期目标将集中于发展更好，更有效 这些寄生虫的抗寄生虫药物 - 尤其是机会性病原体 与艾滋病相关。 干扰嘧啶合成的 传统上为临床管理提供了最有效的工具 弓形虫病。 但是，尽管（i）所有这些病原体都是 嘌呤的pulin营养，（ii）现有的颠覆性嘌呤的先例 有效治疗其他寄生虫病，（iii） 靶向嘌呤或的抗寄生虫铅化合物的可用性 嘧啶挽救途径，后两个途径尚未广泛 探索是t. gondii或 其他apicomplexans。 以前通过这项研究开发的试剂 协作包括：（1）核苷酸打捞途径的遗传图 弓形虫； （2）编码T. gondii UPRT，HGXPRT，AK，NTPase， 以及寄生虫的主要腺苷转运蛋白； （3）毫克数量 上述每种可溶性酶纯化为均匀性和异源 转运蛋白表达的系统； （4）高分辨率晶体结构 UPRT，HGXPRT和AK； （5）具有突变的转基因寄生虫（或 上述每个基因的表达改变。 这些试剂是期望的 允许基于结构的发现针对蛋白质的新药类 寄生虫生存所必需的。 目前可用的分子，生化和细胞试剂以及数据 通过这些研究可用的包括： （i）分子克隆。 T. gondii的全长基因组和cDNA序列 尿嘧啶磷酸贝糖基转移酶（UPRT），核苷三磷酸盐水解 （NTPase），低黄嘌呤 - 黄甘氨酸 - 黄氨酸磷酸蛋白酶基转移酶（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。 大肠杆菌过表达T. gondii Uprt， HGXPRT，AK或NTPase，并有效地提供了其中的无限量 蛋白质纯化为同质性。 重组的功能表达 爪蟾卵母细胞测定系统。 （iii）晶体结构。 高分辨率三维晶体 由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-KNOCKOUT转基因技术 由同源基因替代品创建 寄生虫。 AT和黄嘌呤转运蛋白（XT）转基因分离 IMertional诱变。 NTPase缺陷的转基因，其中内生 酶被反义表达下调。 拟议研究的具体目的包括： （1）执行详细的生化和结构表征 T. gondii Uprt和Ak酶的。 T. gondii Uprt和AK的分辨率 酶将扩展并酶 - 基底和酶产物结构 确定为了充分了解其催化 机制，并通过 计算方法（请参阅特定目标2）。 高分辨率晶体 一系列定向突变体UPRT和AK蛋白的结构也将 进行以评估底物特异性中关键残基的作用 和催化。 突变酶将从大肠杆菌中纯化为动力学 评估和晶体结构将通过分子修饰确定 或分子替换以确定结构变化。 表型 感兴趣突变的后果将在完整的寄生虫中测试 用适当的 目标结构； （2）开发用于识别和评估新颖类的屏幕 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 晶体学确定的高分辨率APO-和底物和 产品结合的UPRT和AK结构，以识别可能 与任一酶的活性位点相互作用。 化合物是 在计算上预测将针对T. gondii Uprt或 AK酶将被评估为针对纯化的潜在铅化合物 UPRT或AK酶；表达T. gondii Uprt或Ak cdnas的大肠杆菌；和疯狂 类型，UPRT-或AK-T。文化中的Gondii寄生虫。 晶体结构将 为UPRT或AK共结晶，并与有希望的铅化合物共结晶； （3）在功能上表征，本地化和遗传剖析T. gondii 腺苷转运蛋白（AT）。 在配体特异性和动力学参数将 通过Xenopus中AT cDNA的功能表达详细确定 Laevis卵母细胞和/或核苷转运（NT） - 缺乏利什曼原虫Donovani。 申请人还计划在Xenopus中使用电生理方法 表达系统可以确定AT是质子 - na（+） - 耦合 积极浓缩腺苷的分类者。 针对AT的抗体将是 用于确定免疫荧光和AT的亚细胞位置 免疫电子显微镜。 最后，远期遗传方法将是 实施以启动对AT关键氨基酸的结构功能分析 参与配体认可或管理底物特异性； （4）XT cDNA的分离并表征了T. gondii Xt的性质。 XT基因将通过Marker Rescue和 用于获得全长cDNA克隆。 XT的功能特性将是 在异爪蟾卵母细胞中XT cDNA异源表达后评估， 产生单特异性抗体后，将对转运蛋白进行免疫定位。 和 （5）结晶并求解T. gondii ntpase的X射线结构和 确定如何调节酶活性。 NTPase cDNA已经 过表达大肠杆菌，提供大量和可补充的数量 用于初始结晶试验的单体重组蛋白。 在 酶上的平行，生产，纯化和结晶 将追求主动寡聚NTPase。 最终，这些结晶 实验将导致NTPase的X射线结构确定 多态替代。 并发实验将决定如何 NTPase在AK HGXPRT-中的功能，在寄生虫下，识别蛋白质 调节NTPase酶活性，并评估 废除寄生虫生存能力上的NTPase表达。