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Polyamine Metabolism in Leishmania

利什曼原虫的多胺代谢

基本信息

批准号：
9110116
负责人：
PHILLIP A YATES
金额：
$ 38.5万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-07-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9110116
关键词：
Address Adenosylmethionine Decarboxylase Affect American Anabolism Biochemical Biochemistry Cells Chemicals Communicable Diseases Complement Complex Data Disease Enzymes Foundations Funding Future Genes Genetic Genetic Screening Growth Health Human In Vitro Infection Institution Investigation Knock-out Laboratories Lead Leishmania Leishmania donovani Leishmaniasis Lesion Libraries Liver Metabolism Methods Molecular Biology Techniques Mus Nutrition Assessment Nutritional Ornithine Decarboxylase Parasites Parasitic Diseases Pathway interactions Peritoneal Macrophages Pharmacology Pharmacotherapy Phenotype Polyamines Protocols documentation Putrescine Reagent Recombinants Recovery Research Role S-Adenosylmethionine Source Spermidine Spermidine Synthase Spleen Testing Therapeutic Transgenic Organisms Validation Visceral Visceral Leishmaniasis Wild Type Mouse arginase chemical genetics cost effective design drinking water drug development drug discovery enzyme pathway follow-up gene replacement high throughput screening inhibitor/antagonist killings macrophage mutant novel novel therapeutics nutritional supplementation overexpression permease prospective research study small molecule small molecule inhibitor small molecule libraries therapeutic target tool uptake

项目摘要

DESCRIPTION (provided by applicant): Amalgamating the tools and techniques of molecular biology, genetics, biochemistry, and pharmacology, this competing renewal application offers an experimental paradigm to further validate key components of the polyamine biosynthetic pathway of Leishmania donovani, the causative agent of visceral leishmaniasis. The application focuses on the role of polyamine acquisition from the host in the establishment of parasite infection] and implements a strategy directed toward drug discovery for the treatment of visceral and perhaps other forms of leishmaniasis. The polyamine biosynthetic pathway of Leishmania consists of four enzymes: arginase (LdARG), ornithine decarboxylase (LdODC), S-adenosylmethionine decarboxylase (LdADOMETDC), and spermidine synthase (LdSPDSYN). [The parasite can also salvage host polyamines and their precursors via uptake mechanisms, one of which is the POT1 putrescine-spermidine transporter. The application will address this intricate and largely uncharacterized relationship between the host and parasite polyamine pathways] and is a logical extension of the fundamental observations that �ldarg, �ldodc, �ldadometdc, and �ldspdsyn lesions, all of which confer polyamine axotrophy, impact the capacity of L. donovani to infect the mammalian host to dramatically different extents. Key reagents available for these investigations include: 1) genes encoding each of the polyamine enzymes and [the POT1 permease]; 2) �ldarg, �ldodc, �ldadometdc, and �ldspdsyn null mutants of L. donovani; and 3) Arg1 flox/flox; Tie2cre mice that lack ARG1 in macrophages and their Tie2cre Cre deleter controls. [Specific Aim I has three components: 1) to ascertain the role of polyamine or polyamine precursor salvage from the host by comparing parasite loads in livers and spleens of both control and Arg1 flox/flox ; Tie2cre mice that have been inoculated with either wild type, �ldarg, �ldodc, or �ldspdsyn L. donovani followed by appropriate nutritional supplementation protocols to test mechanism; 2) to perform complementary infectivity studies with the wild type and mutant parasites in peritoneal macrophages derived from the Arg1flox/flox; Tie2cre and Tie2cre strains; 3) to establish the role of POT1 in modulating parasite infection by testing whether POT1 overexpression boosts parasite burdens when wild type mice are inoculated with either �ldodc or �ldspdsyn L. donovani and by ascertaining whether LdPOT1 is the sole polyamine permeation mechanism in L. donovani via the creation and phenotypic assessment of a �ldpot1 knockout.] Specific Aim 2 is a logical step in drug development and offers a blueprint to discover small molecule inhibitors of the genetically validated polyamine pathway of L. donovani. A simple, cost-effective, and inclusive reverse chemical genetic screen of 5,000 - 6,000 proven anti-leishmanial compounds that have emerged from two comprehensive high throughput forward chemical genetic screens of structurally and chemically diverse small molecule libraries will be performed in order to identify chemicals that target any o the four enzymes of the L. donovani polyamine pathway.

描述(由申请人提供)：融合了分子生物学、遗传学、生物化学和药理学的工具和技术，这项竞争性的更新申请提供了一个实验范例，进一步验证内脏利什曼病的病原体杜氏利什曼原虫多胺生物合成途径的关键成分。申请的重点是从宿主获取多胺在确定寄生虫感染的过程中的作用]，并实施一项旨在发现治疗内脏利什曼病和可能其他形式的利什曼病的药物的战略。利什曼原虫多胺生物合成途径由精氨酸酶(LdARG)、鸟氨酸脱羧酶(LdODC)、S腺苷蛋氨酸脱羧酶(LdADOMETDC)和亚精胺合成酶(LdSPDSYN)4种酶组成。[寄生虫也可以通过摄取机制挽救宿主多胺及其前体，其中之一是POT1腐胺-亚精胺转运蛋白。该应用程序将解决宿主和寄生虫多胺途径之间这种复杂且基本上未描述的关系)，并是基本观察结果的逻辑延伸，即�ldarg、�ldodc、�ldadometdc和�ldspdsyn损伤，所有这些损伤都导致多胺轴营养不良，在极大不同的程度上影响杜诺瓦尼乳杆菌感染哺乳动物宿主的能力。可用于这些研究的关键试剂包括：1)编码每种多胺酶和[POT1通透酶]的基因；2)杜氏乳杆菌的�ldarg、�ldodc、�ldadometdc和�ldspdsyn零突变体；以及3)Arg1Flox/Flox；Tie2cre小鼠及其Tie2cre Cre Deleter对照。[特定目标I]有三个组成部分：1)通过比较对照和Arg1 Flox/Flox的肝脏和脾中的寄生虫负载，确定从宿主中抢救多胺或多胺前体的作用；Tie2cre小鼠目的：1)研究野生型、�ldarg、�ldodc或�ldspdsyn L.donovani野生型、�ldarg、�ldodc或�ldspdsyn L.donovani在适当营养补充方案下的致病机制；2)对来源于Arg1 flx/flx的野生型和突变株的腹膜巨噬细胞中的寄生虫进行互补的感染性研究；3)通过检测pot1的过表达是否增加了野生型小鼠感染�ldodc或�ldspdsyn L.donovani时的寄生虫负担，并通过对�ldpot1基因敲除的表型评估来确定LdPOT1是否是捐赠者体内唯一的多胺渗透机制，以确定POT1在调节寄生虫感染中的作用。特异性目标2是药物开发中合乎逻辑的一步，为发现杜氏乳杆菌多胺途径的小分子抑制剂提供了蓝图。将对5,000-6,000种已证实的抗利什曼类化合物进行简单、成本效益和包容性的反向化学遗传筛选，这些化合物是从结构和化学上不同的小分子库的两个全面的高通量正向化学遗传筛选中出现的，以识别针对杜诺瓦尼乳杆菌多胺途径的四种酶中的任何一种的化学物质。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani.

整合核糖体启动子载体，提供杜氏利什曼原虫组成型表达谱的选择。

DOI：
10.1016/j.molbiopara.2016.01.008
发表时间：
2015
期刊：
Molecular and biochemical parasitology
影响因子：
1.5
作者：
Soysa,Radika;Tran,KhoaD;Ullman,Buddy;Yates,PhillipA
通讯作者：
Yates,PhillipA

Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

克氏锥虫的腐胺-尸胺渗透酶的结构模型预测了对转运和配体结合至关重要的残基。

DOI：
10.1042/bj20130350
发表时间：
2013
期刊：
The Biochemical journal
影响因子：
0
作者：
Soysa,Radika;Venselaar,Hanka;Poston,Jacqueline;Ullman,Buddy;Hasne,Marie-Pierre
通讯作者：
Hasne,Marie-Pierre

S-adenosylmethionine decarboxylase from Leishmania donovani. Molecular, genetic, and biochemical characterization of null mutants and overproducers.

来自杜氏利什曼原虫的 S-腺苷甲硫氨酸脱羧酶。

DOI：
10.1074/jbc.m110118200
发表时间：
2002
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Roberts,SigridC;Scott,Jerry;Gasteier,JudithE;Jiang,Yuqui;Brooks,Benjamin;Jardim,Armando;Carter,NicolaS;Heby,Olle;Ullman,Buddy
通讯作者：
Ullman,Buddy

Leishmania donovani polyamine biosynthetic enzyme overproducers as tools to investigate the mode of action of cytotoxic polyamine analogs.

杜氏利什曼原虫多胺生物合成酶过量生产者作为研究细胞毒性多胺类似物作用模式的工具。

DOI：
10.1128/aac.01193-06
发表时间：
2007
期刊：
Antimicrobial agents and chemotherapy
影响因子：
4.9
作者：
Roberts,SigridC;Jiang,Yuqui;Gasteier,Judith;Frydman,Benjamin;Marton,LaurenceJ;Heby,Olle;Ullman,Buddy
通讯作者：
Ullman,Buddy

Inhibition profile of Leishmania mexicana arginase reveals differences with human arginase I.

墨西哥利什曼原虫精氨酸酶的抑制谱揭示了与人精氨酸酶 I 的差异。