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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，这种竞争性更新申请提供了一种实验范式，以进一步验证内脏利什曼病病原体杜氏利什曼原虫多胺生物合成途径的关键组分。该申请集中于从宿主获得多胺在建立寄生虫感染中的作用，并实施了一种针对用于治疗内脏和其他形式的利什曼病的药物发现的策略。利什曼原虫的多胺生物合成途径由四种酶组成：精氨酸酶（LdARG）、鸟氨酸脱羧酶（LdODC）、S-腺苷甲硫氨酸脱羧酶（LdADOMETDC）和亚精胺合酶（LdSPDSYN）。[The寄生虫还可以通过摄取机制挽救宿主多胺及其前体，其中之一是POT 1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表征的关系，并且是基本观察结果的逻辑延伸，即ldarg，ldodc，ldadometdc和ldspdsyn病变，所有这些都赋予多胺轴向营养，影响L的能力。donovani感染哺乳动物宿主的程度截然不同。可用于这些研究的关键试剂包括：1）编码每种多胺酶和[POT 1通透酶]的基因; 2）L. ldarg，ldodc，ldadometdc和ldspdsyn无效突变体。donovani;和3）在巨噬细胞中缺乏ARG 1的Arg 1 flox/flox; Tie 2cre小鼠及其Tie 2cre Cre删除器对照。[具体目的I有三个组成部分：1）通过比较对照组和Arg 1 flox/flox组肝脏和脾脏中的寄生虫负荷，确定多胺或多胺前体从宿主中挽救的作用; wild type，ldarg，ldodc，or ldspdsyn L. donovani，随后进行适当的营养补充方案以测试机制; 2）在来源于Arg 1flox/flox、Tie 2cre和Tie 2cre菌株的腹膜巨噬细胞中用野生型和突变型寄生虫进行互补感染性研究;第三章通过测试当野生型小鼠接种ldodc或ldodc时，POT 1过表达是否增加寄生虫负荷，来确定POT 1在调节寄生虫感染中的作用。�ldspdsyn L. donovani的多胺渗透机制，并确定LdPOT 1是否是唯一的多胺渗透机制。donovani通过创建和表型评估的ldpot 1敲除。Specific Aim 2是药物开发中的一个逻辑步骤，并为发现遗传验证的多胺途径的小分子抑制剂提供了蓝图。donovani。将对5，000 - 6，000种已证实的抗利什曼原虫化合物进行简单、具有成本效益且具有包容性的反向化学遗传筛选，所述化合物来自结构和化学多样性小分子文库的两个全面高通量正向化学遗传筛选，以鉴定靶向L. Donovani多胺途径

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

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

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

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。