Polyamine Metabolism in Leishmania

利什曼原虫的多胺代谢

• 批准号: 8471488
• 负责人: BUDDY ULLMAN
• 金额: $ 36.19万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471488
• 关键词: ARG1 deficiency; 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; 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; Pharmaceutical Preparations; Pharmacology; Phenotype; Polyamines; Protocols documentation; Putrescine; Reagent; Recombinants; Recovery; Research; Role; Source; Spermidine; Spermidine Synthase; Spleen; Supplementation; 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; overexpression; permease; prospective; public health relevance; research study; small molecule; small molecule libraries; therapeutic target; tool; uptake

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（由应用程序提供）：合并分子生物学，遗传学，生物化学和药理学的工具和技术，这种竞争性更新应用提供了实验性范式，以进一步验证利什马尼亚·多诺瓦尼（Leishmania donovani）的多胺生物合成途径的关键组成部分，即donovani of Visceral of visceral leishmaniasis。该应用的重点是从宿主那里获得多胺在寄生虫感染中的作用]，并实施了针对药物发现的策略，以治疗内脏和其他形式的利什曼尼亚。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-腺苷甲硫代氨基氨酸脱羧酶（LDADOMTDC）和精子合酶（LDSPDSyn）。 [寄生虫还可以通过摄取机制来挽救宿主的多胺及其前体，其中之一是Pot1腐烂 - 胸膜转运蛋白。该申请将解决宿主和寄生虫多胺途径之间的这种复杂且在很大程度上没有特色的关系]，这是基本观察的逻辑扩展，即``ldarg，ldarg，ldoc，ldometDC和„ ldspdsyn病变都会影响多胺轴的能力。范围。这些投资可用的关键试剂包括：1）编码每种多胺酶和[POT1 Persease]的基因； 2）。 3）arg1 flox/flox;在巨噬细胞中缺少ARG1的TIE2CRE小鼠及其TIE2CRE CRE DELETER控制。 [特定目的I具有三个组成部分：1）通过比较对照和Arg1 Flox的肝脏和脾脏中的寄生虫负荷来确定宿主中多胺或多胺前体拯救的作用；已接种两者的TIE2CRE小鼠 野生型，�ldarg，�LDoc或�LDSPDSYNL. donovani，然后进行适当的营养补充方案以测试机制； 2）在腹膜巨噬细胞中对源自Arg1flox/Flox衍生的野生型寄生虫进行互补感染研究； TIE2CRE和TIE2CRE菌株； 3）确定POT1在调节寄生虫感染中的作用，通过测试POT1过表达是否会在野生型小鼠接种时是否会增强寄生虫燃烧，以接种w ldoc或ldspdsyn l. donovani和ldspdsyn l. donovani，并且通过ldpot1是l. donovanimin pivaine creation in creation in creation of creation and creatiq five creatiq five cretiq five creatiq five。敲除。]特定的目标2是药物发育的逻辑步骤，并提供了蓝图，以发现遗传学验证的多胺途径的小分子抑制剂。一个简单，成本效益且包含的反向化学遗传筛选，为5,000-6,000个经过证明的抗莱什人类化合物，这些化合物从两个综合的高吞吐量前向化学遗传筛选中出现，这些化合物的结构和化学上多样的小分子库将执行，以识别ot o的四烯二氧酸盐的二氧化酶的化学品。