Purine Salvage Pathway of Cryptosporidium Parvum

小隐孢子虫的嘌呤回收途径

• 批准号: 7495950
• 负责人: BUDDY ULLMAN
• 金额: $ 38.16万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495950
• 关键词: AMP Deaminase; Acquired Immunodeficiency Syndrome; Address; Adenosine Kinase; Applications Grants; Biochemical; Biochemistry; Biological; Biological Assay; Categories; Cells; Cellular biology; Chemicals; Cryptosporidiosis; Cryptosporidium parvum; Cultured Cells; Disease; Dissection; Drug Delivery Systems; Eligibility Determination; Employee Strikes; Enzymes; Escherichia coli; Fluorescence; GMP synthase; Gene Expression; Genes; Genetic; Genome; Genomics; Goals; Growth; Growth and Development function; Human; Immunoblot Analysis; Immunocompromised Host; Indirect Immunofluorescence; Individual; Infection; Intention; Investigation; Kinetics; Knock-out; Lesion; Life Cycle Stages; Ligase; Localized; Location; Mammals; Metabolic; Microscopy; Molecular; Molecular Biology; Monitor; Mutate; National Institute of Allergy and Infectious Disease; Nucleosides; Nutritional; Opportunistic Infections; Organism; Parasites; Parasitic Diseases; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacology; Property; Propionibacterium acnes; Protein Overexpression; Proteins; Public Health; Purine Nucleotides; Purines; Purpose; Reaction; Reagent; Recombinant Proteins; Recombinants; Research; Research Project Grants; Resolution; Role; Saccharomyces cerevisiae; Scheme; Structure; Structure-Activity Relationship; Substrate Specificity; Technology; Testing; Therapeutic; Therapeutic Corynebacterium Parvum; Toxoplasma gondii; Transgenic Organisms; Vaccines; Validation; base; biodefense; chemotherapy; cytotoxic; drug discovery; enzyme structure; gene function; genetic selection; guanylate; high throughput screening; human disease; in vivo; inhibitor/antagonist; kinase inhibitor; molecular modeling; mouse model; mutant; novel; nucleobase analog; nucleoside analog; pathogen; purine; purine analog; repository; small molecule libraries; structural biology; therapeutic target; three dimensional structure; tool; waterborne

DESCRIPTION (provided by applicant): Amalgamating the tools of molecular biology, biochemistry, cell biology, genetics, structural biology, and pharmacology, this proposal offers an interdisciplinary dissection of the purine salvage pathway of Cryptosporidium parvum, a protozoan parasite that is an opportunistic pathogen of people with AIDS and an NIAID Biodefense Research Category B Priority Pathogen. C. parvum is the etiologic agent of cryptosporidiosis, a debilitating and potentially fatal disease for which no consistently effective chemotherapies exist, especially for immunocompromised individuals. Thus, more efficacious drugs, particularly those that target unique features in C. parvum, are urgently needed. Among the more striking metabolic discrepancies between C. parvum and its human host are the pathways for purine acquisition. C. parvum, unlike mammals, cannot synthesize purines de novo and is dependent upon a unique purine salvage mechanism to fulfill its nutritional needs. Inhibition of purine salvage, therefore, presents a selective therapeutic paradigm for treating C. parvum infections. In this proposal, we will investigate three imperative components of purine salvage; adenosine kinase (CpAK), adenylate deaminase (CpAMPDA), and guanylate synthetase (CpGMPS). There are two Specific Aims. Multi-component Specific Aim I delineates a detailed biochemical, biological, and functional characterization of all three enzymes and a structural analysis of CpAK. We will clone all three genes and functionally evaluate their gene products through biochemical studies of their encoded products and by rescue or complementation of genetic lesions in other organisms. The expression patterns of the CpAK, CpAMPDA, and CpGMPS genes and the levels and location of the three proteins in intact C. parvum parasites will be ascertained throughout biologically accessible life cycle stages. Finally, structure-function studies based upon a molecular model of CpAK will test the roles of key residues on CpAK that are projected to be involved in the catalytic mechanism, and crystallographic studies on CpAK will be initiated with the ultimate intention of determining its three-dimensional structure. The second Specific Aim has two parts. First, we will perform a structure-activity relationship analysis of CpAK against a battery of available nucleoside analogs. Second, we will optimize and implement a high throughput screen of a structurally diverse chemical repository using an E. coli-based assay with the purpose of discovering novel inhibitors of CpAK that could be potentially used for treating the parasitic disease. The "hits" from the screen will be further evaluated using target-based and cell-based assays and eventually in a mouse model that mimics the human disease. Overall, the proposed investigations constitute a logical step in the validation of these enzymes as potential therapeutic targets and in the implementation of a rational strategy of drug discovery for treatment of cryptosporidiosis. PUBLIC HEALTH RELEVANCE The overall purpose of this proposal is to investigate components of the purine salvage pathway of Cryptosporidium parvum, a waterborne parasite that causes a devastating opportunistic infection in people with AIDS and is also a Category B priority pathogen of the National Institute of Allergy and Infectious Disease because the organism can be maliciously manipulated as a biological weapon. There is currently no vaccine and no consistently effective chemotherapy, so there is an urgent need for new drugs and new drug targets. Because purine salvage is essential for Cryptosporidium parvum and not for humans, our studies address the vital issue of validating new targets and discovering potentially novel drugs.

描述（由申请人提供）：结合分子生物学，生物化学，细胞生物学，遗传学，结构生物学和药理学的工具，本提案提供了小隐孢子虫嘌呤拯救途径的跨学科解剖，隐孢子虫是一种原生动物寄生虫，是艾滋病患者的机会性病原体，也是NIAID生物防御研究B类优先病原体。隐孢子虫是隐孢子虫病的病原，隐孢子虫病是一种使人衰弱和可能致命的疾病，目前尚无持续有效的化疗方法，特别是对免疫功能低下的个体。因此，迫切需要更有效的药物，特别是那些针对小梭菌独特特征的药物。在小孢子虫和它的人类宿主之间的代谢差异中，最显著的是嘌呤获取途径。与哺乳动物不同，小孢子虫不能从头合成嘌呤，并依赖于独特的嘌呤回收机制来满足其营养需求。因此，抑制嘌呤打捞，提出了一种选择性治疗模式，以治疗细小梭菌感染。在本提案中，我们将研究嘌呤救助的三个必要组成部分；腺苷激酶（CpAK）、腺苷酸脱氨酶（CpAMPDA）和鸟苷酸合成酶（CpGMPS）。有两个具体目标。多组分特异性目标I描述了所有三种酶的详细生化，生物学和功能特征以及CpAK的结构分析。我们将克隆这三个基因，并通过对其编码产物的生化研究和对其他生物遗传病变的修复或互补来对其基因产物进行功能评估。CpAK、CpAMPDA和CpGMPS基因的表达模式，以及这三种蛋白在完整的小虫虫体内的表达水平和位置，将在整个生物可达的生命周期阶段被确定。最后，基于CpAK分子模型的结构-功能研究将测试CpAK上预计参与催化机制的关键残基的作用，并将启动CpAK的晶体学研究，最终目的是确定其三维结构。第二个具体目标包括两个部分。首先，我们将针对一系列可用的核苷类似物对CpAK进行结构-活性关系分析。其次，我们将利用大肠杆菌为基础的试验优化和实现高通量筛选结构多样化的化学库，目的是发现可能用于治疗寄生虫病的新型CpAK抑制剂。筛选出来的“命中”将通过基于靶标和基于细胞的分析进一步评估，最终在模拟人类疾病的小鼠模型中进行评估。总的来说，拟议的研究构成了验证这些酶作为潜在治疗靶点和实施治疗隐孢子虫病的合理药物发现策略的逻辑步骤。该提案的总体目的是研究细小隐孢子虫嘌呤回收途径的组成部分，隐孢子虫是一种水生寄生虫，可引起艾滋病患者毁灭性的机会性感染，也是国家过敏和传染病研究所的B类优先病原体，因为该生物体可以被恶意操纵作为生物武器。目前没有疫苗，也没有持续有效的化疗，因此迫切需要新的药物和新的药物靶点。因为嘌呤回收对细小隐孢子虫是必需的，而不是对人类，我们的研究解决了验证新靶点和发现潜在新药的关键问题。