Purine Salvage Pathway of Cryptosporidium Parvum

小隐孢子虫的嘌呤回收途径

• 批准号: 8212107
• 负责人: BUDDY ULLMAN
• 金额: $ 37.4万
• 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/8212107
• 关键词: AMP Deaminase; Acquired Immunodeficiency Syndrome; Address; Adenosine Kinase; Applications Grants; Biochemical; Biochemistry; Biological; Biological Assay; Categories; Cell Culture Techniques; Cells; Cellular biology; Chemicals; Cryptosporidiosis; Cryptosporidium parvum; Development; Disease; Dissection; Drug Delivery Systems; Eligibility Determination; Employee Strikes; Enzymes; Escherichia coli; Fluorescence; GMP synthase; Genes; Genetic; Genome; Genomics; Goals; Health; Human; Immunocompromised Host; Individual; Infection; Intention; Investigation; Kinetics; Knock-out; Lesion; Life Cycle Stages; Ligase; Location; Mammals; Metabolic; Molecular; Molecular Biology; Molecular Models; Mutate; National Institute of Allergy and Infectious Disease; Nutritional; Opportunistic Infections; Organism; Parasites; Parasitic Diseases; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacology; Property; Proteins; Purine Nucleotides; Purines; Reaction; Reagent; Recombinant Proteins; Recombinants; Research; Research Project Grants; Resolution; Role; Saccharomyces cerevisiae; Scheme; Structure; Structure-Activity Relationship; Substrate Specificity; Technology; Testing; Therapeutic; Toxoplasma gondii; Transgenic Organisms; Vaccines; Validation; base; biodefense; chemotherapy; drug discovery; enzyme structure; gene function; genetic selection; guanylate; high throughput screening; human disease; in vivo; inhibitor/antagonist; molecular modeling; mouse model; mutant; novel; nucleobase analog; nucleoside analog; overexpression; pathogen; purine; purine analog; repository; structural biology; therapeutic target; three dimensional structure; tool; waterborne; weapons

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，本提案提供了隐孢子虫嘌呤补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致病菌，也是NIAID生物防御研究类别B优先致病菌。C.细小孢子虫是隐孢子虫病的病原体，隐孢子虫病是一种使人衰弱的和潜在致命的疾病，对于这种疾病，特别是对于免疫功能低下的个体，不存在持续有效的化疗。因此，更有效的药物，特别是那些针对C.目前，急需。在C.细小病毒及其人类宿主是嘌呤获得的途径。C.与哺乳动物不同，细小哺乳动物不能从头合成嘌呤，并且依赖于独特的嘌呤补救机制来满足其营养需要。因此，抑制嘌呤补救提供了治疗C的选择性治疗范例。细小病毒感染在这个建议中，我们将研究嘌呤补救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、生物学和功能表征以及CpAK的结构分析。我们将克隆所有三个基因，并通过对其编码产物的生化研究以及对其他生物体中遗传病变的拯救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C.将在整个生物学可及的生命周期阶段确定微小寄生虫。最后，基于CpAK分子模型的结构-功能研究将测试CpAK上的关键残基的作用，这些残基预计将参与催化机制，并将启动CpAK的晶体学研究，最终目的是确定其三维结构。第二个具体目标有两个部分。首先，我们将进行CpAK对电池的可用核苷类似物的结构-活性关系分析。第二，我们将优化和实施一个高通量筛选的结构多样的化学品库使用E。大肠杆菌为基础的测定，目的是发现新的CpAK抑制剂，可能用于治疗寄生虫病。来自筛选的“命中”将使用基于靶点和基于细胞的测定进一步评估，并最终在模拟人类疾病的小鼠模型中进行评估。总的来说，拟议的调查构成了一个合乎逻辑的步骤，这些酶作为潜在的治疗靶点的验证，并在实施一个合理的策略，用于治疗隐孢子虫病的药物发现。公共卫生相关性本提案的总体目的是研究微小隐孢子虫嘌呤补救途径的组成部分，微小隐孢子虫是一种水传播寄生虫，可导致艾滋病患者的毁灭性机会性感染，也是国家过敏和传染病研究所的B类优先病原体，因为该生物体可被恶意操纵为生物武器。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。由于嘌呤补救对于微小隐孢子虫而不是人类是必不可少的，因此我们的研究解决了验证新靶点和发现潜在新药的重要问题。