Purine Salvage Pathway of Cryptosporidium Parvum

小隐孢子虫的嘌呤回收途径

基本信息

批准号：
8015247
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
项目类别：
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/8015247
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

项目摘要

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）：该提案结合了分子生物学、生物化学、细胞生物学、遗传学、结构生物学和药理学的工具，对小隐孢子虫的嘌呤回收途径进行了跨学科剖析，隐孢子虫是一种原生动物寄生虫，是一种机会性病原体艾滋病患者和 NIAID 生物防御研究 B 类优先病原体。小隐孢子虫病是隐孢子虫病的病原体，隐孢子虫病是一种使人衰弱且可能致命的疾病，目前尚无持续有效的化疗方法，尤其是对于免疫功能低下的个体。因此，迫切需要更有效的药物，特别是针对微小念珠菌独特特征的药物。小隐孢子虫与其人类宿主之间最显着的代谢差异是嘌呤获取途径。与哺乳动物不同，小隐球菌不能从头合成嘌呤，而是依赖独特的嘌呤回收机制来满足其营养需求。因此，抑制嘌呤补救为治疗小隐孢子虫感染提供了一种选择性治疗范例。在本提案中，我们将研究嘌呤回收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。多组分特定目标 I 描述了所有三种酶的详细生化、生物和功能特征以及 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通过拯救或补充其他生物体中的遗传损伤来功能评估其基因产物。 CpAK、CpAMPDA 和 CpGMPS 基因的表达模式以及完整小隐孢子虫寄生虫中三种蛋白质的水平和位置将在整个生物学可及的生命周期阶段确定。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，并且将启动对 CpAK 的晶体学研究，最终目的是确定其三-维度结构。第二个具体目标有两个部分。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，我们将使用基于大肠杆菌的测定法对结构多样化的化学储存库进行优化和实施高通量筛选，目的是发现可用于治疗寄生虫病的新型 CpAK 抑制剂。屏幕上的“命中”将使用基于目标和基于细胞的测定进行进一步评估，并最终在模拟人类疾病的小鼠模型中进行评估。总体而言，所提出的研究构成了验证这些酶作为潜在治疗靶点以及实施治疗隐孢子虫病的合理药物发现策略的合理步骤。公共卫生相关性本提案的总体目的是研究小隐孢子虫的嘌呤补救途径的组成部分，隐孢子虫是一种水生寄生虫，可导致艾滋病患者发生毁灭性的机会性感染，也是美国国家过敏症研究所的 B 类优先病原体。传染病，因为该生物体可以被恶意操纵作为生物武器。目前还没有疫苗，也没有持续有效的化疗，因此迫切需要新药和新的药物靶点。由于嘌呤回收对于隐孢子虫而非人类至关重要，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。