Purine Salvage Pathway of Cryptosporidium Parvum

描述(申请人提供)：这项建议融合了分子生物学、生物化学、细胞生物学、遗传学、结构生物学和药理学的工具，对微小隐孢子虫的嘌呤挽救途径进行了跨学科的剖析，微小隐孢子虫是一种原生动物寄生虫，是艾滋病患者的一种机会性病原体，也是NIAID生物防御研究B类优先病原体。微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虚弱并具有潜在致命性的疾病，目前还没有持续有效的化疗方法，特别是对免疫功能低下的人。因此，迫切需要更有效的药物，特别是那些针对微小弧菌独特特征的药物。在微小隐孢子虫和它的人类宿主之间更显著的代谢差异中，有一种是获得嘌呤的途径。与哺乳动物不同，微小隐孢子虫不能从头合成嘌呤，并依赖于一种独特的嘌呤回收机制来满足其营养需求。因此，抑制嘌呤挽救为治疗微小弧菌感染提供了一种选择性治疗范例。在这个提案中，我们将研究嘌呤回收的三个必需成分：腺苷激酶(CPAK)、腺苷脱氨酶(CpAMPDA)和鸟苷合成酶(CpGMPS)。有两个具体目标。多组分特异性目标I详细描述了这三种酶的生化、生物学和功能特性，并对CPAK进行了结构分析。我们将克隆这三个基因，并通过对它们的编码产物进行生化研究，以及通过挽救或补充其他生物的遗传损伤来对它们的基因产物进行功能评估。Cpak、CpAMPDA和CpGMPS基因在完整的微小隐孢子虫中的表达模式以及这三种蛋白的水平和位置将在整个生物可及生命周期阶段得到确定。最后，基于CPAK分子模型的结构-功能研究将检验CPAK上的关键残基在催化机制中的作用，并将启动对CPAK的结晶学研究，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，我们将针对一系列可用的核苷类似物进行CPAK的构效关系分析。其次，我们将使用基于大肠杆菌的检测来优化和实施结构多样化的化学信息库的高通量筛选，目的是发现可能用于治疗寄生虫病的新的CPAK抑制剂。屏幕上的“点击”将使用基于靶点和基于细胞的分析进行进一步评估，最终将在模拟人类疾病的小鼠模型中进行评估。总体而言，拟议的研究是确认这些酶作为潜在治疗目标的合乎逻辑的步骤，并在实施治疗隐孢子虫病的合理药物发现战略方面是合乎逻辑的步骤。这项建议的总体目的是调查微小隐孢子虫的嘌呤挽救途径的组成部分。微小隐孢子虫是一种水媒寄生虫，会在艾滋病患者中造成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，因为这种生物体可以被恶意操纵为生物武器。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药靶点。由于嘌呤回收对微小隐孢子虫是必不可少的，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。

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.