Targeting isoprenoid biosynthesis in Plasmodium falciparum

描述（由申请人提供）：与NIAID使命的相关性：本申请描述了一个为期5年的培训计划，用于发展儿科传染病的学术生涯，目标是独立指导寄生虫生物学、发病机制和治疗研究。研究设计和方法：由于病原体恶性疟原虫耐药性的传播，迫切需要新的抗疟药。了解恶性疟原虫的基本生物学是这些药物开发工作的关键。所有生物体中的一个重要代谢途径是类异戊二烯分子的生物合成，类异戊二烯分子是对细胞呼吸、膜结构和信号传导至关重要的多种细胞化合物的基本构件。我们推测，这一途径也是恶性疟原虫的正常发育和繁殖所必需的。在疟疾物种中，类异戊二烯通过非甲羟戊酸（DXP）途径产生。寄生虫DXP途径在生物化学上不同于人类中的甲羟戊酸途径，并且证据表明该途径是寄生虫存活所必需的。研究将集中在这一途径的两种酶，脱氧木酮糖磷酸还原异构酶（DXR）和甲基环二磷酸合酶（IspF）。为了研究DXR和IspF的生物学和生化特性，我们提出了一个双管齐下的生化和遗传方法。具体目标包括以下：（1）DXR和IspF的异源表达，开发适合于高通量筛选的体外测定，以及两种酶的生物化学表征;（2）通过开发表达DXR和IspF的GFP融合体的恶性疟原虫转基因株，在寄生虫内定位DXR和IspF;（3）如果可能的话，产生恶性疟原虫的DXR和IspF破坏菌株，并详细分析在寄生虫破坏菌株和用DXR的小分子抑制剂磷霉素处理的菌株中抑制类异戊二烯生物合成的表型效应。与公共卫生的相关性：这些实验探索了恶性疟原虫的基本代谢途径，类异戊二烯生物合成的基础生物学。类异戊二烯化合物，包括醌，光合色素和甾醇，对细胞功能至关重要。这一领域的研究预计将提供深入了解寄生虫的发病机制，并最终治疗。

DESCRIPTION (provided by applicant): Relevance to NIAID mission: This application describes a 5-year training program for the development of an academic career in Pediatric Infectious Diseases, with a goal of independently directing research into parasite biology, pathogenesis, and therapy. Research design and methods: New antimalarial agents are urgently needed due to the spread of drug resistance in the pathogen Plasmodium falciparum. Understanding the fundamental biology of P. falciparum is key to these drug development efforts. An important metabolic pathway in all organisms is the biosynthesis of isoprenoid molecules, fundamental building blocks for diverse cellular compounds vital for cellular respiration, membrane structure, and signaling. We hypothesize that this pathway is also essential for the normal development and reproduction of Plasmodium falciparum. In malaria species, isoprenoids are made via the non-mevalonate (DXP) pathway. The parasite DXP pathway is biochemically distinct from the mevalonate pathway in humans, and evidence suggests this pathway is required for parasite survival. Research will focus on two enzymes of this pathway, deoxyxylulose phosphate reductoisomerase (DXR) and methylerythritol cyclodiphosphate synthase (IspF). To study the biological and biochemical characteristics of DXR and IspF, we propose a dual-pronged biochemical and genetic approach. The specific aims include the following: (1) Heterologous expression of DXR and IspF, development of in vitro assays suitable for high-throughput screening, and biochemical characterization of both enzymes; (2) Localization of DXR and IspF within the parasite by development of transgenic strains of P. falciparum that express GFP-fusions of DXR and IspF; (3) Generation of DXR and IspF disruption strains of P. falciparum, if possible, and detailed analysis of the phenotypic effects of inhibition of isoprenoid biosynthesis in both parasite disruption strains and strains treated with a small-molecule inhibitor of DXR, fosmidomycin. Relevance to public health: These experiments explore the basic biology of a fundamental metabolic pathway, isoprenoid biosynthesis, of Plasmodium falciparum. Isoprenoid compounds, which include quinones, photosynthetic pigments, and sterols, are vital to cellular function. This area of research is expected to provide insights into parasite pathogenesis, and ultimately therapeutics.