Investigating the shikimate pathway in Plasmodium falciparum

说明（由申请人提供）：疟原虫是疟疾的病原体。在感染人类的疟原虫种类中，恶性疟原虫是最致命的，它占全世界疟疾感染的大多数。不幸的是，这些感染的负担在非洲、南美洲和亚洲的贫穷和不发达国家尤其沉重。目前，只有少数代谢过程已被证实为治疗疟疾的靶点，并且越来越需要确定针对其他基本途径的药物。七步莽草酸途径对细菌和植物中芳香氨基酸、泛醌和叶酸的产生至关重要。虽然莽草酸途径也被认为对顶复合体（如恶性疟原虫）至关重要，但在其基因组中无法确定完整的途径。最近，一项生物信息学研究在恶性疟原虫中发现了两种预测蛋白，它们与莽草酸途径中的后三种酶同源。尽管我们无法通过生物信息学分析确定该途径的其余部分，但有令人信服的证据表明，恶性疟原虫莽草酸途径存在，并且是一个可行的药物靶点。植物中已知的针对莽草酸途径酶的分子被发现可以抑制恶性疟原虫的生长。此外，预测的莽草酸途径酶的基因在恶性疟原虫生命周期的所有阶段都具有转录活性。我们拟从以下几个方面对恶性疟原虫的莽草酸途径进行研究：(1)对预测的恶性疟原虫莽草酸途径酶（莽草酸激酶、5-烯醇丙酮酰莽草酸-3-磷酸合成酶和甘草酸合成酶）进行生化表征。(2)通过基因互补筛选和代谢物分析，确定恶性疟原虫中参与莽草酸生物合成的其他基因。(3)通过小分子筛选筛选途径特异性抑制剂，构建基因敲除，确定shikimate通路是否对恶性疟原虫生长至关重要。我们认为，这些实验对于确定莽草酸途径是否是治疗疟疾的可行治疗靶点至关重要。

DESCRIPTION (provided by applicant): The parasite Plasmodium is the causative agent of the disease malaria. Among the species of Plasmodium that infects humans, Plasmodium falciparum is the deadliest, and it accounts for the majority of malaria infections worldwide. Unfortunately, the burden of these infections is particularly heavy in poor and undeveloped countries in Africa, South America and Asia. Currently, only a handful of metabolic processes have been verified as targets to treat malaria, and there is a growing need to identify agents that address other essential pathways. The seven-step shikimate pathway is essential to the production of aromatic amino acids, ubiquinone, and folate in bacteria and plants. While the shikimate pathway is also believed to be essential for apicomplexans, such as P. falciparum, the complete pathway cannot be identified in its genome. Recently, a bioinformatics study identified two predicted proteins in P. falciparum that are homologous to the last three enzymes in the shikimate pathway. Despite our inability to identify the rest of the pathway through bioinformatics analysis, there is compelling evidence that the P. falciparum shikimate pathway exists and is a viable drug target. Molecules known to target shikimate pathway enzymes in plants were found to inhibit P. falciparum growth. Additionally, the genes for the predicted shikimate pathway enzymes are transcriptionally active during all stages of the P. falciparum lifecycle. We propose to investigate the shikimate pathway in P. falciparum with the following specific aims: (1) Biochemically characterize the predicted P. falciparum shikimate pathway enzymes (shikimate kinase, 5-enolpyruvylshikimate-3-phosphate synthase, and chorismate synthase). (2) Identify other genes involved in shikimate biosynthesis in P. falciparum via a genetic complementation screen and metabolite analysis. (3) Determine if the shikimate pathway is essential to P. falciparum growth by identification of pathway specific inhibitors through small molecule screens, and the construction of genetic knockouts. We believe that these experiments are essential to determine if the shikimate pathway is a viable therapeutic target for the treatment of malaria. PUBLIC HEALTH RELEVANCE: Malaria is a life-threatening disease that is caused by the Plasmodium parasite. I plan to investigate aromatic amino acid biosynthesis in Plasmodium falciparum, the deadliest species of the parasite, to evaluate the potential of the pathway as a drug target to treat malaria.