Function and Inhibition of Plasmodium Lipid Decarboxylases

描述（由申请方提供）：在人红细胞内的生命周期中，恶性疟原虫经历主要的发育和代谢变化，并繁殖产生多达36个新的子寄生虫。这种快速增殖需要对许多基本寄生虫功能重要的结构和信号脂质的主动合成，例如寄生虫增殖后新膜的产生，以及用于激活寄生虫激酶的二酰基甘油的合成，仅举几例。控制这些大分子合成的代谢机制由从宿主中清除的前体如丝氨酸、乙醇胺和脂肪酸提供燃料。这些机制长期以来一直被认为是开发新型抗疟药物的极好靶点。迄今为止，只有少数这些机制在疟原虫寄生虫中得到了彻底的定性，针对其中一些机制的药理学研究成功地导致了高效抗疟药物的生产。从宿主获得的丝氨酸作为主要磷脂磷脂酰胆碱和磷脂酰乙醇胺合成的主要前体。丝氨酸被寄生虫特异性丝氨酸脱羧酶（PfSD）脱羧以形成乙醇胺，其随后用作合成磷脂酰胆碱和磷脂酰乙醇胺的前体。丝氨酸还通过寄生虫磷脂酰丝氨酸脱羧酶（PfPSD）催化的反应掺入磷脂酰丝氨酸中，磷脂酰丝氨酸作为合成磷脂酰乙醇胺的替代前体。由于PfSD和PfPSD具有预期的基本功能，因此被认为是开发新的抗疟药物的潜在靶点。此外，人类细胞不含SD酶，从而使SD成为寄生虫的物种特异性脆弱性。利用构建在酵母表达载体中的疟原虫cDNA文库，我们成功地补充了缺乏PSD活性的酵母突变体，并鉴定了疟疾PSD基因。已有的数据表明，疟疾PSD在疟原虫的红细胞内生活周期中起着重要作用，并且是开发新的抗疟药物的极好靶点。然而，疟疾SD基因仍有待确定。这项资助申请的总体目标是完成恶性疟原虫PfPSD基因的生物化学和遗传学表征（目标1）;利用新开发的和成功的功能互补测定，使用酵母作为替代系统来筛选抗疟活性化合物的文库，以寻找PfPSD活性的抑制剂（目标2）;并采用遗传和生物化学分析来鉴定疟疾丝氨酸脱羧酶基因并表征其在恶性疟原虫红细胞内发育和存活中的重要性（目的3）。这些研究有可能阐明PfSD和PfPSD的重要性，以及在恶性疟原虫发育过程中磷脂代谢的一般情况，以及促进特异性抑制剂的设计。这项工作将提供新的治疗见解， 防治一种影响全世界2.5亿人并每年造成100万人死亡的疾病。

DESCRIPTION (provided by applicant): During its life cycle within human erythrocytes, Plasmodium falciparum undergoes major developmental and metabolic changes and multiplies to produce up to 36 new daughter parasites. This rapid multiplication requires an active synthesis of structural and signaling lipids important for many essential parasite functions such as the production of new membranes following parasite multiplication, and the synthesis of diacylglycerol for activation of parasite kinases, to name only a few. The metabolic machineries that govern the synthesis of these macromolecules are fueled by precursors such as serine, ethanolamine and fatty acids scavenged from the host. These machineries have long been regarded as excellent targets for the development of novel antimalarial drugs. To date only a few of these machineries have been thoroughly characterized in Plasmodium parasites and pharmacological studies targeting some of them have successfully resulted in the production of highly potent antimalarial drugs. Serine obtained from the host serves as the primary precursor for the synthesis of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Serine is decarboxylated by a parasite specific serine decarboxylase (PfSD) to form ethanolamine, which is subsequently used as a precursor for the synthesis of both phosphatidylcholine and phosphatidylethanolamine. Serine is also incorporated into phosphatidylserine, which serves as an alternate precursor for the synthesis of phosphatidylethanolamine, via a reaction catalyzed by a parasite phosphatidylserine decarboxylase (PfPSD). Because of their predicted essential functions, PfSD and PfPSD are regarded as potential targets for the development of new antimalarial drugs. Moreover, human cells do not contain SD enzymes thereby making SD a species-specific vulnerability of the parasite. Using a Plasmodium cDNA library constructed in a yeast expression vector we have successfully complemented a yeast mutant lacking PSD activity and identified the malarial PSD gene. Available data suggest that the malarial PSD plays an essential role in the intraerythrocytic life cycle of the parasite and is an excellent target for the development of nove antimalarial drugs. The malarial SD gene, however, remains to be identified. The overall objectives of this grant application are to complete the biochemical and genetic characterization of the PfPSD gene in P. falciparum (Aim 1); to take advantage of the newly developed and successful functional complementation assay using yeast as a surrogate system to screen a library of antimalarial active compounds to search for inhibitors of PfPSD activity (Aim 2); and employ genetic and biochemical analyses to identify the malarial serine decarboxylase gene and characterize its importance in P. falciparum intraerythrocytic development and survival (Aim 3). These studies hold the potential for elucidating the importance of PfSD and PfPSD specifically, and phospholipid metabolism in general during P. falciparum development as well as fostering the design of specific inhibitors. This work will provide new therapeutic insights for combating a disease that affects 250 million people worldwide and causes 1 million deaths each year.