PHOSPHATIDYLCHOLINE BIOSYNTHESIS AND ANTICANCER AGENT MILTEFOSINE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 米替福辛是一种具有抗癌和抗微生物活性的磷胆碱类似物。尽管目前它在医学上的应用，但它的作用模式仍然鲜为人知。我们将使用人类寄生虫利什曼原虫作为模式系统，以加深我们对米替福辛的作用模式的理解。磷脂酰胆碱是哺乳动物细胞和利什曼原虫中含量最丰富的细胞脂类物质，由胞外胆碱摄取的从头途径和一个或几个磷脂酰乙醇胺甲基转移酶(S)对磷脂酰乙醇胺的三次甲基化产生。与癌细胞类似，利什曼对米替福辛高度敏感，米替福辛可减少磷脂酰乙醇胺生物合成磷脂酰胆碱。 在癌细胞和利什曼病中都存在。本应用的目的是鉴定参与从磷脂酰乙醇胺生物合成磷脂酰胆碱的利什曼酶，并评估它们是否是米替福辛的靶标。虽然胆碱对于利什曼原虫的生存是必不可少的，但我们的初步数据表明，杜氏利什曼原虫的两个磷脂酰乙醇胺甲基转移酶同源基因LdPEM1和LdPEM2是必不可少的。我们的假设是，LdPEM1和LdPEM2的甲基化途径是磷脂酰胆碱生物合成的主要途径，其中至少一个途径被米替福辛抑制。我们的假设将通过执行以下特定目标来验证：1，通过米替福辛测定LdPEM1和LdPEM2的酶活性和抑制谱；2，确定LdPEM1是否过表达 和/或LdPEM2调节(S)利什曼原虫对米替福辛的敏感性；以及3，证实LdPEM1和LdPEM2在利什曼原虫存活中的重要性。这些研究有望显著提高我们对利什曼原虫磷脂酰胆碱生物合成和米替福辛作用方式的认识，并为开发更有效的抗癌药物提供分子基础。该项目的目标是更好地了解抗癌药物米替福辛的作用机制。拟议的研究可能会对癌症的治疗产生关键影响，因此， 直接关系到公众健康。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Miltefosine is a phosphocholine analog with anticancer and antimicrobial activities. Despite its current medical application, its mode of action is still poorly understood. We will use the human parasite Leishmania as a model system to deepen our understanding of miltefosine's mode of action. Phosphatidyicholine, the most abundant cellular lipid in mammalian cells and in Leishmania, is produced from the de novo pathway from the uptake of extracellular choline, and from the three-fold methylation of phosphatidylethanolamine by one or several phosphatidylethanolamine methyltransferase(s). Similar to cancer cells, Leishmania is highly sensitive to miltefosine, which decreases phosphatidyicholine biosynthesis from phosphatidylethanolamine in both cancer cells and Leishmania. The goal of this application is to identify the leishmanial enzymes involved in phosphatidylcholine biosynthesis from phosphatidylethanolamine and assess whether they are the targets of miltefosine. While choline is dispensable for Leishmania viability, our preliminary data suggest that two phosphatidylethanolamine methyltransferase orthologs, LdPEM1 and LdPEM2 of L. donovani, are essential. Our hypothesis is that the methylation pathway made by LdPEM1 and LdPEM2 represents the primary route for phosphatidyicholine biosynthesis, and that at least one of them is inhibited by miltefosine. Our hypothesis will be tested by performing the following Specific Aims: 1, determine LdPEM1 and LdPEM2 enzymatic activities and inhibition profiles by miltefosine; 2, determine whether overexpression of LdPEM1 and/or LdPEM2 modulate(s) Leishmania sensitivity to miltefosine; and 3, confirm the importance of LdPEM1 and LdPEM2 in Leishmania viability. These studies are expected to significantly enhance our knowledge on phosphatidyicholine biosynthesis in Leishmania and on miltefosine's mode of action, and to provide the molecular basis for the development of more potent anticancer drugs. The goal of this project is to gain a better understanding of the mechanism of action of the anticancer drug miltefosine. The proposed studies may have critical implications for the treatment of cancer, and thus, relates directly to public health.