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.
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