The Neddylation Pathway in Leishmania donovani - A High Opportunity Target

利什曼原虫和其他着丝体寄生虫会导致毁灭性疾病，折磨数百万人，而多诺瓦氏l通常会导致致命的内脏利什曼病。由于目前的药物治疗方案严重不足，因此确定可用于开发新型口服生物有效药物的特定靶点非常重要，这些药物将改善治疗选择。这些寄生虫中的类化修饰途径具有多种酶，这些酶可能是细胞内致病寄生虫生存所必需的。该途径使用特定的E1、E2和E3激活酶将小的泛素样蛋白NEDD8附着在各种细胞底物上，如cullin连接酶，这是泛素化途径的重要组成部分。这些底物的类黄酮化通常激活它们的功能，从而调节相关的下游途径。值得注意的是，这三种类化酶中的每一种都已经成为开发人类抗癌药物的目标，其中一种实验性药物正在进行三期临床试验。这些成功表明，每种酶都是可药物化的，对同源但序列高度分化的L. donovani酶的平行研究可能会发现寄生虫特异性药物样抑制剂，这些抑制剂会灭活类化化途径，对寄生虫具有致命作用，但对宿主细胞的影响很小。本项目的目的是在L. donovani中分别删除这些酶的3个基因UBA3、DCN1和CSN5，并确定这些缺失是否会严重损害寄生虫生命周期细胞内致病阶段的生长。成功证明部分或全部这些类化修饰酶的重要性将证实它们作为药物靶点，通过寄生虫选择性小分子抑制它们将为这种沉重的全球传染病提供新的治疗方式。这些结果将为后续针对这一途径的药物开发计划提供动力。此外，对零突变体的表型研究将揭示由类化修饰途径控制的重要生物学过程，从而为这种病原体的基本生物学提供有力的见解。

Leishmania and other kinetoplastid parasites cause devastating diseases that afflict millions of people, and L. donovani typically causes fatal visceral leishmaniasis. Because current drug regimens are woefully inadequate, it is important to identify specific targets that can be exploited for development of novel orally bioavailable drugs that will improve therapeutic options. The neddylation pathway in these parasites represents a high opportunity target with multiple enzymes that are likely essential for survival of intracellular disease-causing parasites. This pathway uses specific E1, E2, and E3 activating enzymes to attach the small ubiquitin -like protein, NEDD8, onto various cellular substrates, such as the cullin ligases that are important components of the essential ubiquitination pathway. Neddylation of these substrates typically activates their functions and thus regulates the relevant downstream pathway. Significantly, each of 3 neddylation enzymes has already been targeted for development of anti-cancer drugs in humans, with one experimental drug undergoing phase 3 clinical trials. These successes indicate that each enzyme is druggable and that parallel studies on the orthologous but highly sequence divergent enzymes from L. donovani is likely to identify parasite-specific drug- like inhibitors that will inactivate the neddylation pathway with lethal effects for the parasite but with minimal effects on host cells. The purpose of this project is to delete in L. donovani each of the 3 genes for these enzymes, called UBA3, DCN1, and CSN5, and determine whether such deletions strongly impair growth of the intracellular disease-causing stage of the parasite life cycle. Successful demonstration of essentiality for some or all of these neddylation enzymes will validate them as drug targets whose inhibition by parasite-selective small molecules would provide novel therapeutic modalities for this burdensome global infectious disease. These results will provide the impetus for a subsequent drug development program to target this pathway. In addition, phenotypic studies on the null mutants will reveal important biological processes governed by the neddylation pathway and will thus provide powerful insights into the basic biology of this pathogen.