The Neddylation Pathway in Leishmania donovani - A High Opportunity Target

利什曼原虫和其他动质体寄生虫会导致毁灭性的疾病，困扰数百万人，而杜诺瓦尼利什曼原虫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可利用药物以改善治疗选择的特定靶点非常重要。这些寄生虫中的neddylation途径代表了多种酶的高机会靶点，这些酶可能对细胞内致病寄生虫的生存至关重要。该途径使用特定的 E1、E2 和 E3 激活酶将小泛素样蛋白 NEDD8 连接到各种细胞底物上，例如作为基本泛素化途径重要组成部分的 cullin 连接酶。这些底物的 Neddylation 通常会激活它们的功能，从而调节相关的下游途径。值得注意的是，3 种 neddylation 酶中的每一种都已被用于开发人类抗癌药物，其中一种实验药物正在进行 3 期临床试验。这些成功表明每种酶都是可药物化的，并且对来自杜氏乳杆菌的直系同源但高度序列差异的酶进行的平行研究可能会鉴定出寄生虫特异性药物样抑制剂，这些抑制剂将使neddylation途径失活，对寄生虫具有致命作用，但对宿主细胞的影响最小。该项目的目的是删除杜氏乳杆菌中这些酶的 3 个基因（UBA3、DCN1 和 CSN5）中的每一个，并确定这些删除是否会严重损害寄生虫生命周期的细胞内致病阶段的生长。成功证明部分或全部 neddylation 酶的重要性将验证它们作为药物靶点的有效性，其通过寄生虫选择性小分子的抑制将为这种繁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该途径的药物开发计划提供动力。此外，对无效突变体的表型研究将揭示由neddylation途径控制的重要生物过程，从而为该病原体的基础生物学提供强有力的见解。

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.