The Neddylation Pathway in Leishmania donovani - A High Opportunity Target

利什曼原虫和其他动体寄生虫会导致毁灭性的疾病，折磨着数百万人，而多诺瓦尼利什曼原虫通常会导致致命的内脏利什曼病。由于目前的药物方案严重不足，重要的是确定可用于开发新的口服生物可用药物的特定靶点，这些药物将改善治疗选择。这些寄生虫中的脱氢酶途径代表了一个具有多种酶的高机会靶标，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激活酶将小的泛素样蛋白NEDD8连接到各种细胞底物上，例如作为基本泛素化途径的重要组成部分的cullin连接酶。这些底物的去核化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种脱氢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3期临床试验。这些成功表明，每一种酶都是可用药的，对杜诺氏乳杆菌的同源但高度序列差异的酶进行的平行研究很可能识别出寄生虫特异性的类药物抑制剂，它将使死亡途径失活，对寄生虫具有致死作用，但对宿主细胞的影响最小。这个项目的目的是在杜诺瓦尼乳杆菌中删除这些酶的3个基因，称为UBA3，DCN1和CSN5，并确定这些缺失是否强烈地阻碍了寄生虫生命周期的细胞内致病阶段的生长。部分或全部这些脱氢酶的重要性的成功证明将使它们成为药物靶点，其对寄生虫选择性小分子的抑制将为这种负担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，对缺失突变体的表型研究将揭示由突变途径控制的重要生物学过程，从而为深入了解这种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.