Trehalose-6-phosphate phosphatase: a target for anti-onchocerciasis therapeutics

描述(申请人提供)：寄生线虫是非洲、亚洲和美洲许多慢性丧失能力和致残疾病的罪魁祸首。淋巴丝虫病是这些疾病中的一种，这是一种蚊子传播的疾病，在81个国家流行。据估计，有1.2亿人感染了这种疾病。对寄生线虫至关重要但没有人类同源物的酶是治疗干预的潜在药物靶点。马来丝虫是丝虫的代表生物，其基因组的可获得性使这种寄生生物的潜在药物靶标的排名成为可能。其中一种酶是海藻糖-6-磷酸磷酸酶(T6PP)，它是生物合成海藻糖所必需的。在寄生线虫常用的线虫模型中，T6PP活性的减弱最终导致生物体死亡，可能是由于海藻糖6-磷酸(T6P)的积累。由于T6PP是卤代烷酸脱卤酶超家族中的一员，有关该家族结构/功能关系的知识可用于定义T6PP用于药物开发。这项拟议的研究的目的是确定T6PP活性部位区域的空间和静电特征，这些特征可用于铅抑制剂的设计。由于酶的两个结构域可以旋转以打开配体交换的活性部位，因此可以通过确定BOT开放和闭合构象的结构来获得可能被抑制剂靶向的表面积。该研究计划集中于一个目标：确定马来芽胞杆菌海藻糖-6-磷酸酶类药物抑制剂开发的目标部位。用马来丝虫和线虫的重组酶测定铅的T6PP X射线晶体结构，以确定蛋白质的总体结构。共结晶和晶体浸泡实验将捕捉闭合状态(使用不活跃的T6PP突变体和T6P或使用T6PP加惰性底物类似物)和过渡态构象(T6PP和海藻糖加钒、氟化铍或氟化铝)。结构-活性关系的分析将被用来定义决定配体结合能贡献的结合相互作用。将确定开帽构象中酶的结构(使用apo T6PP和T6PP结构域结合突变体)，以便设计和评估能够填充和补充开帽构象扩展的结合缝隙的双齿抑制剂。这项工作将为开发一种治疗疾病的药物(S)奠定基础，这种疾病使世界上大部分人口受到寄生线虫的感染。

DESCRIPTION (provided by applicant): Parasitic nematodes are responsible for numerous chronically incapacitating and deforming diseases in Africa, Asia, and the Americas. Among these diseases is lymphatic filariasis, which is a mosquito-transmitted disease, endemic to 81 countries. It is estimated that 120 million people are infected with this disease. Enzymes that are essential for the parasitic nematodes but that do not have a human homologue are potential drug targets for therapeutic intervention. The availability of the genome from B. malayi, the representative organism for filarial nematodes, has enabled the ranking of potential drug targets from this parasitic organism. One such enzyme is trehalose-6-phosphate phosphatase (T6PP), which is required for the biosynthesis of trehalose. The oblation of T6PP activity in the C. elegans model commonly used for parasitic nematodes ultimately leads to organism death, probably due to the accumulation of trehalose 6-phosphate (T6P). Because T6PP is a member of the haloalkanoate dehalogenase superfamily of phosphatases, knowledge about the structure/function relationships in this family can be used to define T6PP for drug development. The objective of the proposed study is to identify the steric and electrostatic features of the T6PP active-site region that can be exploited in the design of lead inhibitors. Because the two domains of the enzyme can rotate to open the active site for ligand exchange, the surface area that can be potentially targeted by an inhibitor will be obtained by determining structures of bot open and closed conformers. The research plan is focused on a single Aim: Define the Target Site for the Development of Drug-like Inhibitors of B. malayi Trehalose-6-phosphate Phosphatase. The ¿lead¿ T6PP X-ray crystal structure will be determined using the recombinant enzyme from B. malayi and C. elegans to define the overall structure of the protein. Co-crystallization and crystal soaking experiments will capture the closed state (using an inactive T6PP mutant and T6P or using T6PP plus an inert substrate analog) and transition-state conformations (T6PP and trehalose plus vanadate, beryllium fluoride, or aluminum fluoride). Analysis of structure-activity relationships will be used to define the binding interactions which dominate the contributions to the ligand binding energy. The structure of the enzyme in the cap-open conformation will be determined (using apo T6PP and T6PP domain-domain binding mutants) in order to design and evaluate bidentate inhibitors that can fill and complement the expanded binding crevice of the cap-open conformer. This work will deliver the foundation for the development of a drug for the treatment of disease(s) inflicting the large segment of the world's population suffering from infection by parasitic nematodes.