Trehalose-6-phosphate phosphatase inhibitors as anti-helminthics

项目总结/摘要 寄生线虫是许多慢性失能和变形疾病的原因， 非洲、亚洲和美洲。在这些疾病中，淋巴丝虫病是一种蚊子传播的疾病， 流行于81个国家。据估计，有1.2亿人感染了这种疾病。 寄生线虫必需的酶，但没有人类同源物是潜在的 治疗干预的药物靶点。来自B的基因组的可用性。马来西亚代表 针对丝虫线虫的生物体的研究已经使得能够对来自这种寄生生物体的潜在药物靶标进行排序。 一种这样的酶是海藻糖-6-磷酸磷酸酶（T6 PP），其是生物合成 海藻糖T6 PP活性在C.线虫模型常用于寄生线虫 最终导致生物体死亡，可能是由于海藻糖6-磷酸（T6 P）的积累。在我们 我们已经解决了B的结构。马来T6 PP，测定这些残留物 对于T6 P结合至关重要，开发了底物类似物，建立了抑制剂的高通量测定， 筛选并利用该筛选发现了两种纳摩尔亲和的先导抑制剂。其中一种先导化合物 通过筛选发现，Closantel显示出抗B的体内活性。马来人，特别是对蠕虫 这是目前的治疗方法无法实现的。拟议的目标是开发先进的铅的基础上， 已经通过筛选鉴定的化合物，以提高效力和可用性，并确定 T6 P毒性机制该研究计划的重点是四个目标。 目标1（R21）将提供1）原则上命中Closantel的结构-活性关系分析， 头孢菌素C通过合成和体外测试对B。malayi T6 PP和体内C. 2）体外 大鼠血浆和肝脏S9组分中的稳定性试验3）B的X射线晶体结构。马来T6 PP复合 抑制剂，4）在基于SAXS的筛选中测试主要命中和变体，作为对SAR的额外洞察， 作用机制。目标2（R21）将1）提供针对来自相同的磷酸酶的反筛选， 2）开发一组来自其他磷酸酶家族的正磷酸酶T6 PP酶， 寄生线虫的抑制动力学（和可能的结晶学），以提供广泛的基础， 广谱驱虫药目的3（R33）将1）在B中检测体内抑制活性。马来语2）表演 代谢，初步的药代动力学和细胞毒性研究3）确定抗- 通过定量伴随毒性的T6 P水平和评估T6 P的作用， 对主要代谢酶的影响目标4（R33）将测试B中的高级电极导线。马来沙鼠感染模型。 这项拟议的研究将提供先进的线索，显示出选择性杀死行动的治疗， 使世界上大部分人口遭受寄生线虫感染的疾病。

PROJECT SUMMARY/ABSTRACT 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 to 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). In our collaborative research project we have solved the structure of B. malayi T6PP, determined those residues critical to T6P binding, developed substrate analogs, established a high-throughput assay for inhibitor screening and utilized that screen to find two nanomolar-affinity lead inhibitors. One of the lead compounds discovered by screening, Closantel, shows in vivo activity against B. malayi, and notably against adult worms which current treatments fail to achieve. The proposed objective is to develop advanced leads based on the compounds already identified via screening to enhance potency and availability and to determine the mechanism of T6P toxicity. The research plan is focused on four Aims. Aim 1 (R21) will deliver 1) structure-activity relationship analysis on principle hits Closantel and Cephalosporin C via synthesis and testing in vitro on B. malayi T6PP and in vivo on C. elegans 2) in vitro stability tests in rat plasma and liver S9 fractions 3) X-ray crystal structures of B. malayi T6PP complexed with inhibitors, 4) tests of principle hits and variants in a SAXS-based screen as additional insight into SAR and mechanism of action. Aim 2 (R21) will 1) provide counter-screening against phosphatases from the same and other phosphatase families to ensure selectivity, 2) develop a panel of orthologous T6PP enzymes from other parasitic nematodes for inhibition kinetics (and possibly crystallography) to provide the basis for broad- spectrum anti-helminthics. Aim 3 (R33) will 1) test for inhibitory activity in vivo in B. malayi 2) perform metabolism, preliminary pharmacokinetic and cytotoxicity studies 3) determine the mechanism of the anti- metabolite activity of T6P by quantifying levels of T6P accompanying toxicity and assessing the effect of T6P on primary metabolic enzymes. Aim 4 (R33) will test the advanced leads in a B. malayi gerbil infection model. The proposed research will deliver advanced leads that show selective killing action for the treatment of disease(s) inflicting the large segment of the world’s population suffering from infection by parasitic nematodes.