Trehalose-6-phosphate phosphatase inhibitors as anti-helminthics

海藻糖-6-磷酸磷酸酶抑制剂作为抗蠕虫药

• 批准号: 9222517
• 负责人: Karen N. Allen
• 金额: $ 26.46万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-09 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9222517
• 关键词: Ablation; Adult; Affect; Affinity; Africa; Americas; Anabolism; Ancylostoma (genus); Animal Model; Anisakis Simplex; Anthelmintics; Antimetabolites; Ascaris suum; Asia; Binding; Caenorhabditis elegans; Cephalosporins; Cessation of life; Chronic; Complex; Country; Critical Pathways; Crystallization; Crystallography; Culicidae; Data; Detection; Development; Disease; Drug Kinetics; Drug Targeting; Energy-Generating Resources; Ensure; Enzymes; Family; Filarial Elephantiases; Genes; Genome; Genus Mycobacterium; Gerbils; Goals; Growth; Homologous Gene; Human; In Vitro; Infection; Intervention; Kinetics; Laboratories; Lead; Libraries; Life Cycle Stages; Link; Liver; Low Income Population; Lymphatic; Mass Spectrum Analysis; Metabolic; Metabolism; Mitochondria; Modeling; Necator americanus; Nematoda; Onchocerca volvulus; Organism; PTPN1 gene; PTPN6 gene; Parasitic infection; Parasitic nematode; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphoric Monoester Hydrolases; Plasma; Population; Probability; Proxy; Rattus; Research; Research Personnel; Research Project Grants; Roentgen Rays; Scheme; Severities; Stress; Strongyloides stercoralis; Structure; Structure-Activity Relationship; Testing; Therapeutic Intervention; Toxic effect; Trehalose; Trichinella spiralis; Trichocephalus trichiura; Variant; X-Ray Crystallography; analog; base; cytotoxicity; high throughput screening; in vivo; inhibitor/antagonist; inorganic phosphate; insight; killings; knock-down; man; nanomolar; phosphatase inhibitor; screening; stability testing; transmission process

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.

项目概要/摘要 寄生线虫是导致许多慢性失能和变形疾病的原因 非洲、亚洲和美洲的疾病包括淋巴丝虫病，它是一种由蚊子传播的疾病。 这种疾病在 81 个国家流行，估计有 1.2 亿人感染这种疾病。 寄生线虫必需但不具有人类同源物的酶是潜在的 治疗干预的药物靶点。来自马来芽孢杆菌的基因组的可用性。 丝虫线虫生物体，使得能够对这种寄生生物体的潜在药物靶标进行排序。 其中一种酶是海藻糖-6-磷酸磷酸酶 (T6PP)，它是生物合成 海藻糖通常用于寄生线虫的线虫模型中 T6PP 活性的消除。 最终导致生物体死亡，可能是由于海藻糖 6-磷酸 (T6P) 在我们体内的积累。 合作研究项目我们解决了 B. malayi T6PP 的结构，确定了那些残基 对 T6P 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 筛选并利用该筛选找到了两种纳摩尔亲和力的先导抑制剂。 通过筛选发现 Closantel 对马来亚细线虫具有体内活性，特别是对成虫 目前的治疗方法未能实现这一目标。 已通过筛选鉴定出的化合物，以增强效力和可用性，并确定 T6P毒性机制研究计划集中于四个目标。 目标 1 (R21) 将提供 1) 结构-活性关系分析原则上命中 Closantel 和 头孢菌素 C 通过马来芽孢杆菌 T6PP 体外合成和测试以及秀丽隐杆线虫体内测试 2) 体外 大鼠血浆和肝脏 S9 组分的稳定性测试 3) 马来芽孢杆菌 T6PP 复合物的 X 射线晶体结构 抑制剂，4) 在基于 SAXS 的屏幕中测试主要命中和变体，作为对 SAR 和 作用机制 目标 2 (R21) 将 1) 提供针对相同和 的磷酸酶的反筛选。 其他磷酸酶家族以确保选择性，2) 开发一组来自其他磷酸酶的直系同源 T6PP 酶 寄生线虫的抑制动力学（以及可能的晶体学）为广泛的 谱抗蠕虫药 目标 3 (R33) 将 1) 测试马来巴氏蠕虫体内的抑制活性 2) 执行 代谢、初步药代动力学和细胞毒性研究3)确定抗- 通过量化 T6P 伴随毒性的水平并评估 T6P 的作用来评估 T6P 的代谢活性 目标 4 (R33) 将测试马来沙鼠感染模型中的高级线索。 拟议的研究将提供先进的线索，显示出选择性杀灭作用，用于治疗 使世界上很大一部分人口遭受寄生线虫感染的疾病。