Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis

开发动质体寄生虫多重测定法来鉴定糖酵解探针

• 批准号: 10382390
• 负责人: KENNETH A CHRISTENSEN
• 金额: $ 49.75万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-03 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10382390
• 关键词: Address; Adopted; Antiparasitic Agents; Area; Biological Assay; Biology; Biosensor; Cells; Cellular Assay; Chemicals; Chemistry; Collection; Development; Disease; Eukaryota; Evaluation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fructose; Future; Gluconeogenesis; Glucose; Glycolysis; Glycosome; Goals; Health; Human; In Vitro; Individual; Lead; Leishmania; Leishmania donovani; Libraries; Link; Maps; Measures; Metabolic; Metabolic Pathway; Metabolism; Methodology; Methods; Molecular Genetics; Monitor; Nature; Organism; Outcome; Outcomes Research; Parasites; Parasitic infection; Pathway interactions; Permeability; Phenotype; Proteins; Proteomics; Pyruvate; Recombinants; Reporter; Role; Series; Solubility; Time; Trypanosoma brucei brucei; Validation; Work; World Health Organization; base; carbohydrate metabolism; cell type; cellular targeting; chemical property; cytotoxicity; genetic approach; glucose metabolism; glucose uptake; high throughput screening; improved; inhibitor; innovation; insight; member; multiplex assay; neglected tropical diseases; novel; novel strategies; pathogen; reverse genetics; screening; sensor; small molecule; tool

Project Summary/Abstract Parasite infection with members of the class Kinetoplastea, including Trypanosoma brucei and Leishmania spp., places a tremendous burden on human health, with an estimated ~1.4 million cases of disease recorded in 2015 (World Health Organization). Despite the widespread impact of these organisms, there remain major gaps in our understanding of fundamental parasite biology. For example, it is appreciated that glucose is a critical metabolite that can also serve to regulate important developmental pathways in the parasites. However, our understanding of these areas, particularly in the context of living parasites, is extremely limited. We propose to elucidate key mechanistic aspects of kinetoplastid glucose metabolism in kinetoplastids using a validated, yet unprecedented, screening assay platform based on live parasites expressing protein sensors that measure glucose uptake, distribution, and metabolism. We will adopt these cells to a novel and effective multiplexed high-throughput screening assay to identify small molecule probes that disrupt glucose uptake, distribution and metabolism in live parasites. This unique approach will simultaneously identify inhibitors of glycolysis that possess chemical properties required for delivery to the cellular target. Hits from the screening of a structurally diverse 100,000 compound collection will be validated by resynthesis, reconfirmation and counter- screening. Target identification, which will be facilitated by mapping the impact of the inhibitor on the pathway using the read-out from the assay, will be confirmed using reverse genetic and proteomic approaches. Last, the identified small molecule glycolytic probe inhibitors will be improved by SAR and their activity against parasites scored. Expected outcomes include development of a screening strategy that will be applicable to many types of cells, along with the identification of validated probes that dissect an essential parasite metabolic pathway.

项目总结/摘要 动原体类成员的寄生虫感染，包括布氏锥虫和利什曼原虫属， 给人类健康带来巨大负担，2015年估计有约140万例疾病病例 （世界卫生组织）。尽管这些生物体的影响广泛，但我们的研究仍存在重大差距。 了解基本的寄生虫生物学。例如，人们认识到葡萄糖是一种关键代谢产物 这也可以用来调节寄生虫的重要发育途径。然而，我们对 这些领域，特别是在活寄生虫的情况下，是非常有限的。 我们建议使用一种新的方法来阐明动质体中动质体葡萄糖代谢的关键机制。 基于表达蛋白质传感器的活寄生虫的经验证但前所未有的筛选测定平台， 测量葡萄糖摄取、分布和代谢。我们将采用这些细胞， 多重高通量筛选测定以鉴定破坏葡萄糖摄取的小分子探针， 分布和代谢。这种独特的方法将同时识别 糖酵解具有递送至细胞靶点所需的化学性质。从一个筛选的点击 结构多样化10万个化合物集合将通过再合成、再确认和反确认进行验证， 筛选靶点识别，通过绘制抑制剂对通路的影响来促进 将使用反向遗传学和蛋白质组学方法来确认。最后 已鉴定的小分子糖酵解探针抑制剂将通过SAR及其抗寄生虫活性得到改进 得分了预期成果包括制定一项适用于许多类型的 细胞，沿着鉴定出有效的探针，该探针剖析了一种重要的寄生虫代谢途径。