Selective Inhibitors of the Hexose Transporter from African Trypansomes

描述（由申请方提供）：布氏锥虫引起非洲锥虫病，估计有50，000 - 70，000人患病，偶尔大流行导致多达800，000人死亡。用于治疗非洲锥虫病的药物数量有限，存在重大缺陷，包括高毒性、高成本和耐药性的发展。因此，人们普遍认为迫切需要发现新的抗锥虫药物。锥虫严重依赖于导致疾病的血流形式（BF）中葡萄糖的摄取和代谢，并且在BF寄生虫中表达的己糖转运蛋白THT 1已被鉴定并在多个出版物中实验验证为必需的通透酶和非常有前途的药物靶标。尽管意识到靶向THT 1的前景，但基本上没有做任何关于鉴定选择性抑制这种寄生虫通透酶的化合物的工作。该项目的新奇在于，它将对选择性抑制THT 1而不抑制哺乳动物己糖转运蛋白的化合物进行筛选。这些化合物最终可用于开发改进的抗锥虫药物。已经建立了一种细胞生长试验，并充分验证了高通量筛选（HTS），以确定THT 1的选择性抑制剂。本试验采用相关寄生虫墨西哥利什曼原虫的葡萄糖转运蛋白无效突变体，该突变体已与编码THT 1的基因或主要人葡萄糖转运蛋白GLUT 1的基因互补。在目标1中，将筛选来自圣裘德儿童研究医院CBT库的约600，000种药物样化合物，以确定其抑制转基因L。表达THT 1的墨西哥卷叶螟。然后将来自该初步筛选的命中物进行二次筛选以选择不抑制转基因L.表达GLUT 1的墨西哥小球藻。这种两步筛选将鉴定可能是THT 1选择性抑制剂的化合物。在目标2中，这些命中化合物将在采用[3 H] D-葡萄糖的摄取测定中进行测试，以鉴定那些是THT 1而不是GLUT 1摄取葡萄糖的高亲和力选择性抑制剂。还将测试化合物抑制T.布氏杆菌BFs体外和体内 一种已建立的非洲锥虫病小鼠模型。将对几种人类细胞系进行毒性试验，以评估命中化合物的治疗指数，并确定代谢稳定性。该项目利用了PI实验室Scott Landfear博士（一位广泛研究寄生虫转运蛋白的分子寄生虫学家）和Kip Guy博士（他在St. Jude经营着一家国际知名的化合物筛选和药物化学设施，并且在筛选抗寄生虫化合物方面有着良好的记录）之间的合作。

DESCRIPTION (provided by applicant): Trypanosoma brucei causes the disease African trypanosomiasis that afflicts an estimated 50,000-70,000 individuals with occasional pandemics resulting in deaths of up to 800,000 individuals. The limited number of drugs for treatment of African trypanosomiasis suffer from major deficiencies, including high toxicity, high cost, and development of drug resistance. Hence, there is a widely recognized urgent need for discovery of novel anti-trypanosomal drugs. Trypanosomes are critically dependent upon uptake and metabolism of glucose in the bloodstream form (BF) that causes disease, and the hexose transporter THT1 that is expressed in BF parasites has been identified and experimentally validated as an essential permease and highly promising drug target in multiple publications. Despite awareness of the promise of targeting THT1, essentially nothing has been done regarding identifying compounds that selectively inhibit this parasite permease. The novelty of this project is that it will carry out a screen for compounds that selectively inhibit THT1 and do not inhibit mammalian hexose transporters. These compounds can ultimately be employed to develop improved anti- trypanosomal drugs. A cell growth assay has been established and fully validated for high-throughput screening (HTS) to identify selective inhibitors of THT1. This assay employs a glucose transporter null mutant of the related parasite Leishmania mexicana that has been complemented with either the gene encoding THT1 or the gene for a major human glucose transporter GLUT1. In Aim 1, ~600,000 drug-like compounds from the St. Jude Children's Research Hospital CBT library will be screened for ability to inhibit growth of the transgenic L. mexicana expressing THT1. Hits from this primary screen will then be secondarily screened to select those that do not inhibit growth of transgenic L. mexicana expressing GLUT1. This two-step screen will identify compounds that are likely selective inhibitors of THT1. In Aim 2, these hit compounds will be tested in uptake assays employing [3H]D-glucose to identify those that are high affinity selective inhibitors of glucose uptake by THT1 but not by GLUT1. Compounds will also be tested for ability to inhibit growth of T. brucei BFs in vitro and in an established mouse model of African trypanosomiasis. Toxicity tests against several human cell lines will be performed to assess the therapeutic index of hit compounds, and metabolic stability will also be determined. This project takes advantage of an established collaboration between the laboratory of the PI Dr. Scott Landfear, a molecular parasitologist who has studied parasite transporters extensively, and that of Dr. Kip Guy, who operates an internationally renowned compound screening and pharmaceutical chemistry facility at St. Jude and who has a strong track record of screening for anti-parasitic compounds.