Selective Inhibitors of the Hexose Transporter from African Trypansomes

描述(申请人提供)：布氏锥虫引起非洲锥虫病，估计有50,000-70,000人患有间歇性大流行，导致多达800,000人死亡。治疗非洲锥虫病的药物数量有限，存在着主要不足，包括高毒性、高成本和产生抗药性。因此，人们普遍认为迫切需要发现新的抗锥虫药物。锥虫体严重依赖于血液中葡萄糖的摄取和代谢而致病，而在血液中表达的己糖转运蛋白THT1已被鉴定和实验证实为一种重要的通透酶和极具前景的药物靶标。尽管人们意识到靶向THT1的前景，但基本上没有采取任何行动来识别选择性抑制这种寄生虫渗透酶的化合物。该项目的新奇之处在于，它将对选择性抑制THT1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。细胞生长试验已经建立，并完全验证了高通量筛选(HTS)，以确定THT1的选择性抑制剂。这种检测方法使用了相关寄生虫墨西哥利什曼原虫的葡萄糖转运蛋白缺失突变体，该突变体已与编码THT1的基因或人类主要葡萄糖转运蛋白GLUT1的基因互补。在目标1中，将从圣犹大儿童研究医院CBT文库中筛选出约60万种类药物化合物，以检测其对表达THT1的转基因墨西哥乳杆菌的生长抑制能力。然后，将对来自该初级筛选的命中进行二次筛选，以选择不抑制表达GLUT1的转基因墨西哥乳杆菌生长的那些。这两个步骤的筛选将识别可能是THT1选择性抑制剂的化合物。在目标2中，这些HIT化合物将在使用[~3H]D-葡萄糖的摄取试验中进行测试，以确定那些高亲和力选择性抑制THT1而不是GLUT1摄取葡萄糖的化合物。此外，还将测试化合物在体外和体外抑制布氏毛滴虫生长的能力。 已建立的非洲锥虫病小鼠模型。将对几种人类细胞株进行毒性测试，以评估HIT化合物的治疗指数，并确定代谢稳定性。该项目利用了PI的Scott LandFear博士和Kip Guy博士的实验室之间建立的合作，Scott LandFear博士是一名分子寄生虫学家，对寄生虫转运体进行了广泛的研究，Kip Guy博士在圣裘德经营着一家国际知名的化合物筛选和药物化学设施，他在筛选抗寄生虫化合物方面有着良好的记录。

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.