Selective Inhibitors of the Hexose Transporter from African Trypansomes

描述（由申请人提供）：布鲁氏锥虫引起的疾病是非洲锥虫病，估计有5万至7万人患病，偶尔发生的大流行导致多达80万人死亡。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、高成本和产生耐药性。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，在BF寄生虫中表达的己糖转运体THT1已被确定并实验验证为一种必需的渗透酶和极有希望的药物靶点。尽管人们意识到靶向THT1的前景，但在识别选择性抑制这种寄生虫渗透酶的化合物方面，基本上没有任何进展。这个项目的新颖之处在于，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充分验证了高通量筛选（HTS），以确定THT1的选择性抑制剂。该试验采用了相关寄生虫墨西哥利什曼原虫的葡萄糖转运蛋白零突变体，该突变体已与编码THT1的基因或主要人类葡萄糖转运蛋白GLUT1的基因互补。在Aim 1中，将筛选St. Jude儿童研究医院CBT文库中的约60万种药物样化合物，以抑制表达THT1的转基因L. mexicana的生长能力。然后从这个初级筛选中选择那些不抑制表达GLUT1的转基因L. mexicana生长的片段进行二次筛选。这两步筛选将确定可能是THT1选择性抑制剂的化合物。在目标2中，这些命中的化合物将在使用[3H] d -葡萄糖的摄取试验中进行测试，以确定那些是THT1而不是GLUT1摄取葡萄糖的高亲和力选择性抑制剂。化合物还将在体外和体内测试抑制布鲁氏杆菌BFs生长的能力

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.