Development of Assay for High Throughput Screen of Parasite Glucose Transporter

描述（由申请人提供）：寄生虫病，包括疟疾、锥虫病和利什曼病，在全世界造成巨大的疾病负担，但迫切需要的新药的开发进展缓慢。寄生虫葡萄糖转运蛋白对于疟疾、非洲锥虫和利什曼原虫的感染阶段是必不可少的，并且是用于药物开发的遗传或生物学验证的靶标。在本申请中，将开发一种用于寄生虫葡萄糖转运蛋白抑制剂的高通量形式的测定方法，该方法可能代表开发新型抗寄生虫药物的线索。该测定利用墨西哥利什曼原虫的葡萄糖转运蛋白无效突变体，其在适当的培养基中依赖于异源葡萄糖转运蛋白转基因的功能表达而生长。该无效突变体将用编码恶性疟原虫己糖转运蛋白PfHT的转基因进行补充，并使用alamarBlue荧光试验和SYBR绿色I荧光试验测定转基因寄生虫的生长。PfHT的抑制剂将抑制PfHT转基因利什曼原虫寄生虫的生长。将采用已建立的统计学标准，针对基于高通量微量滴定板的形式对该测定进行优化，并随后将其用于中等大小化学文库的自动化筛选，以确定其用于较大化学文库的最终高通量筛选的实用性。将进一步表征从该中等通量筛选中鉴定的任何抑制剂，以确定它们是否是PfHT而不是哺乳动物葡萄糖转运蛋白的特异性抑制剂，以及它们是否杀死培养物中的疟疾寄生虫但对哺乳动物细胞显示低毒性。该项目将开发一种高通量筛选方法，可用于未来的研究，以筛选来自疟疾，非洲锥虫和利什曼原虫的葡萄糖转运蛋白的高亲和力，高特异性抑制剂的大型文库。长期的承诺是，有可能鉴定出先导化合物，用于开发对促进全球公共卫生具有巨大潜在效用的新型抗寄生虫药物。公共卫生相关性。寄生虫疟疾、利什曼原虫和非洲锥虫在全球范围内造成巨大的疾病负担，迫切需要鉴定新药。本申请的目的是筛选抑制这些寄生虫摄取糖葡萄糖的化合物。由于葡萄糖是这些寄生虫的必需营养素，这些化合物可以杀死寄生虫，并可以作为治疗它们引起的疾病的新药。

DESCRIPTION (provided by applicant): Parasitic diseases, including malaria, trypanosomiasis, and leishmaniasis, produce an enormous disease burden throughout the world, yet development of urgently needed new drugs has proceeded at a slow pace. Parasite glucose transporters are essential for the infectious stages of malaria, African trypanosomes, and Leishmania and are genetically or pharmacologically validated targets for development of drugs. In this application, an assay will be developed for high throughput format for inhibitors of parasite glucose transporters that could represent leads for development of novel anti-parasitic drugs. This assay utilizes a glucose transporter null mutant of Leishmania mexicana that is dependent for growth, in the appropriate medium, upon functional expression of a heterologous glucose transporter transgene. This null mutant will be complemented with a transgene encoding the Plasmodium falciparum hexose transporter, PfHT, and the transgenic parasites will be assayed for growth using the alamarBlue fluorescence assay and the SYBR Green I fluorescence assay. Inhibitors of PfHT will inhibit growth of the PfHT transgenic Leishmania parasites. The assay will be optimized for a high throughput microtiter plate based format employing established statistical criteria and will subsequently be employed in an automated screen of a modest size chemical library to establish its utility for eventual high throughput screening of larger chemical libraries. Any inhibitors identified from this medium throughput screen will be further characterized to determine whether they are specific inhibitors of PfHT but not mammalian glucose transporters and whether they kill malaria parasites in culture yet display low toxicity for mammalian cells. This project will develop a high throughput screening method that can be employed in future studies to screen large libraries for high affinity, high specificity inhibitors of glucose transporters from malaria, African trypanosomes, and Leishmania parasites. The long-term promise is to make possible the identification of lead compounds for development of novel anti-parasitic drugs of great potential utility for promoting global public health. Public Health Relevance. The parasites malaria, Leishmania, and the African trypanosomes cause an enormous disease burden on a global scale, and the identification of new drugs is urgently needed. The objective of this application is to develop a screen for compounds that inhibit the uptake of the sugar glucose by these parasites. Since glucose is an essential nutrient for these parasites, such compounds would kill the parasite and could act as novel drugs for treatment of the diseases they cause.