Compound library screening using Giardia labmblia high throughput viability assay

描述(申请人提供)：蓝氏贾第鞭毛虫是一种困扰贫穷国家的人类病原体，也是美国爆发腹泻的最常见原因。贾第鞭毛虫已被疾控中心列为B类生物恐怖主义生物。在美国，最受欢迎的抗贾第鞭毛虫病药物是甲硝唑或替硝唑，这是一种有不良副作用的化合物。此外，对药物制度的抗药性增加和复发是一个令人关切的问题。因此，很明显，需要替代药物治疗。我们已经建立了一种基于滋养体ATP含量的生物发光Giarida活性检测方法，适用于高通量筛选，并将其用于1536孔板上化合物筛选的小型化。我们建议筛选NIH化合物文库，并研究最初使用CHO细胞和后来的CACO-2细胞对HIT化合物的选择性。具有低IC50的选择性化合物的最低致死浓度将通过再生长实验来确定。对于几个热门目标，将进行贾第虫目标的识别，如果成功，将推进基于结构的化合物优化。 公共卫生意义：蓝氏贾第鞭毛虫是一种人类病原体，每年困扰数十亿人，是一种B类生物恐怖主义生物。贾第鞭毛虫病的治疗有不良的副作用、复发和增加耐药性。为了便于发现更好的药物，我们开发了一种贾第虫活性测定方法，并将其微型化，用于高通量化合物筛选。这项试验将用于筛选NIH化合物文库，并鉴定杀死贾第虫滋养体的化合物。

DESCRIPTION (provided by applicant): Giardia lamblia is a human pathogen afflicting impoverished nations, and the most common cause of outbreaks of diarrhea in the United States. Giardia has been classified by the CDC as a category B bioterrorism organism. The favored anti giardiasis drugs in the US are metronidazole or tinidazole, compounds that have undesirable side effects. Moreover, increasing resistance to drug regimes and recurrence are a concern. It is thus clear that alternative drug treatments are needed. We have developed a bioluminescence Giarida viability assay based on the trophozoites ATP content that is suitable for high throughput screening and miniaturized the assay for compound screening in 1536-well plates. We propose to screen the NIH compound library and investigate the selectivity of hit compounds using initially CHO cells and later CACO-2 cells. The minimum lethal concentration of selective compounds exhibiting low IC50 will be determined by re- growth experiments. For a few top hits, identification of the Giardia target will be undertaken and if successful, advanced to structure-based compound optimization. PUBLIC HEALTH RELEVANCE: Giardia lamblia is a human pathogen afflicting billions of people annually and a category B bioterrorism organism. Giardiasis treatments have undesirable side effects, recurrence, and increasing drug resistance. To facilitate discovery of better drugs, we have developed a Giardia viability assay and miniaturized it for high throughput compound screening. This assay will be used to screen the NIH compound library and identify compounds that kill Giardia trophozoites.