High-throughput screening for new inhibitors of Giardia lamblia

说明(申请人提供)：甲硝唑通常用于治疗贾第鞭毛虫病，这是一种由原虫寄生虫蓝氏贾第鞭毛虫引起的肠道感染。临床对该药物的耐药性和复发感染的报告强调了替代疗法的必要性。由于肠道中蓝氏菌滋养体的增殖与贾第鞭毛虫病的症状密切相关，我们开发了一种适合于高通量筛选抑制滋养体增殖的化合物的微型增殖试验。对大约1500种生物活性化合物进行的中试筛选发现了50种小分子，它们显著抑制或阻止了生长。这些化合物中有许多是未知的抑制蓝氏菌的。我们建议使用这种简单、高通量的方法来筛选大量的化合物。这一筛选将与两个兰氏革兰氏菌组合(基因类型)同时进行，以鉴定抑制这两个组合的化合物，以及不同干扰组合A或B组合增殖的化合物。在二次筛选中确认的抑制剂将根据其分子靶标进行分类，从而识别兰氏革兰氏菌中新的可用药途径。双重筛选将能够识别对感染人类的革兰氏杆菌组合有不同影响的化合物。基于这些化合物的作用方式，我们将能够在分子基础上提出组装之间的表型差异的假设。本提案是对R21开发/探索性赠款征集的回应，该征集旨在支持处于早期阶段并以新的实验系统为基础的项目。具体地说，我们将使用滋养体增殖试验，在384孔板中筛选具有集合体A和集合体B的蓝氏菌小分子文库。在高通量筛选中确定的抑制剂将与其他组合A和组合B分离株进行验证。显微镜和流式细胞术将用于研究选定的、已确认的抑制剂的靶标。公共卫生相关性：蓝氏贾第鞭毛虫是肠道感染的常见原因。我们建议筛选大量的小分子，以确定未来针对这种寄生虫的药物开发的新线索。

DESCRIPTION (provided by applicant): Metronidazole is commonly used for the treatment of giardiasis, an intestinal infection caused by the protozoan parasite Giardia lamblia. Reports of clinical resistance to this drug and recurrent infections underscore the need for alternative therapeutics. Since G. lamblia trophozoite proliferation in the intestine is intimately associated with the symptoms of giardiasis, we developed a miniaturized proliferation assay amenable to high-throughput screening for compounds which inhibit trophozoite proliferation. A pilot screen of ~1500 bioactive compounds uncovered 50 small molecules which significantly depressed or arrested growth. Many of these compounds were not known to inhibit G. lamblia. We propose to use this simple, high-throughput assay to screen a large collection of compounds. This screen will be conducted simultaneously with two G. lamblia assemblages (genotypes) to identify compounds which inhibit both assemblages, and compounds which differentially perturb proliferation of assemblage A or B. Inhibitors confirmed in a secondary screen will be categorized with respect to their molecular target, thus identifying new druggable pathways in G. lamblia. The dual screening will enable the identification of compounds which differentially affect G. lamblia assemblages infecting humans. Based on the mode of action of such compounds, we will be able to formulate hypotheses on the molecular basis of phenotypic differences between assemblage. This proposal is submitted in response to the R21 Developmental/Exploratory grant solicitation which is intended to support projects which are in their early phase and are based on novel experimental systems. Specifically, we will use a trophozoite proliferation assay to screen, in 384-well plates, small molecule libraries with an assemblage A and an assemblage B isolate of G. lamblia. Inhibitors identified in the high-throughput screen will be verified with additional assemblage A and assemblage B isolates. Microscopy and flow cytometry will be used to investigate the target of selected, confirmed inhibitors. PUBLIC HEALTH RELEVANCE: Giardia lamblia is a common cause of intestinal infections. We propose to screen large collections of small molecules to identify new leads for future development of drugs against this parasite.