High-throughput screening for new inhibitors of Giardia lamblia

描述（由申请方提供）：甲硝唑通常用于治疗贾第虫病，这是一种由原生动物寄生虫贾第虫引起的肠道感染。对这种药物的临床耐药性和复发性感染的报告强调了对替代疗法的需求。自G.肠内的兰氏贾第虫滋养体增殖与贾第虫病的症状密切相关，我们开发了一种小型化增殖测定法，该测定法适于高通量筛选抑制滋养体增殖的化合物。对约1500种生物活性化合物进行了初步筛选，发现了50种显著抑制或抑制生长的小分子。这些化合物中有许多是未知的抑制G。兰布利亚我们建议使用这种简单的，高通量的检测来筛选大量的化合物。该筛选将与两个G同时进行。Lamblia集合（基因型）以鉴定抑制这两种集合的化合物，以及差异地干扰集合A或B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点进行分类，从而确定G.兰布利亚双重筛选将能够鉴定差异影响G.感染人类的兰伯氏菌基于这种化合物的作用模式，我们将能够在组合之间表型差异的分子基础上制定假设。该提案是为了响应R21开发/探索资助申请而提交的，该申请旨在支持处于早期阶段并基于新型实验系统的项目。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G的集合体A和集合体B分离物的小分子文库。兰布利亚高通量筛选中鉴定的抑制剂将用额外的集合A和集合B分离株进行验证。将使用显微镜检查和流式细胞术研究选定的、确认的抑制剂的靶点。公共卫生相关性：贾第虫是肠道感染的常见原因。我们建议筛选大量的小分子，以确定未来开发抗这种寄生虫药物的新线索。

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.