High-throughput screening for new inhibitors of Giardia lamblia

描述（由申请人提供）：甲硝唑通常用于治疗贾第虫病，一种由原生动物寄生虫贾第虫引起的肠道感染。临床对该药的耐药和复发性感染的报告强调了替代治疗的必要性。由于兰氏梭菌滋养体在肠道中的增殖与贾第虫病的症状密切相关，我们开发了一种小型化的增殖试验，适用于高通量筛选抑制滋养体增殖的化合物。对约1500种生物活性化合物的初步筛选发现了50种显著抑制或阻止生长的小分子。这些化合物中的许多都不知道能抑制G. lamblia。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。该筛选将与两种G. lamblia组合（基因型）同时进行，以确定抑制两种组合的化合物，以及不同地干扰组合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，从而确定新的G. lamblia药物通路。双重筛选将使鉴定不同的影响兰氏螺旋体组合感染人类的化合物成为可能。基于这些化合物的作用模式，我们将能够在组合之间表型差异的分子基础上提出假设。该提案是为了响应R21发展/探索性拨款招标而提交的，该招标旨在支持处于早期阶段的基于新型实验系统的项目。具体来说，我们将使用滋养体增殖试验在384孔板中筛选含有G. lamblia和B分离物的小分子文库。在高通量筛选中鉴定的抑制剂将用额外的装配A和装配B分离物进行验证。显微镜和流式细胞术将用于研究选定的、确认的抑制剂的靶标。公共卫生相关性：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集合，以确定未来开发针对这种寄生虫的药物的新线索。

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.