Novel whole-animal screens for anti-microbials

描述（由申请方提供）：拟议项目的目标是在96孔板中开发秀丽隐杆线虫全动物高通量筛选试验，以鉴定新型抗菌或免疫增强化合物。以前的研究表明，许多人类病原体感染并杀死线虫C。优雅提出的总体策略是确定治疗C的天然或合成化合物。一种持续性细菌感染。我们预计，这项工作将建立解决当前抗菌药物发现中一些主要障碍的方法-寻找新的化合物类别，解决毒性/功效测试的瓶颈，并将药物递送到目标病原体。初步结果表明，C. elegans致病性模型可用于测定具有抗微生物活性的化合物。开发了用于筛选抗感染或免疫增强化合物的原型测定法，其利用革兰氏阳性人类机会性病原体粪肠球菌在C.线虫肠该测定用于筛选6000种小分子和1224种天然产物提取物，从而鉴定出在测定中具有活性的16种化合物和9种提取物。此外，MDR泵抑制剂显示与抗菌剂在C. elegans固化试验。初步数据表明，线虫模型将产生在常规体外筛选中被忽略的命中，并且线虫是体外筛选和哺乳动物测试之间的有用中间体。提出的屏幕开发项目的一个主要目标是增加C的吞吐量。elegans-E.粪便全动物筛选试验，以允许筛选数十万或数百万的化合物和提取物。梭elegans筛选方法也将扩展到包括革兰氏阴性病原体肠道沙门氏菌，并适用于鉴定多药耐药（MDR）泵抑制剂。具体目标有三：1）开发高通量的大肠杆菌;粪便治愈测定部分地通过自动化蠕虫活力的评分来实现。2)开发高通量C. elegans - S.肠固化试验3)建立一种高通量筛选方法，用于鉴定C. elegans固化测定。

DESCRIPTION (provided by applicant): The goal of the proposed project is to develop Caenorhabditis elegans whole-animal high-throughput screening assays in 96-well plates that lead to the identification of novel classes of antimicrobial or immune- enhancing compounds. Previous work has shown that many human pathogens infect and kill the nematode C. elegans. The proposed overall strategy is to identify natural or synthetic compounds that cure C. elegans of a persistent bacterial infection. We anticipate that this work will establish approaches that solve some of the main obstacles in current antimicrobial discovery - finding new classes of compounds, solving the bottleneck of toxicity/efficacy testing, and delivering the drug to the target pathogen. Preliminary results demonstrate that the C. elegans pathogenicity model can be used to assay compounds with antimicrobial activity. A prototype assay for screening for anti-infective or immune enhancing compounds was developed that takes advantage of the fact that the Gram positive human opportunistic pathogen, Enterococcus faecalis, forms a persistent infection in the C. elegans intestine. The assay was used to screen 6000 small molecules and 1224 natural product extracts, leading to the identification of 16 compounds and 9 extracts that were active in the assay. In addition MDR pump inhibitors were shown to act in synergy with antimicrobials in the C. elegans curing assay. The preliminary data indicate that a nematode model will produce hits that are overlooked in a conventional in vitro screen and that the nematode is a useful intermediate between an in vitro screen and testing in mammals. A major goal of the proposed screen development project is to increase the throughput of the C. elegans-E. faecalis whole-animal screening assay to allow screening of hundreds of thousands or millions of compounds and extracts. The C. elegans screening methodology will also be extended to include the Gram negative pathogen Salmonella enterica and adapted to enable the identification of multi-drug resistant (MDR) pump inhibitors. There are three specific aims: 1) Develop a high throughput E. faecalis curing assay in part by automating the scoring of worm viability. 2) Develop a high throughput C. elegans - S. enterica curing assay. 3) Develop a high throughput screen for identifying MDR pump inhibitors in the C. elegans curing assays.