A C.elegans high-throughput assay for the identification of new antifungal agents

描述(由申请人提供)：本提案的主要目标是开发自动化、高通量、基于秀丽线虫的分析方法，可用于筛选化合物并鉴定具有抗真菌活性的化合物。这种完整的动物方法在蠕虫的存活/死亡中提供了一个明确的分析终点，允许使用液体处理机器人来填充分析板，并允许使用平板阅读器和成像显微镜自动读出化合物从库存板到分析板的针状转移。在初步研究中，我们发现哺乳动物中白色念珠菌和新生隐球菌致病的关键成分也参与了线虫的杀灭。我们利用这些观察结果设计出了全动物线虫-C。白色念珠菌和线虫-C。使用96孔板技术进行的新生杆菌检测，对非浮游形式的真菌细胞的研究，以及在病原体和宿主都可以通过基因操作的系统中鉴定抗真菌化合物。对1,266种已知药理活性的化合物进行了中试筛选，确定了15种(~1.2%)能够延长感染白色念珠菌的线虫的存活时间，并抑制体内白色念珠菌的丝状生长。通过这一筛选鉴定的化合物在小鼠身上显示出抗真菌活性。我们最近扩展了这个系统，设计了一种线虫-C。可在液体介质中进行的新生杆菌检测。具体目标如下：目标1.线虫-C的自动化和扩展。白念珠菌检测。目标2.线虫-C的标准化、自动化和扩展。新生杆菌试验。目的3.验证白色念珠菌和新生念珠菌的实验方法：a.确认抗真菌活性，b.建立药物活性的定量读数，c.评估“Hit”化合物对多种真菌的MIC，d.评估化合物对哺乳动物细胞的毒性，即优先选择化合物，以及f.评估选定化合物在线虫免疫反应中的作用。化合物文库的体内评价可以解决目前抗真菌药物发现中的一些主要障碍，如寻找新的化合物类别，解决毒性/有效性测试的瓶颈。除了具有直接抗真菌活性的化合物外，基于线虫的检测可能有助于识别影响毒力因子或免疫调节宿主对真菌反应的进化保存元件的化合物。此外，开发这两种抗真菌试验将使我们能够在未来对白色念珠菌和新生念珠菌筛查中鉴定的化合物进行比较分析，并鉴定具有广泛抗真菌活性的化合物。公共卫生相关性：迫切需要开发新的抗真菌药物，以应对日益增加的真菌感染和抗真菌耐药性的发展。这一应用的主要目标是开发自动化、高通量、全动物秀丽隐杆线虫检测方法，可用于筛选化合物并鉴定具有抗真菌活性的化合物。一种简便的体内评估化合物文库的模型可以解决当前抗真菌发现中的一些主要障碍，如发现新的化合物类别，解决毒性/有效性测试的瓶颈。

DESCRIPTION (provided by applicant): The main objective of this proposal is the development of automated, high-throughput, Caenorhabditis elegans-based assays that can be used to screen chemical compounds and identify those with antifungal activity. This whole animal approach provides an unambiguous assay endpoint in the survival/death of the worms, allows the use of liquid handling robots for filling assay plates and for pin transfer of compounds from library stock plates to assay plates, and permits automated readouts using plate readers and imaging microscopes. In preliminary studies, we found that key components of Candida albicans and Cryptococcus neoformans pathogenesis in mammals are also involved in nematode killing. We used these observations to devise whole-animal C. elegans-C. albicans and C. elegans-C. neoformans assays that are performed using 96-well plate technology and study of fungal cells that are in non-planktonic form and identification of antifungal compounds in a system where both the pathogen and the host can be genetically manipulated. A pilot screen of 1,266 compounds with known pharmaceutical activities identified 15 (~1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Compounds identified through this screen exhibited anti-fungal activity in mice. We recently expanded this system and devised a C. elegans-C. neoformans assay that can be performed in liquid media. The SPECIFIC AIMS are as follows: Aim 1. Automate and expand the C. elegans-C. albicans assay. Aim 2. Standardize, automate and expand the C. elegans-C. neoformans assay. Aim 3. Validate the C. albicans and C. neoformans assays: a. Confirm antifungal activity, b. Develop a quantitative read-out of drug activity, c. Evaluate the MIC of "hit" compounds against a variety of fungi, d. Evaluate the toxicity of compounds against mammalian cells, e. Prioritize compounds, and, f. Evaluate the role of selected compounds in C. elegans immune response. In vivo evaluation of libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery, such as finding new classes of compounds and solving the bottleneck of toxicity/efficacy testing. In addition to the compounds that have direct antifungal activity, the C. elegans- based assays may help identify compounds that affect virulence factors or immuno-modulate evolutionary preserved elements of the host response to fungi. Moreover, developing these two antifungal assays will enable us in the future to perform comparative analyses between compounds identified in C. albicans and C. neoformans screens and to identify compounds that have broad antifungal activity. PUBLIC HEALTH RELEVANCE: There is an urgent need for the development of new antifungal agents to combat the increasing number of fungal infections and the development of antifungal resistance. The main objective of this application is the development of automated, high throughput, whole animal Caenorhabditis elegans-based assays that can be used to screen chemical compounds and identify those with antifungal activity. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery, such as finding new classes of compounds and solving the bottleneck of toxicity/efficacy testing.