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

描述（由申请人提供）：本申请的主要目标是开发自动化，高通量，基于秀丽隐杆线虫的检测方法，可用于筛选化合物并鉴定具有抗真菌活性的化合物。这种全动物方法为蠕虫的生存/死亡提供了明确的分析终点，允许使用液体处理机器人填充分析板，并将化合物从库库板转移到分析板，并允许使用板读取器和成像显微镜自动读取数据。在初步研究中，我们发现白色念珠菌和新型隐球菌在哺乳动物中致病的关键成分也参与了线虫的杀伤。我们利用这些观察结果设计了全动物秀丽隐杆线虫。白念珠菌和秀丽隐杆线虫；使用96孔板技术进行新形态分析，研究非浮游形式的真菌细胞，并在病原体和宿主都可以基因操纵的系统中鉴定抗真菌化合物。在对1,266种已知药物活性化合物的初步筛选中，发现15种（约1.2%）延长了感染白色念珠菌的线虫的存活时间，并抑制了白色念珠菌的体内丝化。通过该筛选鉴定的化合物在小鼠中表现出抗真菌活性。我们最近扩展了这个系统，设计了C. elegans-C。可在液体介质中进行的Neoformans试验。具体目标如下：目标1。自动化和扩展C. elegans-C。白色的测定。目标2。标准化、自动化和扩展C. elegans。neoformans化验。目标3。验证白色念珠菌和新生念珠菌的检测：a.确认抗真菌活性，b.开发药物活性的定量读出，c.评估“命中”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，e.对化合物进行优先排序，f.评估选定化合物在秀丽隐杆线虫免疫应答中的作用。化合物文库的体内评价可以解决当前抗真菌发现的一些主要障碍，如发现新的化合物类别和解决毒性/药效测试的瓶颈。除了具有直接抗真菌活性的化合物外，基于秀丽隐杆线虫的检测可能有助于识别影响毒力因子的化合物或宿主对真菌反应的免疫调节进化保存元素。此外，开发这两种抗真菌检测方法将使我们将来能够在白色念珠菌和新生念珠菌筛选中鉴定的化合物之间进行比较分析，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来对抗越来越多的真菌感染和抗真菌耐药性的发展。该应用程序的主要目的是开发自动化，高通量，基于全动物秀丽隐杆线虫的检测方法，可用于筛选化合物并鉴定具有抗真菌活性的化合物。一个简单的体内模型可以评估化合物的文库，从而解决当前抗真菌发现的一些主要障碍，如寻找新的化合物类别和解决毒性/功效测试的瓶颈。

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.