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

用于鉴定新型抗真菌药物的线虫高通量测定

• 批准号: 7473741
• 负责人: ELEFTHERIOS MYLONAKIS
• 金额: $ 43.94万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7473741
• 关键词: Affect; Animals; Antifungal Agents; Automation; Biological Assay; Caenorhabditis elegans; Candida; Candida albicans; Candidiasis; Cell Membrane Permeability; Cells; Cessation of life; Characteristics; Chemicals; Class; Condition; Cryptococcus neoformans; Cytochrome P450; Defecation; Detection; Development; Disease; Dose; Elements; End Point Assay; Enoxacin; Enzyme Stability; Evaluation; Exhibits; Figs - dietary; Filament; Fluoroquinolones; Fungal Drug Resistance; Future; Generations; Genes; Image; Immune response; In Vitro; Infection; Intestines; Laccase; Lead; Libraries; Liquid substance; London; Mammalian Cell; Mammals; Microbial Biofilms; Microscope; Modeling; Molecular; Molecular Weight; Mus; Mycoses; Nematoda; Numbers; Pathogenesis; Pharmaceutical Preparations; Pharmacologic Substance; Play; Production; Propolis; PubMed; Public Health; Reader; Reading; Robot; Role; Signal Transduction Pathway; Standards of Weights and Measures; System; Technology; Testing; Toxic effect; Virulence; Virulence Factors; base; caffeic acid phenethyl ester; comparative; cost; fungus; high throughput screening; in vivo; in vivo Model; killings; mutant; pathogen; reproductive; response; size; small molecule libraries; tool; trait

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.

描述（由申请方提供）：本提案的主要目的是开发自动化、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。这种整体动物方法在蠕虫的存活/死亡方面提供了明确的测定终点，允许使用液体处理机器人来填充测定板和将化合物从文库储备板销转移到测定板，并允许使用读板器和成像显微镜进行自动读数。在前期研究中，我们发现哺乳动物中白色念珠菌和新型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了整体动物C。秀丽隐杆藻白色念珠菌和C.秀丽隐杆藻使用96孔板技术进行的新形式真菌测定和对非嗜热形式的真菌细胞的研究以及在病原体和宿主都可以被遗传操纵的系统中鉴定抗真菌化合物。对1，266种具有已知药物活性的化合物进行了初步筛选，确定了15种（约1.2%）可延长C.白念珠菌感染线虫，抑制白念珠菌的体内增殖。白色念珠菌。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，设计了一个C。秀丽隐杆藻可在液体培养基中进行的新生儿试验。具体目标如下：目标1。自动化和扩展C.秀丽隐杆藻白色念珠菌测定目标二。标准化，自动化和扩展C。秀丽隐杆藻neoformans测定。目标3。C.白色念珠菌和C.新生儿测定：a.确认抗真菌活性，B。开发药物活性的定量读数，c.评估“命中”化合物对多种真菌的MIC，d.评价化合物对哺乳动物细胞的毒性，优先考虑化合物，和，f。评价所选化合物在C. elegans免疫反应。化合物库的体内评价可以解决当前抗真菌发现中的一些主要障碍，例如发现新的化合物类别和解决毒性/功效测试的瓶颈。除了具有直接抗真菌活性的化合物外，C.基于线虫的分析可能有助于鉴定影响毒力因子或免疫调节宿主对真菌反应的进化保留元件的化合物。此外，开发这两种抗真菌试验将使我们能够在未来对C.白色念珠菌和C.筛选新型真菌并鉴定具有广泛抗真菌活性的化合物。公共卫生关系：迫切需要开发新的抗真菌剂以对抗日益增加的真菌感染和抗真菌耐药性的发展。本申请的主要目的是开发基于秀丽隐杆线虫的自动化、高通量、全动物检测方法，可用于筛选化合物并鉴定具有抗真菌活性的化合物。一种评估化合物库的简单体内模型可以解决当前抗真菌发现中的一些主要障碍，例如寻找新的化合物类别和解决毒性/功效测试的瓶颈。