Assays for compounds that block or stimulate yeast cell death

阻止或刺激酵母细胞死亡的化合物的测定

基本信息

批准号：
7169693
负责人：
KYLE W CUNNINGHAM
金额：
$ 16.25万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-05 至 2007-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7169693
关键词：
NIH Roadmap Initiative tag cell death chemical registry /resource drug discovery /isolation flow cytometry free radical oxygen fungal genetics high throughput technology inhibitor /antagonist mycosis stimulant /agonist technology /technique development yeasts

项目摘要

DESCRIPTION (provided by applicant): Yeast Cell Death (YCD) is a phenomenon in fungi that is possibly related to programmed cell death in animals. Recent studies show that common antifungal drugs activate a pro-YCD regulatory pathway but its effects are totally blocked by the simultaneous activation of an anti-YCD regulatory pathway. We have identified reactive oxygen species as a key component of the pro-YCD pathway and several components of the anti-YCD pathway. Natural compounds that specifically block one component of the anti-YCD pathway (such as Cyclosporin and FK506) greatly increase the potency of existing antifungal drugs by unmasking their fungicidal activity. Here we propose to develop assays of YCD suitable for high-through screening of chemical libraries to reveal new compounds that modulate pro- and anti-YCD factors. The assays under development must differentiate between dead cells and live non-proliferating cells and they must be applicable to diverse species of yeasts such as Saccharomyces cerevisiae and Candida albicans. The most direct assay will utilize high-throughput flow cytometry available at one MLSCN center whereas two other indirect assays will utilize instrumentation available at all MLSCN centers. We propose to develop all three assays for HTS format and evaluate the effectiveness of each using a variety of control strains and test conditions. Using the best assay and optimized conditions, a pilot screen will be performed to assess the frequencies of false and true positives in a broad chemical library. Secondary assays to rapidly identify and eliminate false positives will be developed for high-throughput application, if necessary. Finally, tertiary assays will be developed to rapidly map the point of action of each new chemical to the known factors in the anti-YCD pathway. These studies will greatly impact the final design and implementation of high-throughput screens for modulators of YCD. Such modulators will enable studies of YCD, programmed cell death, and lifespan control in fungi that are not genetically tractable. They will also provide new opportunities for control of fungal infections.

描述（由申请人提供）：酵母细胞死亡（YCD）是真菌中的一种现象，可能与动物的程序性细胞死亡有关。最近的研究表明，常见的抗真菌药物会激活前 YCD 调节途径，但其作用会被同时激活的抗 YCD 调节途径完全阻断。我们已经确定活性氧是 pro-YCD 途径的关键成分和抗 YCD 途径的几个成分。特异性阻断抗 YCD 途径之一成分的天然化合物（例如环孢菌素和 FK506）通过揭示其杀菌活性，极大地提高了现有抗真菌药物的效力。在这里，我们建议开发适用于化学库高通量筛选的 YCD 检测方法，以揭示调节亲 YCD 因子和抗 YCD 因子的新化合物。正在开发的检测方法必须区分死细胞和活的非增殖细胞，并且它们必须适用于不同种类的酵母，例如酿酒酵母和白色念珠菌。最直接的测定将利用一个 MLSCN 中心提供的高通量流式细胞术，而其他两种间接测定将利用所有 MLSCN 中心提供的仪器。我们建议开发 HTS 格式的所有三种测定法，并使用各种对照菌株和测试条件评估每种测定法的有效性。使用最佳测定和优化条件，将进行试点筛选，以评估广泛化学库中假阳性和真阳性的频率。如有必要，将为高通量应用开发快速识别和消除假阳性的二次测定方法。最后，将开发三级测定法，以快速将每种新化学物质的作用点映射到抗 YCD 途径中的已知因子。这些研究将极大地影响 YCD 调制器高通量筛选的最终设计和实现。此类调节剂将使研究 YCD、程序性细胞死亡和非遗传可控真菌的寿命控制成为可能。它们还将为控制真菌感染提供新的机会。