Assays for compounds that block or stimulate yeast cell death

描述(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，常见的抗真菌药物激活了一条前yCD调节通路，但其作用被同时激活的一条抗yCD调节通路完全阻断。我们已经确定，活性氧是前yCD途径的关键成分，也是抗yCD途径的几个成分。专门阻断抗yCD途径的一种成分的天然化合物(如环孢素和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，我们建议开发适合于高通量筛选化学文库的yCD分析方法，以揭示调节前yCD因子和抗yCD因子的新化合物。正在开发的分析方法必须区分死亡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，如酿酒酵母和白色念珠菌。最直接的分析将利用一个MLSCN中心可用的高通量流式细胞术，而另外两种间接分析将利用所有MLSCN中心可用的仪器。我们建议开发HTS格式的所有三种检测方法，并使用各种对照菌株和测试条件来评估每种方法的有效性。使用最佳的分析和优化的条件，将进行中试筛选，以评估广泛的化学资料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分析。最后，将开发第三级检测方法，以快速将每种新化学物质的作用点与抗yCD途径中的已知因素相对应。这些研究将极大地影响用于yCD调制器的高通量屏幕的最终设计和实现。这样的调节剂将使对yCD、程序性细胞死亡和真菌寿命控制的研究成为可能，这些真菌在遗传上不容易处理。它们还将为控制真菌感染提供新的机会。

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.