Cellular Basis for the Antifungal Activity of Amiodarone

描述（由申请人提供）：本提案的目的是阐明一种新型抗真菌药物引起的程序性细胞死亡途径，并探索其作为抗真菌辅助药物的用途。胺碘酮是一种有效的抗真菌药物，最近发现具有有效和广泛的杀真菌活性。我们已经表明，胺碘酮在酵母酿酒酵母中的毒性是通过破坏钙稳态介导的，随后出现凋亡标志物和细胞死亡。在目的1中，我们建议使用生物化学和细胞生物学方法的组合，以确定身份和时间顺序的事件导致从细胞溶质钙的初始爆发细胞死亡。我们将寻求验证致病性酵母菌白色念珠菌中的关键发现，作为胺碘酮杀真菌机制普遍性的原理证明。在目标2中，我们将使用各种全基因组方法来鉴定有助于胺碘酮诱导细胞死亡的基因和信号通路。通过这些研究确定的基因将被组织成生物模块，并通过整合来自高通量生化测定的实验数据与来自在线数据库的信息来放置在细胞通路中，以提供药物毒性的全局视图。我们已经证明，低剂量的胺碘酮与现有的抗真菌药对念珠菌、隐球菌和曲霉菌的致病性真菌种表现出有效的协同作用。来自这些体外研究的药物协同作用将形成将在系统性疟原虫病的鼠模型中探索的联合治疗的基础（目的3）。总之，这些研究将开发钙介导的细胞死亡途径作为一个主要的新药物发现的目标机会。胺碘酮将作为靶向该途径的模型试验化合物，并用于验证一类新的抗真菌增效剂的潜力。 该项目的公共卫生相关性源于新的真菌病原体和耐药真菌的出现，以及对真菌感染管理替代策略的迫切需求。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the pathway of programmed cell death by a novel antifungal agent and explore its use as an antimycotic adjunct. Amiodarone is an effective antiarrhythmic drug that was recently discovered to have potent and broad range fungicidal activity. We have shown that amiodarone toxicity in the yeast Saccharomyces cerevisiae is mediated by disruption of calcium homeostasis, followed by the appearance of apoptosis markers and cell death. In Aim 1, we propose to use a combination of biochemical and cell biological approaches to determine the identity and temporal order of events leading from the initial burst of cytosolic calcium to cell death. We will seek to validate key findings in the pathogenic yeast Candida albicans as proof-of-principle for the universality of the fungicidal mechanism of amiodarone. In Aim 2, we will use a variety of genome-wide approaches to identify the genes and signaling pathways that contribute to amiodarone-induced cell death. Genes identified by these studies will be organized into biomodules and placed in cellular pathways by integrating experimental data from high-throughput biochemical assays with information from online databases to give a global view of drug toxicity. We have shown that low doses of amiodarone exhibit potent synergism with existing antifungals against pathogenic fungal species of Candida, Cryptococcus and Aspergillus. Drug synergy from these in vitro studies will form the basis for combination therapy that will be explored in a murine model of systemic Candidiasis (Aim 3). Taken together these studies will develop the calcium-mediated cell death pathway as a major new drug discovery target opportunity. Amiodarone will serve as a model test compound for targeting this pathway and for validating the potential of a new class of antifungal potentiating agents. The public health relevance of this project arises from the emergence of new fungal pathogens and drug resistant fungi, and the urgent need for alternative strategies in the management of fungal infections.