Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois, United States of America.
PLoS One. 2011;6(9):e25136. doi: 10.1371/journal.pone.0025136. Epub 2011 Sep 29.
Resistance to traditional antifungal drugs has increased significantly over the past three decades, making identification of novel antifungal agents and new targets an emerging priority. Based on the extraordinary zinc requirement of several fungal pathogens and their well-established sensitivity to zinc deprivation, we developed an efficient cell-based screen to identify new antifungal drugs that target the zinc homeostasis machinery. The screen is based on the zinc-regulated transcription factor Zap1 of Saccharomyces cerevisiae, which regulates transcription of genes like the high-affinity zinc transporter ZRT1. We generated a genetically modified strain of S. cerevisae that reports intracellular zinc deficiency by placing the coding sequence of green fluorescent protein (GFP) under the control of the Zap1-regulated ZRT1 promoter. After showing that the GFP fluorescence signal correlates with low intracellular zinc concentrations in this strain, a protocol was developed for screening small-molecule libraries for compounds that induce Zap1-dependent GFP expression. Comparison of control compounds and known modulators of metal metabolism from the library reveals a robust screen (Z' = 0.74) and validates this approach to the discovery of new classes of antifungal compounds that interfere with the intracellular zinc homeostasis. Given that growth of many pathogenic organisms is significantly impaired by zinc limitation; these results identify new types of antifungal drugs that target critical nutrient acquisition pathways.
过去三十年来，真菌对传统抗真菌药物的耐药性显著增加，因此确定新型抗真菌药物和新靶标成为当务之急。基于几种真菌病原体对锌的特殊需求以及它们对锌剥夺的固有敏感性，我们开发了一种高效的基于细胞的筛选方法，旨在鉴定靶向锌稳态机制的新型抗真菌药物。该筛选基于酿酒酵母的锌调控转录因子 Zap1，它调节高亲和力锌转运蛋白 ZRT1 等基因的转录。我们通过将绿色荧光蛋白 (GFP) 的编码序列置于 Zap1 调控的 ZRT1 启动子的控制下，生成了一种报告细胞内锌缺乏的酿酒酵母遗传修饰株。在证明该菌株中的 GFP 荧光信号与低细胞内锌浓度相关后，开发了一种用于筛选小分子文库中诱导 Zap1 依赖性 GFP 表达的化合物的方案。比较文库中的对照化合物和已知的金属代谢调节剂，揭示了一个强大的筛选（Z'=0.74），并验证了这种方法可以发现干扰细胞内锌稳态的新型抗真菌化合物。鉴于许多致病生物的生长受到锌限制的显著影响，这些结果确定了靶向关键营养获取途径的新型抗真菌药物。
