Identification and Characterization of Antifungal Compounds Using a Saccharomyces cerevisiae Reporter Bioassay
Identification and Characterization of Antifungal Compounds Using a Saccharomyces cerevisiae Reporter Bioassay
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DOI：
10.1371/journal.pone.0036021
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发表时间：
2012-05-04
期刊：
影响因子：
3.7
通讯作者：
Klein, Bruce S.
中科院分区：
文献类型：
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作者：
Tebbets, Brad;Stewart, Douglas;Klein, Bruce S.
由于目前抗真菌药物的选择有限，耐药性的出现，以及几种常用药物的毒性，迫切需要新的抗真菌药物。为了鉴定药物先导物，我们使用酿酒酵母报告基因生物测定筛选小分子，其中S。酿酒酵母异源表达Hik 1，一种来自稻瘟病菌的III组杂合组氨酸激酶（HHK）。第III组HHK是真菌细胞生理学中不可或缺的，并且在整个王国中高度保守;它们在哺乳动物中不存在，使其成为有吸引力的药物靶标。我们的筛选鉴定了化合物13和33，它们显示出对许多真菌属包括假丝酵母属，隐球菌属和霉菌如烟曲霉和根霉。来自患者的耐药白色念珠菌也对化合物13和33高度敏感。虽然化合物不直接作用于HHK，但微阵列分析显示化合物13诱导与氧化应激相关的转录物，化合物33诱导与重金属应激相关的转录物。这两种化合物对C.白念珠菌生物膜，在体外和体内，并发挥协同作用，与氟康唑，这是无活性的单独。因此，我们使用高通量S。酿酒酵母报告基因生物测定。
New antifungal drugs are urgently needed due to the currently limited selection, the emergence of drug resistance, and the toxicity of several commonly used drugs. To identify drug leads, we screened small molecules using a Saccharomyces cerevisiae reporter bioassay in which S. cerevisiae heterologously expresses Hik1, a group III hybrid histidine kinase (HHK) from Magnaporthe grisea. Group III HHKs are integral in fungal cell physiology, and highly conserved throughout this kingdom; they are absent in mammals, making them an attractive drug target. Our screen identified compounds 13 and 33, which showed robust activity against numerous fungal genera including Candida spp., Cryptococcus spp. and molds such as Aspergillus fumigatus and Rhizopus oryzae. Drug-resistant Candida albicans from patients were also highly susceptible to compounds 13 and 33. While the compounds do not act directly on HHKs, microarray analysis showed that compound 13 induced transcripts associated with oxidative stress, and compound 33, transcripts linked with heavy metal stress. Both compounds were highly active against C. albicans biofilm, in vitro and in vivo, and exerted synergy with fluconazole, which was inactive alone. Thus, we identified potent, broad-spectrum antifungal drug leads from a small molecule screen using a high-throughput, S. cerevisiae reporter bioassay.