Novel Antifungal Drug Discovery Based on Targeting Pathways Regulating the Fungus-Conserved Upc2 Transcription Factor
Novel Antifungal Drug Discovery Based on Targeting Pathways Regulating the Fungus-Conserved Upc2 Transcription Factor
复制标题
DOI：
10.1128/aac.01677-13
复制
发表时间：
2014-01-01
影响因子：
4.9
通讯作者：
Nickels, Joseph T., Jr.
中科院分区：
文献类型：
--
作者：
Gallo-Ebert, Christina;Donigan, Melissa;Nickels, Joseph T., Jr.
白色念珠菌和相关真菌病原体的感染对免疫功能低下的患者造成严重的健康问题。唑类药物是最常用的抗感染药物，靶向固醇生物合成途径。随着药物应激细胞通过上调途径中的几个基因来补偿，对唑类药物治疗的适应性发展，这一过程部分由Upc 2转录因子介导。我们已经实施了一个基于细胞的高通量筛选，以确定小分子抑制剂的Upc 2依赖性诱导固醇基因表达的唑类药物治疗。该测定法不仅用于鉴定Upc 2 DNA结合抑制剂，还用于鉴定阻碍Upc 2激活基因表达的化合物。开发了AlphaScreen测定以确定所鉴定的化合物是否与Upc 2直接相互作用并抑制DNA结合。通过基于细胞的测定鉴定的三种化合物抑制Upc 2蛋白水平和UPC 2-LacZ基因表达，以响应甾醇生物合成的阻断。这些化合物在体内具有生长抑制作用和减弱抗真菌诱导的甾醇基因表达。他们通过降低药物存在下Upc 2蛋白和Upc 2 DNA结合的水平来实现这一点。化合物限制Upc 2 DNA结合的机制不是通过直接相互作用，如使用AlphaScreen测定缺乏DNA结合抑制活性所证明的。相反，它们可能会抑制一种新的途径，激活Upc 2，以响应甾醇生物合成的阻断。我们认为所鉴定的化合物代表了合成新型抗真菌药物的潜在前体。
Infections by Candida albicans and related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level and UPC2-LacZ gene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expression in vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.