Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.
Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.
复制标题
使用新的高通量筛选测定法鉴定细菌西格玛因子的抑制剂。
DOI：
10.1128/aac.03979-14
复制
发表时间：
2015
影响因子：
4.9
通讯作者：
Keiler,KC
中科院分区：
文献类型：
--
作者：
El-Mowafi,SA;Sineva,E;Alumasa,JN;Nicoloff,H;Tomsho,JW;Ades,SE;Keiler,KC
革兰氏阴性细菌是可怕的病原体，因为它们的细胞膜对环境和宿主介导的挑战构成了一种适应性屏障。由另一种Sigma因子σ控制的应激反应通路对于维持细胞膜是至关重要的。由于σ是几种革兰氏阴性病原体毒力或生存所必需的，因此它可能是抗生素开发的有用靶点。为了确定小分子是否可以抑制σE途径，并允许高通量筛选抗生素先导化合物，开发并验证了一种与高通量筛选兼容的σE活性检测方法。该筛查采用了具有阳性读数的生物检测。一株大肠杆菌在σE途径的负调控下表达黄色荧光蛋白，该途径的抑制剂可增加黄色荧光蛋白的产量。为了验证筛选，报告菌株被用来从环肽文库中鉴定σE途径抑制剂。其中一个抑制环肽的生化性质表明，它在体外与σE结合，抑制σ聚合酶全酶的形成，并抑制RNAE依赖的转录。这些结果表明，替代的西格玛因子可以被小分子抑制，并使高通量筛选σE途径的抑制剂成为可能。
Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σEis critical for maintenance of the cell envelope. Because σEis required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σEpathway, and to permit high-throughput screening for antibiotic lead compounds, a σEactivity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σEpathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σEpathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σE, inhibits RNA polymerase holoenzyme formation, and inhibits σE-dependent transcriptionin vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σEpathway.