Fan Jun, de Jonge Boudewijn L M, MacCormack Kathy, Sriram Shubha, McLaughlin Robert E, Plant Helen, Preston Marian, Fleming Paul R, Albert Robert, Foulk Melinda, Mills Scott D
Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom.
Antimicrob Agents Chemother. 2014 Dec;58(12):7264-72. doi: 10.1128/AAC.03475-14. Epub 2014 Sep 22.
Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.
细菌生物传感器菌株可成为发现和表征抗菌化合物的有用工具。将一个包含recA启动子与绿色荧光蛋白基因之间转录融合的基于质粒的报告载体导入大肠杆菌ΔtolC菌株，以创建一个通过SOS反应选择性感知DNA代谢抑制剂的生物传感器菌株。该菌株被用于开发一种高通量测定法，以鉴定DNA代谢的新抑制剂。用该菌株对阿斯利康化合物文库进行筛选，鉴定出了已知的DNA代谢抑制剂以及新型化学类型。通过对实验室产生的抗性突变体进行遗传表征，随后在DNA回旋酶活性测定中测量50%抑制浓度，阐明了一个新系列的细胞靶点为DNA回旋酶。这些研究验证了使用这种抗生素生物传感器菌株通过高通量筛选来鉴定DNA代谢新型选择性抑制剂的方法。
