Abu Mraheil M, Heisig A, Heisig P
Pharmaceutical Biology and Microbiology, University of Hamburg, Germany.
Pharmazie. 2013 Jul;68(7):541-8.
Due to the increasing prevalence of antibiotic resistance and the yet low output of the genomics-based drug discovery approach novel strategies are urgently needed to detect new antibiotics. One such strategy uses known ubiquitous targets like DNA topoisomerases. However, to detect inhibitors of these enzymes by an in vitro assay time-consuming isolation of enzymes and DNA followed by electrophoretic separation of topoisomers are required. Instead, this study aimed at developing an in vivo assay for the detection of alterations in DNA supercoiling indicative of topoisomerase inhibition by a reporter gene assay. A pair of plasmids was developed which carry the reporter gene luc for firefly luciferase under control of either promoter ptopA (pPHB90) or pgyrA (pPHB91), whose activities are reciprocally affected by alterations of the supercoiling degree. Each plasmid is individually transferred into E. coli cells. The quotient of the luciferase activities determined using cells with either plasmid was taken as relative measure of the global supercoiling degree Qsc (quotient of supercoiling). Using isogenic reference strains with known alterations of the global DNA supercoiling degree due to mutations in either gyrB or topA, the reporter gene system was able to detect both a decrease and an increase of the negative supercoiling degree compared to the isogenic parent strain. Treating cells with known inhibitors of DNA gyrase, like fluoroquinolones, novobiocin as well as simocyclinone D8 from Streptomyces antibioticus which has been identified as an inhibitor of DNA gyrase in vitro, also caused decreases of the Qsc value in vivo. The suitability of this reporter gene system to screen for anti-topoisomerase I and II compounds from various natural sources like plant extracts by sensing alterations of the DNA supercoiling was demonstrated and offers a new application to identify novel compounds active against bacterial topoisomerases I and gyrase.
由于抗生素耐药性日益普遍，且基于基因组学的药物发现方法产量仍然较低，因此迫切需要新的策略来发现新的抗生素。一种这样的策略是使用已知的普遍存在的靶点，如DNA拓扑异构酶。然而，要通过体外测定来检测这些酶的抑制剂，需要耗时地分离酶和DNA，然后对拓扑异构体进行电泳分离。相反，本研究旨在开发一种体内测定方法，通过报告基因测定来检测指示拓扑异构酶抑制的DNA超螺旋变化。构建了一对质粒，它们携带萤火虫荧光素酶的报告基因luc，分别受启动子ptopA（pPHB90）或pgyrA（pPHB91）的控制，其活性会受到超螺旋程度变化的相互影响。每个质粒分别转入大肠杆菌细胞。使用含有任一质粒的细胞测定的荧光素酶活性的商作为全局超螺旋程度Qsc（超螺旋商）的相对度量。使用由于gyrB或topA突变而导致全局DNA超螺旋程度已知变化的同基因参考菌株，该报告基因系统能够检测到与同基因亲本菌株相比负超螺旋程度的降低和增加。用已知的DNA回旋酶抑制剂处理细胞，如氟喹诺酮类、新生霉素以及来自抗生链霉菌的西莫环素D8（已在体外被鉴定为DNA回旋酶抑制剂），也会导致体内Qsc值降低。通过检测DNA超螺旋的变化，证明了该报告基因系统适用于从各种天然来源（如植物提取物）中筛选抗拓扑异构酶I和II的化合物，并为鉴定对细菌拓扑异构酶I和回旋酶有活性的新型化合物提供了新的应用。
