Elitra Pharmaceuticals, San Diego, California 92121, USA.
Antimicrob Agents Chemother. 2010 Sep;54(9):3659-70. doi: 10.1128/AAC.00308-10. Epub 2010 Jun 14.
The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.
抗生素耐药菌的广泛出现和新药物研发的缺乏，促使人们需要新的创新方法来发现抗生素药物。抗生素发现的一个瓶颈是缺乏快速而全面的方法来确定化合物的作用模式 (MOA)。由于抗生素作用的一个标志是作为必需细胞靶标和过程的抑制剂，我们在临床上重要的病原体金黄色葡萄球菌中鉴定出一组 308 个必需基因。总共构建了 446 株差异表达这些基因的菌株，这些菌株构建在一个敏感和耐药菌株的综合平台上。通过一组四环素调控启动子，一组菌株可以实现目标基因的低表达或过表达。另一组 236 个反义 RNA 表达克隆可以使同源靶基因的表达受到干扰。基因表达的下调赋予抗生素敏感性增加，而过表达赋予耐药性。将反义菌株配置成一个 TargetArray，其中敏感菌株的混合物在适应度测试中受到挑战。一种快速检测方法可以测量菌株对抗生素的反应。TargetArray 抗生素适应度测试结果显示出具有机制信息的生物指纹图谱，从而可以阐明作用模式。
