Inhibitors of Bacterial Protein Synthesis with Novel Modes of Action

描述(申请人提供)：细菌核糖体是天然和临床抗生素的首选靶标。然而，细菌感染的治疗正变得困难，因为抗药性的发展严重削弱了现有抗生素的疗效。大多数已知的翻译抑制剂作用于核糖体中的极少数部位，这些部位会受到微生物群落中循环的特定天然抗性酶的化学修饰。识别通过与新的核糖体结合而干扰翻译的化合物将导致对耐药菌株具有活性的新型抗生素的开发。我们设计了一种实验策略来识别 当与核糖体中四个主要新的靶点结合时抑制翻译的化合物。制备成对的大肠杆菌菌株，它们表达正常的细菌核糖体或杂交核糖体，其中感兴趣的位置发生突变，以模仿人类细胞质核糖体中的对应序列。每种菌株表达一种不同的荧光蛋白，允许监测混合培养中的每种类型的细胞。将对化学库进行筛选，寻找优先抑制野生型细胞生长的化合物，同时允许携带“人源化”核糖体的细胞生长。初步的打击将在剂量依赖的细菌生长抑制试验中得到验证，作用部位将通过体外翻译实验和化学RNA足迹来验证。由于全细胞筛选方法的性质，所鉴定的化合物应该能够通过与预定义的核糖体结合来选择性地抑制细菌的翻译来抑制细菌的生长。

DESCRIPTION (provided by applicant): Bacterial ribosome is a preferred target for both natural and clinical antibiotics. However, treatment of bacterial infections is becoming problematic due to the development of drug resistance that severely impairs the efficacy of currently available antibiotics. Most of known translation inhibitors act upon very few sites in th ribosome and these sites are subject to chemical modification by specific pre-existing natural resistance enzymes circulating within microbial communities. Identification of compounds interfering with translation by binding to new ribosomal sites will lead to development of novel antibiotics active against the resistant strains. We designed an experimental strategy to identify compounds that inhibit translation upon binding to four principally new target sites in the ribosome. Pairs of E. coli strains were prepared such that they express either the normal bacterial ribosome or a hybrid ribosome in which the site of interest was mutated to mimic the counterpart sequence in the human cytoplasmic ribosome. Each strain expresses a different fluorescent protein that allows for monitoring each type of cells in a mixed culture. Chemical libraries will be screened for compounds that preferentially inhibit growth of the wild-type cells while allowing growth of cells carrying "humanized" ribosome. The primary hits will be validated in dose-dependent bacterial growth inhibition assays, and the site of action will be verified with combination of in vitro translation experiments and chemical RNA footprinting. Because of the nature of the whole-cell screening protocol, the identified compounds should be able to inhibit bacterial growth by selectively inhibiting bacterial translation via binding to the predefined ribosomal sites.