Inhibitors of Bacterial Protein Synthesis with Novel Modes of Action

描述（由申请人提供）：细菌核糖体是天然和临床抗生素的首选靶标。然而，由于耐药性的发展严重损害了目前可用抗生素的疗效，细菌感染的治疗正在成为问题。大多数已知的翻译抑制剂作用于核糖体中很少的位点，这些位点受到微生物群落中循环的特定预先存在的天然抗性酶的化学修饰。鉴定通过结合新的核糖体位点干扰翻译的化合物将有助于开发对耐药菌株具有活性的新型抗生素。我们设计了一个实验策略来确定

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.