Inhibitors of Bacterial Protein Synthesis with Novel Modes of Action

具有新颖作用模式的细菌蛋白质合成抑制剂

• 批准号: 8408750
• 负责人: ALEXANDER S MANKIN
• 金额: $ 3.99万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8408750
• 关键词: Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Bacterial Proteins; Bacterial Typing; Binding; Binding Sites; Biological Assay; Cells; Chemicals; Clinical; Development; Dose; Drug resistance; Eligibility Determination; Engineering; Enzymes; Escherichia coli; Experimental Designs; Growth; Health; Human; Hybrids; In Vitro; Lead; Modification; Molecular Bank; Monitor; Mutate; Mutation; Natural Resistance; Nature; Pharmaceutical Preparations; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Public Health; RNA; Resistance; Ribosomes; Site; Specificity; Translations; cell growth; cell type; inhibitor/antagonist; interest; microbial community; novel; pathogen; repository; research study; resistance mechanism; resistant strain; small molecule; small molecule libraries

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.

描述（由申请人提供）：细菌核糖体是天然和临床抗生素的优选靶点。然而，由于耐药性的发展，细菌感染的治疗正变得有问题，这严重损害了目前可用的抗生素的功效。大多数已知的翻译抑制剂作用于核糖体中的非常少的位点，并且这些位点受到在微生物群落中循环的特定的预先存在的天然抗性酶的化学修饰。通过与新的核糖体位点结合来干扰翻译的化合物的鉴定将导致对耐药菌株具有活性的新型抗生素的开发。 我们设计了一个实验策略来识别 在结合核糖体中的四个主要新靶位点后抑制翻译的化合物。对E.制备大肠杆菌菌株，使得它们表达正常细菌核糖体或杂合核糖体，其中感兴趣的位点被突变以模拟人细胞质核糖体中的对应序列。每种菌株表达不同的荧光蛋白，允许监测混合培养物中的每种类型的细胞。将筛选化学文库中优先抑制野生型细胞生长同时允许携带“人源化”核糖体的细胞生长的化合物。将在剂量依赖性细菌生长抑制试验中验证主要命中，并结合体外翻译实验和化学RNA足迹法验证作用位点。由于全细胞筛选方案的性质，所鉴定的化合物应当能够通过结合预定义的核糖体位点选择性抑制细菌翻译来抑制细菌生长。