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Genetically-encoded fluorescent RNA sensors for measuring transport of antibiotics into the cytoplasm of Gram-negative pathogens and development of efflux pump inhibitors

用于测量抗生素转运至革兰氏阴性病原体细胞质的基因编码荧光RNA传感器以及外排泵抑制剂的开发

基本信息

批准号：
10326785
负责人：
STEPHEN LORY
金额：
$ 60.52万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-13 至 2023-07-31
项目状态：
已结题

项目摘要

Project Summary Complications from infections caused by difficult to treat Gram-negative pathogens resistant to multiple antibiotics (the MDR phenotype) has become a major public health threat. The basis of resistance in the most important members of this group, including Pseudomonas aeruginosa, is the poor penetration of the antibiotics through a relatively impermeable bacterial cell envelope and their expulsion (efflux) by various pumps expressed by the microorganisms. Consequently, the intracellular concentrations of antibiotic are kept below bactericidal levels. This proposal aims at creating new classes of diagnostic tools and therapeutic agents to combat efflux-based resistance by a project organized into two phases. First, we will develop genetically encoded fluorescent sensors activated by binding of antibiotics following their entry into the bacterial cytoplasm. This tool will enable us to measure antibiotic flux under different environmental conditions and on a single cell basis. During the second phase of the project, working with our industrial partner, we will implement a screening program, using a library of several hundred billion small molecules, to identify inhibitors targeting different components of efflux pumps. These inhibitors should activate the fluorescent detector by causing enhanced intracellular accumulation of the antibiotics resulting from inhibition of efflux. Functional studies are proposed to assess the ability of the active compounds to promote killing of P. aeruginosa by antibiotics that are normally expelled by the efflux pumps. Crystal structures of inhibitors bound to target proteins will be determined and these will guide medicinal chemistry efforts with the goal of creating additional derivatives with increased potencies and expanded spectrum, capable of inhibiting efflux pumps of other important Gram-negative pathogens.

项目摘要由难以治疗的革兰氏阴性病原体引起的感染并发症多重抗生素（MDR表型）已成为主要的公共卫生威胁。的基础包括铜绿假单胞菌在内的该组最重要成员的耐药性，抗生素通过相对不渗透的细菌细胞包膜的渗透性差以及它们通过微生物表达的各种泵的排出（流出）。因此，委员会认为，抗生素的细胞内浓度保持低于杀菌水平。这项建议旨在创造新的诊断工具和治疗药物，以对抗基于外排的一个项目分为两个阶段。首先，我们将开发基因编码的在抗生素进入细菌后，通过抗生素的结合激活荧光传感器细胞质该工具将使我们能够测量不同环境下的抗生素通量条件下，在单细胞的基础上。在项目的第二阶段，与我们的作为工业合作伙伴，我们将使用数百个库实施筛选计划 10亿个小分子，以确定针对外排泵不同组分的抑制剂。这些抑制剂应该通过引起增强的细胞内荧光来激活荧光检测器。由外排抑制引起的抗生素蓄积。建议进行功能研究评价活性化合物促进抗生素杀死铜绿假单胞菌的能力通常由外排泵排出。与靶标结合的抑制剂的晶体结构蛋白质将被确定，这些将指导药物化学的努力，目标是产生具有增加的效力和扩展的谱的另外的衍生物，其能够抑制其他重要革兰氏阴性病原体的外排泵。