Combating resistant superbugs by understanding the molecular determinants of target site penetration and binding

项目摘要/摘要 严重缺乏针对多药耐药(MDR)革兰氏阴性菌的有效抗生素治疗选择 细菌(即“超级细菌”)正在造成世界上三个最严重的人类健康威胁之一。加剧 这是对鲍曼不动杆菌多药耐药有效的新抗生素数量的急剧下降， 肺炎克雷伯菌和铜绿假单胞菌。这些超级细菌会导致严重的血液循环，呼吸系统- 胆道、尿路、伤口和其他感染的发病率很高，死亡率高达80%。许多 抗生素对靶部位的渗透性极差，尤其是对鲍曼不动杆菌和铜绿假单胞菌。 对于这些抗生素，较差的靶点穿透性和广泛的外排共同导致抗生素 目标部位的浓度比胞外抗生素浓度低1000倍以上。 不足为奇的是，许多抗生素候选药物失败是因为它们的渗透性差和/或广泛的外排。 细菌靶点。重要的是，目前对如何 最大化抗生素靶点穿透，避免细菌细胞外流，从而最大化受体 有约束力的。然而，这个多学科的项目将确定如何最大限度地 抗生素靶点浓度和受体结合，以对抗耐药的“超级细菌”。我们的初步数据 模型表明，分子描述符可以预测抗生素靶点的穿透和效果 铜绿假单胞菌的多个外排泵。我们已经开发了一系列分析方法来表征 关键选择的抗生素对其周质或胞浆靶点的渗透性以及抗生素与其 完整细菌中的受体。在目标1中，这些新的分子和表型分析将大大扩展和 应用于所有三种“超级细菌”；此外，还将产生一系列等基因外排泵基因敲除菌株。 所产生的数据将唯一地为新的定量模型(目标2)提供信息，该模型可以预测渗透、外流、 从而基于分子抗生素特性与细菌靶点上的受体结合。这些型号 将能够有针对性地合成关键的选定抗生素探针(目标3)，这些探针用于前瞻性地 验证这些预测模型。这些新的探针将成为创新的抗生素组合的支柱- 国家给药策略将通过量化和系统药理学模型进行合理优化 目的4.免疫系统完整或受损的动态体外和小鼠感染模型将 前瞻性评估这些联合治疗方案。这些模型可以模拟抗生素的浓度-时间。 与患者的个人资料完全一致。总体而言，该项目将提供使药物能够 开发人员设计新的抗生素，使其在细菌目标部位达到高浓度，从而 改善受体结合。该方法和本项目中合成的靶向新型抗生素探针成立 承诺为抗击三种耐多药革兰氏阴性“超级细菌”做出实质性贡献。

Project Summary/Abstract A severe lack of effective antibiotic treatment options against multidrug-resistant (MDR) Gram-negative bacteria (i.e., “superbugs”) is causing one of the world’s three most serious human health threats. Exacerbating this is a dramatic decline in the number of new antibiotics effective against MDR Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. These “superbugs” cause serious bloodstream, respira- tory and urinary tract, wound, and other infections with very high morbidity and up to 80% mortality. Many antibiotics have extremely poor penetration to their target site, especially in A. baumannii and P. aeruginosa. For these antibiotics, the combination of poor target site penetration and extensive efflux causes the antibiotic concentration at the target site to be over 1,000-fold lower than that of the extracellular antibiotic concentration. Unsurprisingly, many antibiotic candidates fail because of poor penetration to and/or extensive efflux from their bacterial target site. Importantly, there are very substantial gaps in the current understanding of how to maximize the antibiotic target site penetration, avoid efflux from bacterial cells, and thereby maximize receptor binding. This multi-disciplinary project, however, will identify the molecular determinants of how to maximize antibiotic target site concentrations and receptor binding to combat resistant “superbugs”. Our preliminary data and models demonstrate that molecular descriptors can predict the antibiotic target site penetration and effect of multiple efflux pumps in P. aeruginosa. We have developed a series of assays that characterize the penetration of key selected antibiotics to their periplasmic or cytosolic target sites and antibiotic binding to their receptors in intact bacteria. In Aim 1, these new molecular and phenotypic assays will be greatly extended and applied to all three “superbugs”; additionally, a series of isogenic efflux pump knockout strains will be created. The resulting data will uniquely inform novel quantitative models (Aim 2) that can predict penetration, efflux, and thus receptor binding at the bacterial target sites based on molecular antibiotic properties. These models will enable the targeted synthesis of key selected antibiotic probes (Aim 3) that are used to prospectively validate these predictive models. These new probes will serve as the backbone of innovative antibiotic combi- nation dosing strategies that will be rationally optimized via Quantitative and Systems Pharmacology models in Aim 4. Dynamic in vitro and murine infection models with an intact or compromised immune system will then prospectively evaluate these combination regimens. These models can simulate antibiotic concentration-time profiles that mirror those in patients. Overall, this project will provide the molecular insights that enable drug developers to design new antibiotics that achieve high concentrations at their bacterial target site and thereby improve receptor binding. This approach and the targeted new antibiotic probes synthesized in this project hold excellent promise to substantially contribute to combating the three MDR Gram-negative “superbugs”.