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

项目概要/摘要 严重缺乏有效的抗生素治疗选择，以对抗多重耐药（MDR）革兰氏阴性菌 细菌（即，“超级细菌”）正在造成世界上三大最严重的人类健康威胁之一。加剧 这是对MDR鲍曼不动杆菌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，呼吸- 泌尿道、伤口和其他感染的发病率非常高，死亡率高达80%。许多 抗生素对它们的靶位点的穿透性极差，尤其是在A.鲍曼不动杆菌和铜绿假单胞菌。 对于这些抗生素，靶部位渗透性差和广泛外排的组合导致抗生素 靶位点处的抗生素浓度比胞外抗生素浓度低超过1，000倍。 毫不奇怪，许多抗生素候选物失败是因为它们的渗透性差和/或大量流出。 细菌靶位点。重要的是，目前对如何 使抗生素靶位点渗透最大化，避免从细菌细胞流出，从而使受体 约束力然而，这个多学科的项目将确定如何最大化 抗生素靶位点浓度和受体结合，以对抗耐药的“超级细菌”。我们的初步数据 和模型表明，分子描述符可以预测抗生素靶位点的渗透和效果， 铜绿假单胞菌中的多个外排泵。我们已经开发了一系列的检测方法， 关键选择的抗生素对其周质或胞质靶位点的渗透以及抗生素对其 完整细菌中的受体。在目标1中，这些新的分子和表型测定将被大大扩展， 应用于所有三种“超级细菌”;此外，将产生一系列同基因外排泵敲除菌株。 由此产生的数据将独特地为新的定量模型（目标2）提供信息，这些模型可以预测渗透，外排， 并因此基于分子抗生素性质在细菌靶位点处结合受体。这些模型 将能够靶向合成关键选定的抗生素探针（Aim 3），用于前瞻性地 验证这些预测模型。这些新的探针将作为创新抗生素组合的支柱， 通过定量和系统药理学模型合理优化国家给药策略， 目标4。然后将具有完整或受损免疫系统的动态体外和鼠感染模型 前瞻性评价这些联合方案。这些模型可以模拟抗生素浓度-时间关系 与病人的情况相对应的资料。总体而言，该项目将提供分子见解，使药物 开发人员设计新的抗生素，在其细菌靶位点达到高浓度， 改善受体结合。这种方法和本项目中合成的靶向新抗生素探针 这是一个很好的承诺，大大有助于对抗三种MDR革兰氏阴性“超级细菌”。

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”.