Exploring the physicochemical properties governing compound efflux in Gram-negative bacteria

项目摘要 多重耐药感染是对全球健康的主要威胁， 每一种临床使用的抗生素，即使是那些被认为是最后一线治疗的抗生素。进一步加剧 抗生素耐药性危机是缺乏新的抗生素进入管道，特别是对革兰氏- 阴性病原体具有不可渗透的外膜，限制小分子积聚，和 混杂的外排泵，它可以识别并排出细胞中的大多数小分子。基础科学 了解促进化合物积累和减少所需的有利化学性质 需要外排倾向来开发对革兰氏阴性菌具有全细胞活性的抗菌候选物 病原体Hergenrother实验室的初步努力已经确定了 E.大肠杆菌，并成功地应用这些指导方针，转化几个革兰氏阳性 从抗生素到广谱药剂虽然这种策略提高了革兰氏阴性抗菌活性， 化合物外排仍然对功效有害，并阻止这些先导物发展成有效的抗生素。 必须了解控制化合物识别和外排的物理化学性质， 提供了一个新的设计平台，以工程外排敏感性的候选药物。的目的 建议是确定定义革兰氏阴性菌中化合物外排的参数，并应用这些参数 研究结果，以消除外排倾向的有前途的抗菌候选人。 本文提出的工作将建立在对约200种化合物的外排倾向的初步研究的基础上 利用一种新的基于LC-MS/MS的积累（外排倾向评价（EXPEL））测定法， 检测外排敏感性的微小变化，而不考虑抗菌活性和化学信息学模型 其可以准确地将50%的化合物分类为外排底物和非底物。具体目标1、 通过随机森林模型确定的对化合物外排重要的其他理化性质 将通过合成并排比较的靶向文库来探测。这些化合物将 添加到化合物合成、EXPEL测定和化学信息学模型的数据集和迭代循环中 将进行验证。利用EXPEL测定和被鉴定为与以下相关的初始性质： 外排率，激动人心的FabI抑制剂的衍生物将被探索，以确定有前途的抗菌药物， 具体目标2中的外排负债减少。这些化合物的治疗潜力将被探索 通过毒性研究、药代动力学特征测定和小鼠感染中的功效评价 模型具体目标1和2将同时进行，这些研究的完成将显著影响 抗菌药物研究和抗菌药物临床管道中的补救磨损点。

Project Summary Multi-drug resistant infections are a major threat to global health and resistance has been observed for every clinically-used antibiotic, even those considered to be the last lines of treatment. Further compounding the antimicrobial resistance crisis is the lack of new antibiotics entering the pipeline, particularly for Gram- negative pathogens which have an impermeable outer membrane, limiting small molecule accumulation, and promiscuous efflux pumps, which can recognize and expel most small molecules from the cell. A basic-science understanding of favorable chemical properties required to enhance compound accumulation and decrease efflux propensity is needed to develop antibacterial candidates with whole-cell activity against Gram-negative pathogens. Initial efforts in the Hergenrother lab have identified the physicochemical traits needed for compound permeation in E. coli and successfully applied these guidelines to convert several Gram-positive only antibiotics to broad-spectrum agents. While this strategy improves Gram-negative antibacterial activity, compound efflux is still detrimental to efficacy and prevents development of these leads into potent antibiotics. It is imperative to understand the physicochemical properties governing compound recognition and efflux to provide a novel design platform to engineer efflux susceptibility out of drug candidates. The objective of this proposal is to identify the parameters that define compound efflux in Gram-negative bacteria and apply these findings to remove efflux liability out of promising antibacterial candidates. Work proposed herein will build upon preliminary studies of the efflux propensity of ~200 compounds utilizing a novel LC-MS/MS-based accumulation (Efflux Propensity EvaLuation (EXPEL)) assay which can detect small changes in efflux susceptibilities irrespective of antibacterial activity and a chemoinformatic model which can accurately classify 50% of compounds as efflux substrates and non-substrates. In Specific Aim 1, additional physicochemical properties determined important for compound efflux by the random forest model will be probed through synthesis of a targeted library of side-by-side comparisons. These compounds will be added to the dataset and iterative cycles of compound synthesis, EXPEL assay, and chemoinformatic model validation will be performed. Utilizing the EXPEL assay and the initial properties identified as correlating to efflux ratios, derivatives of an exciting FabI inhibitor will be explored to identify promising antibacterials with decreased efflux liabilities in Specific Aim 2. The therapeutic potential of these compounds will be explored through toxicity studies, determination of pharmacokinetic profile, and evaluation of efficacy in mouse infection models. Specific Aims 1 and 2 will run concurrently and completion of these studies will significantly impact antibacterial research and remedy attrition points in the antibacterial clinical pipeline.