Multiplexed targeting of Pseudomonas aeruginosa essential outer membrane proteins

铜绿假单胞菌必需外膜蛋白的多重靶向

• 批准号: 8843773
• 负责人: DEBORAH T HUNG
• 金额: $ 40.23万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-15 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8843773
• 关键词: Accounting; Animal Model; Animals; Antibiotics; Bacteremia; Bar Codes; Biological Assay; Candida albicans; Cell membrane; Cells; Chemicals; Chemistry; Clinical; Collection; Couples; Culture Media; Data Set; Development; Dose; Engineering; Environment; Escherichia coli; Essential Genes; Failure; Gene Dosage; Genes; Genetic Engineering; Genomics; Growth; Hypersensitivity; Individual; Infection; Institutes; Intensive Care Units; Lead; Libraries; Literature; Membrane Protein Gene; Membrane Proteins; Methods; Nosocomial pneumonia; Phase; Pneumonia; Proteins; Proteomics; Pseudomonas aeruginosa; Resistance; Resistance development; Serum; Staphylococcus aureus; System; Technology; Urine; Validation; Zebrafish; abstracting; clinically relevant; cost; efflux pump; follow-up; genetic technology; genome-wide; inhibitor/antagonist; killings; knock-down; mouse model; novel; novel strategies; pathogen; response; scaffold; scale up; screening; small molecule; small molecule libraries; success

Abstract Pseudomonas aeruginosa is a major cause of intensive care unit pneumonias and the number two cause of Gram-negative bacteremia and nosocomial pneumonia. Because of its ability to evade current antibiotics or develop resistance, P. aeruginosa clinical strains are increasingly resistant to all current clinically relevant antibiotics. Yet, the current pipeline of antibiotics in general, but anti-pseudomonal agents in particular, is alarmingly empty. Much of this failure is due to the incredible challenge of finding lead compounds against P. aeruginosa for further development because of its intrinsic barriers and resistance to small molecules. Herein, we propose a new method to identify such lead compounds that circumvent these barriers by taking an approach that interfaces genomics and novel high-throughput chemical screening technologies. Using genomics, we will identify essential outer membrane proteins (OMPs) that are valid targets for antibiotic discovery, thus circumventing the need for small molecule intracellular accumulation. We will then perform chemical screening in a multiplexed fashion against strains hypersensitized to inhibitors by controlled low expression of the respective OMP. This multiplexed approach will increase the efficiency and ability to identify small molecule leads for further development. In the R21 phase of the proposal, we will identify essential outer membrane proteins (OMPs) across many different strains of P. aeruginosa under clinically relevant growth conditions using recently developed genome-wide negative selection technology. Combining this dataset with publically available proteomic studies on OMPs, we will select a core set of essential OMPs to be targeted for small molecule discovery. Further, in the R21 phase, we will develop a method for Multiplexed Targeting of Essential Proteins (MTEP), which would allow simultaneous chemical screening against numerous essential targets. We will screen a small molecule library against a pool of bar-coded, genetically engineered target-specific screening strains in which each of the essential OMP genes has been knocked-down. This controlled low expression will confer hypersensitivity to small molecule inhibitors of the respective target. We will screen against 20 OMP targets simultaneously, in contrast to parallel individual screens against each of these strains, which rapidly becomes cost-prohibitive. This multiplexed strategy couples whole cell screening with target identification. Finally, in the R33 phase of the proposal, we propose to scale up MTEP screening against a large, unique collection of diversity-oriented synthetic (DOS) compounds, to identify candidates for further development as novel anti-pseudomonal antibiotics. Thus, we will develop a method for more efficiently identifying lead small molecules against the challenging highly resistant Gram-negative pathogen P. aeruginosa and will develop several candidate scaffolds for ultimate challenge in an animal model.

摘要 铜绿假单胞菌是重症监护室肺炎的主要原因， 导致革兰氏阴性菌血症和医院获得性肺炎。因为它有躲避电流的能力 抗生素或产生耐药性，铜绿假单胞菌临床菌株对目前临床上所有 相关抗生素然而，目前的抗生素，特别是抗假单胞菌药物， 空得惊人这种失败在很大程度上是由于寻找铅化合物的令人难以置信的挑战， P.铜绿假单胞菌的进一步发展，因为其固有的障碍和耐药性的小分子。 在此，我们提出了一种新的方法来确定这样的先导化合物，通过采取这些障碍， 该方法将基因组学与新型高通量化学筛选技术相结合。使用 基因组学，我们将确定必要的外膜蛋白（OMPs）是抗生素的有效靶点 发现，从而避免了对小分子细胞内积累的需要。然后我们将表演 以多重方式对抑制剂超敏菌株进行化学筛选， 表达各自的OMP。这种多路复用的方法将提高识别的效率和能力， 小分子导致进一步发展。 在该提案的R21阶段，我们将确定跨膜的必需外膜蛋白（OMP）。 在临床相关生长条件下，使用最近开发的 全基因组负选择技术将此数据集与生物学上可用的蛋白质组学研究相结合 在OMP方面，我们将选择一组核心的必需OMP作为小分子发现的目标。 此外，在R21阶段，我们将开发一种用于必需蛋白质多重靶向的方法 这将允许针对许多基本目标同时进行化学筛选。我们将 针对条形码化的、遗传工程化的靶特异性筛选池筛选小分子文库 其中每个必需OMP基因都已被敲低的菌株。这种受控的低表达将 赋予对各自靶点的小分子抑制剂的超敏性。我们将对20 OMP进行筛选 同时靶向，与针对这些菌株中的每一种的平行单独筛选相反， 变得成本高昂。这种多重策略将全细胞筛选与靶标鉴定相结合。 最后，在建议的R33阶段，我们建议扩大MTEP筛查， 独特的多样性导向的合成（DOS）化合物的集合，以确定候选人， 开发为新型抗假单胞菌抗生素。因此，我们将开发一种方法， 鉴定针对具有挑战性的高抗性革兰氏阴性病原体P的先导小分子。 并将开发几种候选支架用于动物模型的最终挑战。