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: 8266949
• 负责人: DEBORAH T HUNG
• 金额: $ 24.24万
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8266949
• 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; 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; Screening procedure; Serum; Staphylococcus aureus; System; Technology; Urine; Validation; Zebrafish; 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; small molecule; small molecule libraries; success

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Increasing resistance to our current antibiotics is challenging our ability to manage infections with these resistant pathogens. In this setting, the current antibiotic development pipeline is alarmingly empty due to the challenges of discovering new antibiotic leads. We propose to develop a novel, multiplexed method for identifying new leads against the clinically important pathogen Pseudomonas aeruginosa.

描述（由申请方提供）：铜绿假单胞菌是重症监护病房肺炎的主要原因，也是革兰氏阴性菌血症和医院获得性肺炎的第二大原因。由于铜绿假单胞菌能够逃避当前的抗生素或产生耐药性，因此其临床菌株对当前所有临床相关抗生素的耐药性越来越强。然而，目前的抗生素管道一般，但特别是抗假单胞菌剂，是惊人的空。这种失败主要是由于寻找 由于铜绿假单胞菌固有的屏障和对小分子的抗性，因此其是抗铜绿假单胞菌的先导化合物，以供进一步开发。在此，我们提出了一种新的方法来确定这样的铅化合物，绕过这些障碍，采取的方法，接口基因组学和新的高通量化学筛选技术。使用基因组学，我们将确定必要的外膜蛋白（OMP）是抗生素发现的有效靶点，从而避免了小分子细胞内积累的需要。然后，我们将进行化学筛选，在一个多路复用的方式对菌株超敏抑制剂的控制低表达的相应OMP。这种多重方法将提高识别小分子先导物的效率和能力，以供进一步开发。在该提案的R21阶段，我们将使用最近开发的全基因组阴性选择技术在临床相关生长条件下鉴定许多不同铜绿假单胞菌菌株的必需外膜蛋白（OMP）。将该数据集与实验室可用的OMPs蛋白质组学研究相结合，我们将选择一组核心的必需OMPs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的多重靶向（MTEP）的方法，该方法将允许同时对许多必需靶标进行化学筛选。我们将针对条形码、基因工程靶标特异性筛选菌株池筛选小分子文库，其中每个必需OMP基因都已被敲低。这种受控的低表达将赋予对相应靶标的小分子抑制剂的超敏反应。我们将同时针对20个OMP靶标进行筛选，而不是针对这些菌株中的每一个进行平行的单独筛选，这很快就变得成本高昂。这种多重策略将全细胞筛选与靶标鉴定相结合。最后，在该提案的R33阶段，我们建议针对大量独特的多样性导向合成（DOS）化合物的集合扩大MTEP筛选，以确定进一步开发为新型抗假单胞菌抗生素的候选物。因此，我们将开发一种方法，用于更有效地识别针对具有挑战性的高耐药性革兰氏阴性病原体铜绿假单胞菌的先导小分子，并将开发几种候选支架用于动物模型中的最终挑战。 公共卫生关系：对我们目前抗生素的耐药性不断增加，这对我们管理这些耐药病原体感染的能力提出了挑战。在这种情况下，由于发现新抗生素先导物的挑战，目前的抗生素开发管道是惊人的空。我们建议开发一种新的，多路复用的方法来确定新的线索对临床上重要的病原体铜绿假单胞菌。