Structural Determinants of Permeation Barriers in Escherichia coli

大肠杆菌渗透屏障的结构决定因素

基本信息

批准号：
10749251
负责人：
Marcos M. Pires
金额：
$ 62.32万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-31 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10749251
关键词：
3-Dimensional Academia Agreement Alkynes Antibiotic Therapy Antibiotics Azides Bacteria Binding Sites Biochemical Biological Assay Biology Cells Chemicals Collection Communities Cytoplasm Data Data Set Drug Targeting Epitopes Escherichia coli Fluorescence Foundations Genomics Gram-Negative Bacteria Healthcare Industry Knock-in Knock-out Label Laboratories Libraries Measurable Measures Medical Membrane Metabolic Methodology Methods Modernization Molecular Operative Surgical Procedures Organelles Organic Synthesis Penetration Permeability Pharmaceutical Chemistry Pharmaceutical Preparations Phase Physical condensation Predisposition Proteomics Reaction Reagent Research Resolution Science Specificity Testing Three-Dimensional Imaging VDAC1 gene Work cell envelope cheminformatics cycloaddition design drug discovery efflux pump fluorophore high throughput analysis human disease infection risk innovation instrumentation machine learning algorithm metabolomics minimal risk novel pathogen periplasm screening small molecule small molecule libraries structural determinants subcellular targeting tool ultra high resolution

项目摘要

PROJECT SUMMARY Antibiotics represent the most successful class of drugs developed by modern science. They have spurred numerous medical advances by facilitating invasive surgeries with minimal risk of infection. Today, a major healthcare crisis looms as we are fast approaching a post-antibiotic era. Historically, it has proven to be much more difficult to find agents that are active against Gram-negative pathogens compared to Gram-positive pathogens. The primary reason is that Gram-negative bacteria have a unique asymmetric outer membrane (OM) in addition to an inner membrane. The targets for most antibiotics reside beyond the OM, and thus these molecules need to penetrate the OM to be active. Yet, the OM is uniquely effective in blocking the translocation of small molecules, thus creating a major challenge for the field. The Golden Era of antibiotics leveraged naturally abundant small molecules that were readily identified using traditional methods; however, this methodology has proven to be much more difficult to be further mined for new antibiotics during the past several decades. The next phase of antibiotic drug discovery has the potential to be supported by our increasing collection of proteomics, genomics, and metabolomics data that will reveal promising drug targets. Academia and industry could potentially exploit these data sets to design small molecule agents that are potent and of high specificity. To accomplish this, the field fundamentally requires guiding principles describing the molecular determinants of permeation into bacterial cells akin to the Lipinski’s rules of 5 (Ro5). We propose to develop a novel fluorescence assay that measures the accumulation of small molecules in Gram- negative pathogens based on a combination of HaloTag expression and anchoring of a biorthogonal epitope within HaloTag. Our team will systematically (testing established antibiotics with known permeation profiles) and broadly (screening a unique library of small molecules modified with an azide tag) apply this approach to measurably grow our fundamental understanding of the molecular determinants of permeation. Additionally, we will utilize the same platform to provide unprecedented spatial resolution of the distribution of small molecules (including known antibiotics) in subcellular compartments.

项目概要抗生素代表了现代科学开发的最成功的一类药物。今天，通过促进侵入性手术和最小化感染风险而取得了许多医学进步。随着我们快速接近后抗生素时代，医疗保健危机迫在眉睫。历史证明，这一危机的影响非常大。与革兰氏阳性病原体相比，更难找到对革兰氏阴性病原体有活性的药物主要原因是革兰氏阴性菌具有独特的不对称外膜（OM）。除了内膜之外，大多数抗生素的靶点都位于 OM 之外，因此这些然而，分子需要穿透 OM 才能发挥作用，而 OM 在阻止易位方面具有独特的功效。小分子，从而给该领域带来了重大挑战。抗生素的黄金时代利用了天然丰富的小分子，这些小分子很容易通过使用来识别传统方法；然而，事实证明，这种方法更难以进一步挖掘新的方法。抗生素药物发现的下一阶段有可能是过去几十年的抗生素。在我们不断收集的蛋白质组学、基因组学和代谢组学数据的支持下，这些数据将揭示有希望的学术界和工业界可能会利用这些数据集来设计小分子药物。为了实现这一目标，该领域从根本上需要指导原则。描述了类似于 Lipinski 5 规则 (Ro5) 的渗透到细菌细胞的分子决定因素。我们建议开发一种新颖的荧光测定法来测量革兰氏阴性菌中小分子的积累基于 HaloTag 表达和双正交表位锚定相结合的阴性病原体在 HaloTag 内，我们的团队将系统地（测试具有已知渗透特性的已建立的抗生素）并广泛地（筛选用叠氮化物标签修饰的独特小分子库）将该方法应用于显着增强我们对渗透分子决定因素的基本理解。将利用同一平台提供前所未有的小分子分布空间分辨率（包括已知的抗生素）在亚细胞区室中。