Structural Determinants of Permeation Barriers in Escherichia coli

项目摘要 抗生素是现代科学开发的最成功的一类药物。他们刺激了 许多医疗进步，促进侵入性手术，感染风险最小。今天，一位少校 随着我们迅速进入后抗生素时代，医疗保健危机迫在眉睫。从历史上看， 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试剂 病原体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。大多数抗生素的靶标位于OM之外，因此这些 分子需要穿透有机质才能活跃然而，OM是唯一有效地阻止易位 小分子，从而为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 传统的方法;然而，这种方法已被证明是更难以进一步挖掘新的 抗生素在过去的几十年里抗生素药物发现的下一阶段有可能是 我们越来越多地收集蛋白质组学、基因组学和代谢组学数据， 药物靶点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 它们是有效的并且具有高度特异性。要做到这一点，该领域从根本上需要指导原则 描述了渗透到细菌细胞中的分子决定因素，类似于Lipinski规则5（Ro 5）。 我们建议开发一种新的荧光测定法，测量革兰氏阴性杆菌中小分子的积累， 基于HaloTag表达和双正交表位锚定的组合的阴性病原体 在HaloTag中。我们的团队将系统地（测试具有已知渗透特性的已确立抗生素）和 广泛地（筛选用叠氮化物标签修饰的小分子的独特文库）将该方法应用于 可测量地增长我们对渗透的分子决定因素的基本理解。另外我们 将利用相同的平台提供前所未有的小分子分布的空间分辨率 （包括已知的抗生素）在亚细胞区室。

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.