Identifying and validating new antibiotic targets in cell wall synthesis pathways

描述(申请人提供)：抗生素耐药性对我们的医疗保健系统构成了重大威胁。六种问题病原体，即所谓的ESKAPE细菌，是医院中大多数耐药感染的原因。迫切需要新的策略来治疗这些感染。针对肽聚糖(PG)/细胞壁生物发生的抗生素是治疗细菌感染最有效的药物之一，但目前临床上使用的所有药物都出现了耐药性。这项拟议的工作源于最近的发现，使用β-内酰胺类化合物作为PG生物合成的化学探针。它的目的是在耐甲氧西林金黄色葡萄球菌(MRSA)和大肠杆菌(E.Coli)的细胞壁组装途径中识别和验证新的靶点。耐甲氧西林金黄色葡萄球菌是ESKAPE病原菌中毒力最强的，而大肠杆菌本身也是一种重要的病原菌，是所有致病性革兰氏阴性杆菌PG生物发生的模式系统。我们的前两个目标集中在验证使MRSA对内酰胺类药物重新敏感的抑制剂的新靶点。耐甲氧西林金黄色葡萄球菌已经获得了一种名为PBP2a的PG转肽酶，它可以促进内酰胺类抗生素的耐药性。我们发现PBP2a的功能依赖于糖基转移酶TARS的活性，TARS将-O-GlcNAc残基附着到壁磷壁酸(WTAS)上，WTAS是一种对金黄色葡萄球菌细胞分裂至关重要的细胞壁聚合物。这表明PG和WTA的合成途径在某种程度上是相互关联的。我们将使用遗传和化学方法的组合来揭示这些联系的机制基础，以便我们能够利用它们作为靶点来对抗MRSA中的内酰胺类耐药性。我们还将通过监测小分子β-内酰胺增强剂对其活性的影响以及在有和没有结合抑制剂的情况下解决其结构来探索TARS本身作为药物靶点。我们的第二组目标集中于了解PG合成机的功能，并验证它们作为抗生素靶标的有效性。考虑到它们作为潜在药物靶点的重要性，令人惊讶的是，人们对这些机器组装PG的机制知之甚少。这主要是由于可用于分析其功能的基因分析方法有限。利用大肠杆菌系统的遗传易感性，我们开发出了第一个阳性 根据PG装配机的活性进行选择，PG装配机是细胞伸长所需的高度保守的杆系统。我们利用这种选择来鉴定Rod功能的小分子拮抗剂，并建议确定它们的特定靶点和作用模式。我们还将利用我们的选择来从基因上询问多蛋白Rod复合体的结构，并确定对每个成分的功能至关重要的氨基酸残基。联合的化学遗传分析将帮助我们识别和验证Rod系统功能的一些方面，这些功能符合新的治疗方法的靶向。由于我们将研究的PG和WTA合成机制是高度保守的，我们在MRSA和大肠杆菌中的发现将与我们对其他微生物中细胞壁聚合物生物发生的理解广泛相关，并将显著影响和指导针对MRSA和革兰氏阴性ESKAPE病原体的治疗方法。

DESCRIPTION (provided by applicant): Antibiotic resistance poses a major threat to our healthcare system. Six problem pathogens, the so-called ESKAPE bacteria, are responsible for the majority of drug resistant infections in hospitals. New strategies to treat these infections ar sorely needed. Antibiotics that target peptidoglycan (PG)/cell wall biogenesis are among the most effective drugs for treating bacterial infections, but resistance has emerged to all those currently in clinical use. The proposed work grew out of recent discoveries made using ¿-lactams as chemical probes of PG biosynthesis. It is aimed at identifying and validating new targets in the pathways for cell wall assembly in methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). MRSA is the most virulent of the ESKAPE pathogens, and E. coli, an important pathogen in its own right, is the model system for PG biogenesis in all pathogenic Gram-negative rods. Our first two aims are focused on validating a new target for inhibitors that resensitize MRSA to ¿-lactams. MRSA have acquired a PG transpeptidase called PBP2A that promotes ¿-lactam resistance. We discovered that PBP2A function is dependent on the activity of a glycosyltransferase, TarS, that attaches ¿-O-GlcNAc residues to wall teichoic acids (WTAs), an additional cell wall polymer important for cell division in S. aureus. This suggests that the pathways of PG and WTA synthesis are somehow interconnected. We will use a combination of genetic and chemical approaches to uncover the mechanistic basis for these connections so that we can exploit them as targets to combat ¿-lactam resistance in MRSA. We will also explore TarS itself as a drug target by monitoring the effect of small molecule ¿-lactam potentiators on its activity and solving its structure with and without bound inhibitors. Our second set of aims focus on understanding the function of PG synthesizing machines and validating them as antibiotic targets. Given their importance as potential drug targets, surprisingly little is known about the mechanism of PG assembly by these machines. This has primarily been due to a limited availability of genetic assays to dissect their function. Taking advantage of the genetic tractability of the E. coli system, we developed the first positive selection against the activity of a PG assembly machine, the highly conserved Rod system needed for cell elongation. We used this selection to identify small molecule antagonists of Rod function and propose to determine their specific targets and mode of action. We will also use our selection to genetically interrogate the structure of the multi-protein Rod complex and identify amino acid residues critical for the function of each component. The combined chemical genetic analysis will help us identify and validate aspects of Rod system function amenable to targeting by novel therapeutics. Because the PG and WTA synthesis machineries we will study are highly conserved, our findings in MRSA and E. coli will be broadly relevant to our understanding of cell wall polymer biogenesis in other microorganisms and should significantly impact and inform efforts to generate therapies against MRSA and Gram-negative ESKAPE pathogens.