Identifying and validating new antibiotic targets in cell wall synthesis pathways

识别和验证细胞壁合成途径中的新抗生素靶标

• 批准号: 8659341
• 负责人: Thomas G Bernhardt
• 金额: $ 85.88万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8659341
• 关键词: Accounting; Acinetobacter; Actins; Active Sites; Address; Affect; Affinity; Amino Acids; Anabolism; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Binding; Biogenesis; Biological Assay; Biological Models; Cell Shape; Cell Wall; Cell division; Cells; Chemicals; Clinical; Collection; Complex; Dependence; Development; Drug Targeting; Drug resistance; Elements; Enterobacter; Enterococcus faecium; Enzymes; Escherichia coli; Future; Genetic; Gram-Negative Bacteria; Gram-Positive Bacteria; Healthcare Systems; Hospitals; Individual; Infection; Klebsiella pneumonia bacterium; Laboratories; Lactams; Lead; Methods; Modification; Monitor; Monobactams; Pathway interactions; Peptides; Peptidoglycan; Peptidyltransferase; Pharmaceutical Preparations; Physiological; Polymers; Polysaccharides; Proteins; Pseudomonas aeruginosa; Reagent; Regulation; Resistance; Resistance to infection; Resolution; Shapes; Site; Staphylococcus aureus; Structure; System; Tars; Teichoic Acids; Transferase; United States; Virulent; Walkers; Work; base; chemical genetics; combat; crosslink; genetic analysis; glycosyltransferase; inhibitor/antagonist; insight; methicillin resistant Staphylococcus aureus; microorganism; mutant; new therapeutic target; novel; pathogen; protein complex; research study; retinal rods; small molecule; therapeutic target

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.

描述（由申请人提供）：抗生素耐药性对我们的医疗保健系统构成重大威胁，即所谓的 ESKAPE 细菌，它们是造成医院中大多数耐药感染的原因，迫切需要治疗这些感染的新策略。以肽聚糖（PG）/细胞壁生物发生为目标的抗生素是治疗细菌感染最有效的药物之一，但目前临床使用的所有药物都出现了耐药性。拟议的工作源于最近的发现。使用 ¿ 制作-内酰胺作为 PG 生物合成的化学探针，旨在识别和验证耐甲氧西林金黄色葡萄球菌 (MRSA) 和大肠杆菌 (E. coli) 细胞壁组装途径中的新靶点。病原体，而大肠杆菌本身就是一种重要的病原体，是所有致病性革兰氏阴性菌中 PG 生物发生的模型系统我们的前两个目标是验证使 MRSA 对 ¿ 重新敏感的抑制剂的新靶标。 -内酰胺类细菌获得了一种称为 PBP2A 的 PG 转肽酶，可促进 ¿我们发现 PBP2A 的功能取决于糖基转移酶 TarS 的活性，该酶附着 ¿ -O-GlcNAc 残基到壁磷壁酸 (WTA)，这是一种对金黄色葡萄球菌细胞分裂很重要的额外细胞壁聚合物，这表明 PG 和 WTA 合成途径在某种程度上是相互关联的。揭示这些联系的机制基础的方法，以便我们可以利用它们作为打击目标 ¿ -MRSA 中的内酰胺耐药性。我们还将通过监测小分子的作用来探索 TarS 本身作为药物靶点。我们的第二组目标集中于了解 PG 合成机器的功能并验证它们作为抗生素靶标的作用，但令人惊讶的是，人们对它们作为潜在药物靶标的重要性知之甚少。这些机器组装 PG 的机制主要是由于分析其功能的基因分析有限，我们利用大肠杆菌系统的遗传易处理性开发了第一个阳性结果。 针对细胞延伸所需的高度保守的杆系统的活性进行选择，我们使用这种选择来鉴定杆功能的小分子拮抗剂，并建议确定它们的具体靶点和作用方式。对多蛋白 Rod 复合物的结构进行遗传学研究，并鉴定对每个成分的功能至关重要的氨基酸残基，联合化学遗传分析将帮助我们鉴定和验证适合新型疗法靶向的 Rod 系统功能的各个方面。 PG 和我们将研究的 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，并且应显着影响和指导针对 MRSA 和革兰氏阴性 ESKAPE 的治疗方法的努力病原体。