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: 8279957
• 负责人: Thomas G Bernhardt
• 金额: $ 93.63万
• 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/8279957
• 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 Delivery Systems; 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. PUBLIC HEALTH RELEVANCE: New strategies to treat antibiotic resistant bacterial infections are sorely needed. This project combines small molecules and genetic methods to identify and validate new antibiotic targets in the pathway for assembly of the bacterial cell wall. The proposed work may lead to new therapies against methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative ESKAPE pathogens.

描述（适用提供）：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性感染的原因。迫切需要治疗这些感染的新策略。靶向肽聚糖（PG）/细胞壁生物发生的抗生素是治疗细菌感染的最有效药物之一，但抗药性已经出现在所有目前从事临床使用的抗生素。拟议的工作是由于最近使用� -lactams作为PG生物合成的化学问题而提出的。它旨在鉴定和验证甲氧西林抗葡萄球菌金黄色葡萄球菌（MRSA）和大肠杆菌（大肠杆菌）中细胞壁组装途径中的新靶标。 MRSA是Eskape病原体中最具毒性的一种，大肠杆菌本身就是重要的病原体，是所有致病性革兰氏阴性棒中PG生物发生的模型系统。我们的前两个目的是侧重于验证将MRSA归因于 - lactams的抑制剂的新目标。 MRSA获得了一种称为PBP2A的PG转肽酶，该酶促进了-lactam抗性。我们发现PBP2A函数取决于附着的糖基转移酶Tars的活性，该糖基转移酶（TAR）附着 - O -O -GlCNAC保留在壁teichoic酸（WTAS）上，这是对金黄色葡萄球菌的细胞分裂重要的其他细胞壁聚合物。这表明PG和WTA合成的途径在某种程度上互连。我们将使用遗传和化学方法的组合来揭示这些连接的机械基础，以便我们可以探索它们作为对抗MRSA中的lactam耐药性的目标。我们还将通过监测小分子� -lactam势能对其活性的影响并以有限抑制剂的抑制作用来解决其结构，从而探索焦油本身作为药物靶标。我们的第二组目标专注于理解PG合成机器的功能，并将其验证为抗生素靶标。鉴于它们作为潜在药物靶标的重要性，对这些机器的PG组装机制知之甚少。这主要是由于遗传评估的可用性有限，以剖析其功能。利用大肠杆菌系统的遗传障碍性，我们开发了第一个阳性 针对PG组装机的活性，这是细胞伸长所需的高度组成的杆系统。我们使用此选择来识别杆功能和建议的小分子拮抗剂，以确定其特定目标和作用方式。我们还将使用我们的选择来遗传询问多蛋白棒复合物的结构，并识别氨基酸对每个成分的功能保留至关重要。化学遗传分析的组合将有助于我们识别和验证杆系统功能的各个方面，可通过新颖的治疗进行靶向。由于我们将研究的PG和WTA合成机是高度保守的，因此我们在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，并应显着影响并为对MRSA和革兰氏阴性Eskape Eskape病原体产生疗法的努力。 公共卫生相关性：迫切需要治疗抗生素抗生素感染的新策略。该项目结合了小分子和遗传方法，以识别和验证细菌细胞壁组装途径中的新抗生素靶标。拟议的工作可能导致针对抗甲氧西林的金黄色葡萄球菌（MRSA）和革兰氏阴性埃斯卡普病原体的新疗法。