GENETIC AND CHEMICAL APPROACHES TO NOVEL LIPID II BINDING PEPTIDE ANTIBIOTICS

新型脂质 II 结合肽抗生素的遗传和化学方法

• 批准号: 7904153
• 负责人: XIHOU YIN
• 金额: $ 35.5万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904153
• 关键词: Active Sites; Acute; Address; Affect; Amino Acid Substitution; Amino Acids; Anabolism; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacterial Antibiotic Resistance; Bacterial Infections; Binding; Biochemical; Biochemical Process; Biological; Biological Factors; Cell Wall; Chemicals; Clinical; Communicable Diseases; Complex; Development; Drug resistance; Emerging Communicable Diseases; Evaluation; Family; Frequencies; Gene Cluster; Genes; Genetic; Glycopeptides; Goals; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Hand; Hospitals; Immunity; In Situ; In Vitro; Individual; Infection; Kinetics; Knowledge; Laboratories; Lead; Libraries; Life; Lipid Binding; Lipids; Long-Term Care; Methodology; Methods; Mining; Modification; Molecular; Molecular Genetics; Mutate; Organism; Parents; Pattern; Peptide Antibiotics; Peptides; Peptidoglycan; Pharmaceutical Preparations; Photoaffinity Labels; Physical condensation; Population; Production; Property; Public Health; Reporting; Research Personnel; Resistance; Resources; Role; Side; Streptomyces; Structure; Substrate Specificity; Tail; Therapeutic; Therapeutic Agents; Vancomycin; Vancomycin Resistance; Variant; Work; analog; base; cellular targeting; chemical genetics; combinatorial; community setting; design; drug candidate; drug discovery; epimerization; extracellular; flexibility; genetic element; halogenation; improved; in vitro activity; in vivo; innovation; insight; loss of function; methicillin resistant Staphylococcus aureus; mouse model; mutant; novel; novel therapeutics; peptide synthase; physical property; portability; prevent; programs; ramoplanin; rapid technique; research study; resistance mechanism; scaffold; tool

DESCRIPTION (provided by applicant): The escalating frequency of antibiotic resistant infections combined with the emergence of new infectious diseases underscores the critical need to develop novel antibiotics and better understand mechanisms of antibiotic resistance. This proposal addresses both of these needs through the described studies on enduracidin, a lipopeptide antibiotic produced by Streptomyces fungicidicus and an analog of the drug candidate ramoplanin. These peptides share a similar mechanism of action with vancomycin but bind the Lipid II target at a distinct site and are active against life-threatening vancomycin-resistant bacterial infections. The overall objectives of this proposal are to complete the functional characterization of the cloned enduracidin biosynthetic gene cluster and use this information to generate novel enduracidin analogs and elucidate the mechanism of resistance to the peptide in the producing organism. Functional analysis of the enduracidin biosynthetic gene cluster and flanking regions (aim 1) will employ a novel and rapid method of constructing a S. fungicidicus mutant library in which selected gene in the cluster and flanking regions is individually disrupted. This will permit a comprehensive analysis of individual gene loss-of-function on all aspects of antibiotic biosynthesis. The knowledge and tools acquired in aim 1 will drive the creation of enduracidin analogs using a combination of chemical and molecular genetics methods (aim 2). Novel peptides will be generated through gene disruption and mutational biosynthesis using exogenous and in situ generated precursors. Compounds will be prepared with variations in the lipid side chain that may have improved therapeutic properties or will serve as molecular tools for target analysis and kinetic studies (aim 2a). In aim 2b, gene disruption/complementation will be used to further examine alterations to the lipid tail, the role of peptide halogenation, and the function of various amino acid residues. Substitutions in the peptide core will be accomplished through gene swapping experiments designed to yield unique scaffolds for additional chemical modifications and also to explore the flexibility of the rare dual function epimerization/condensation domains in the enduracidin peptide synthetases. The goal of aim 3 is to identify the genetic elements in S. fungicidicus that confer self-resistance to the peptide and decipher the biochemical mechanism of immunity. In addition to producing novel antibiotics with potential as improved therapeutic agents for treating life- threatening Gram-positive bacterial infections, the knowledge and methods developed in this work will lead to new ways to increase natural product chemical diversity for drug discovery. Understanding the molecular basis of enduracidin resistance in the producing organism will help predict how new antibiotics may be affected by existing resistance mechanisms and provide insight into ways that resistance may emerge.

描述（由申请人提供）：抗生素耐药性感染的频率不断上升，加上新传染病的出现，强调了开发新型抗生素和更好地了解抗生素耐药性机制的迫切需要。该提案通过对enduracidin（一种由杀真菌链霉菌产生的脂肽抗生素和候选药物雷莫拉宁的类似物）的研究来满足这两个需求。这些肽与万古霉素具有相似的作用机制，但在不同的位点结合脂质 II 靶点，并且能够有效对抗危及生命的万古霉素耐药细菌感染。该提案的总体目标是完成克隆的enduracidin生物合成基因簇的功能表征，并利用该信息生成新型enduracidin类似物，并阐明生产生物体对该肽的耐药机制。 enduracidin 生物合成基因簇和侧翼区域的功能分析（目标 1）将采用一种新颖且快速的方法来构建杀真菌链球菌突变体文库，其中簇和侧翼区域中的选定基因被单独破坏。这将允许对抗生素生物合成各个方面的单个基因功能丧失进行全面分析。目标 1 中获得的知识和工具将推动结合化学和分子遗传学方法创建 enduracidin 类似物（目标 2）。新的肽将通过使用外源和原位产生的前体进行基因破坏和突变生物合成来产生。将制备具有脂质侧链变化的化合物，这些变化可能具有改善的治疗特性或将作为靶点分析和动力学研究的分子工具（目标2a）。在目标 2b 中，基因破坏/互补将用于进一步检查脂质尾部的改变、肽卤化的作用以及各种氨基酸残基的功能。肽核心的取代将通过基因交换实验来完成，该实验旨在产生用于额外化学修饰的独特支架，并探索恩尿酸素肽合成酶中罕见的双功能差向异构化/缩合结构域的灵活性。目标 3 的目标是鉴定杀真菌链球菌中赋予肽自我抵抗力的遗传元件，并破译免疫的生化机制。除了生产有潜力作为治疗危及生命的革兰氏阳性细菌感染的改进治疗剂的新型抗生素外，这项工作中开发的知识和方法还将带来增加药物发现天然产物化学多样性的新方法。了解生产生物体中耐尿酸素耐药性的分子基础将有助于预测新抗生素如何受到现有耐药机制的影响，并深入了解耐药性可能出现的方式。