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: 7658090
• 负责人: XIHOU YIN
• 金额: $ 35.86万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7658090
• 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加上新的传染病的出现强调了开发新型抗生素和更好地理解抗生素耐药机制的关键需求。该提案通过对耐酰胺素的描述研究（一种由链霉菌真菌产生的脂蛋白抗生素和候选药物候选拉莫普兰素的类似物）来解决这两种需求。这些肽与万古霉素具有相似的作用机理，但在不同的位点结合脂质II靶标，并且具有危及生命的万古霉素抗性细菌感染的活跃。该提案的总体目标是完成克隆的耐酰胺生物合成基因簇的功能表征，并使用此信息来产生新型的耐耐核苷类似物，并阐明生产生物体中对肽的抗性机制。耐酸生物合成基因簇和侧翼区域的功能分析（AIM 1）将采用一种新颖而快速的方法来构建真菌链球菌突变库文库，其中群集和侧翼区域中选择的基因被单独中断。这将允许对抗生素生物合成各个方面的各个方面的单个基因丧失进行全面分析。 AIM 1中获得的知识和工具将使用化学和分子遗传学方法的组合来驱动耐酰胺类似物的创建（AIM 2）。新型肽将通过使用外源和原位产生的前体来通过基因破坏和突变生物合成产生。化合物将与脂质侧链的变化制备，该链链可能提高了治疗特性，或者将作为靶向分析和动力学研究的分子工具（AIM 2A）。在AIM 2B中，基因破坏/互补将用于进一步检查脂质尾巴的改变，肽卤素的作用以及各种氨基酸残基的功能。肽芯中的取代将通过基因交换实验来实现，旨在产生独特的支架，以进行其他化学修饰，并探索耐耐酸素肽合成酶中罕见的双重功能表达/缩合域的灵活性。目标3的目的是确定杀真菌链球菌中的遗传因素，这些遗传因素赋予肽自我抗性并破译免疫的生化机制。除了产生具有潜力的新型抗生素外，还可以改善治疗生命威胁革兰氏阳性细菌感染的治疗剂外，这项工作中开发的知识和方法还将带来新的方法来增加天然产物化学多样性以发现药物发现。了解生产生物体中耐酸肽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，并提供对抗性可能出现的方式的见解。