GENETIC AND CHEMICAL APPROACHES TO NOVEL LIPID II BINDING PEPTIDE ANTIBIOTICS

描述(由申请人提供)：抗生素耐药感染的频率不断上升，加上新的传染病的出现，突出了开发新的抗生素和更好地了解抗生素耐药机制的迫切需要。这项建议通过对耐尿酸的研究来满足这两种需求，耐酸是一种由真菌链霉菌产生的脂肽抗生素，也是候选药物雷莫拉宁的类似物。这些多肽与万古霉素的作用机制相似，但在不同的位置结合了Lipid II靶标，并对威胁生命的万古霉素耐药细菌感染具有活性。这项建议的总体目标是完成克隆的耐尿酸生物合成基因簇的功能鉴定，并利用这些信息生成新的耐酸类似物，并阐明生产生物体对该肽的抗性机制。耐酸蛋白生物合成基因簇和侧翼区的功能分析(AIM 1)将采用一种新的快速构建真菌链霉菌突变文库的方法，在该库中，簇和侧翼区中的选定基因被分别扰乱。这将使对抗生素生物合成各个方面的单个基因功能丧失的全面分析成为可能。在目标1中获得的知识和工具将推动使用化学和分子遗传学方法的组合来创造耐尿酸类似物(目标2)。利用外源和原位生成的前体，通过基因破坏和突变生物合成，将产生新的多肽。将制备具有不同脂类侧链的化合物，这些化合物可能具有更好的治疗性能，或者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，将使用基因中断/互补来进一步检查脂尾的变化、多肽卤化的作用以及各种氨基酸残基的功能。多肽核心中的取代将通过基因交换实验来完成，这些实验旨在为额外的化学修饰产生独特的支架，并探索耐尿酸多肽合成酶中罕见的双功能异构化/缩聚结构域的灵活性。目标3的目的是鉴定真菌链霉菌中对多肽产生自我抗性的遗传元件，并破译免疫的生化机制。除了生产有潜力作为治疗威胁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导致为药物发现增加天然产品化学多样性的新途径。了解产生耐尿酸的生物体耐药的分子基础将有助于预测新的抗生素可能如何受到现有耐药机制的影响，并提供对可能出现耐药性的方式的洞察。

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.