Chemical tools for perturbing iron homeostasis in P. aeruginosa

扰乱铜绿假单胞菌铁稳态的化学工具

• 批准号: 9158507
• 负责人: Mario Rivera
• 金额: $ 38万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9158507
• 关键词: Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; Benchmarking; Binding; Binding Sites; Biological; Biology; Caenorhabditis elegans; Cells; Chemicals; Chromosomes; Ciprofloxacin; Clinical; Complex; Crystallization; Cytosol; Data; Defect; Development; Electrons; Ferredoxin; Future; Gene Deletion; Genes; Genetic Techniques; Growth; Hand; Homeostasis; Human; Immunity; In Vitro; Infection; Intervention; Invaded; Iron; Learning; Libraries; Microbiology; Modeling; Modern Medicine; Molecular; Mutation; Nosocomial Infections; Nutrient; Nutritional; Organ Transplantation; Organic Synthesis; Oxidative Stress; Pharmaceutical Chemistry; Procedures; Proteins; Pseudomonas aeruginosa; Research Personnel; Roentgen Rays; Siderophores; Source; Structure; Testing; Theft; Virulence; Virulent; Work; analog; base; cancer therapy; chemical genetics; combat; cystic fibrosis patients; design; drug discovery; genetic manipulation; inhibitor/antagonist; insight; iron metabolism; killings; meetings; mortality; multidisciplinary; pathogen; pathogenic bacteria; premature; protein protein interaction; response; screening; small molecule; structural biology; tool

ABSTRACT Antibiotic resistance is a worldwide problem, which threatens to disarm important procedures in modern medicine, such as cancer therapy, organ transplantation, etc. P. aeruginosa is a significant source of hospital acquired infections and the leading cause of mortality in patients with cystic fibrosis. To combat antibiotic resistance new antibiotics and new targets are needed. We propose to validate iron homeostasis as a new target for future development of antibiotics. Bacterial iron homeostasis offers a significant vulnerability because essential iron must be obtained from the host, which makes the nutrient scarce to invading pathogens (nutritional immunity). Pathogens have evolved mechanisms to “steal” iron from their host, but these depend on well-regulated iron homeostasis. We are targeting the protein/protein interactions between the iron storage protein bacterioferritin (BfrB) and its associated ferredoxin (Bfd), which are necessary to regulate cytosolic iron concentrations. Importantly, BfrB and Bfd exist only in bacteria. The proposed work builds from our crystal structure of the BfrB:Bfd complex, and from having shown that blocking the BfrB:Bfd interaction disrupts iron homeostasis, causes P. aeruginosa cells to become iron deficient, and significantly less virulent in C. elegans model of infection. Our approach is multidisciplinary and involves investigators with expertise in bacterial iron metabolism and structural biology (Rivera), organic synthesis/drug discovery (Bunce), chemical biology and medicinal chemistry (Peterson), and microbiology (Chandler). The aims are to: 1) Develop small molecule probes for blocking the BfrB:Bfd interaction in vitro and in P. aeruginosa. (A) We will utilize structure based-design principles to develop promising molecules obtained from screening a small library into potent inhibitors of the BfrB:Bfd interaction. (B) Screen a large library to find additional molecules that bind BfrB. These will be subjected to co-crystallization trials with BfrB, and the emerging structural information used to guide their synthetic elaboration into probes capable of blocking the BfrB:Bfd interaction. 2) Study the consequences of perturbing the BfrB:Bfd interaction in P. aeruginosa using genetic and chemical intervention. We will use genetic techniques to interrogate the effects of blocking the BfrB:Bfd interaction on iron homeostasis in P. aeruginosa. As we learn about these effects using gene deletions and mutations to the chromosome, we will use the emerging information as a benchmark to evaluate the efficacy of the chemical probes developed in Aim 1. The new probes will serve as the first-ever molecular tools for interrogating bacterial iron homeostasis, first in a model organism such as P. aeruginosa PAO1, then in clinical isolates of P. aeruginosa, and ultimately in other pathogenic bacteria where the BfrB:Bfd interaction is conserved.

摘要 抗生素耐药性是一个世界性的问题，它威胁着现代社会解除重要程序的武装 药物，如癌症治疗、器官移植等。铜绿假单胞菌是医院的重要来源 获得性感染是囊性纤维化患者死亡的主要原因。与抗生素作斗争 需要抗药性、新的抗生素和新的靶点。我们建议将铁稳态作为一种新的 抗生素未来发展的目标。细菌铁稳态提供了一个重大的脆弱性，因为 必需的铁必须从宿主那里获得，这使得入侵的病原体缺乏营养。 (营养免疫)。病原体已经进化出从宿主“窃取”铁的机制，但这些机制取决于 调节良好的铁稳态。我们的目标是铁储存之间的蛋白质/蛋白质相互作用 蛋白质细菌铁蛋白(BFRB)及其相关的铁还蛋白(BFD)，这是调节胞质铁所必需的 浓度。重要的是，BFRB和BFD只存在于细菌中。建议的工作建立在我们的水晶基础上 BFRB：BFD复合体的结构，并从已经表明阻止BFRB：BFD相互作用扰乱铁 动态平衡，导致铜绿假单胞菌细胞缺铁，对线虫的毒力显著降低 感染模式。我们的方法是多学科的，涉及具有细菌铁专业知识的调查人员。 代谢和结构生物学(Rivera)、有机合成/药物发现(Bunce)、化学生物学和 药物化学(彼得森)和微生物学(钱德勒)。我们的目标是： 1)研制阻断BFRB：BFD相互作用的小分子探针。(A)我们会 利用基于结构的设计原理开发从筛选小分子中获得的有前途的分子 文库成为BFRB：BFD相互作用的有效抑制剂。(B)筛选大型文库以寻找更多分子 这约束了BFRB。这些将接受与BFRB的共结晶试验，以及新兴的结构 用于指导他们合成精化成能够阻止BFRB：BFD相互作用的探针的信息。 2)从遗传和化学角度研究了干扰BFRB：BFD相互作用对铜绿假单胞菌的影响。 干预。我们将使用基因技术来询问阻止BFRB：BFD相互作用的影响 铜绿假单胞菌中铁的动态平衡。随着我们了解到这些影响，通过基因缺失和突变 染色体，我们将使用新出现的信息作为基准来评估该化学物质的疗效 在目标1中开发的探头。 新的探针将作为有史以来第一个询问细菌铁稳态的分子工具，在一个 模式生物，如铜绿假单胞菌PAO1，然后在铜绿假单胞菌的临床分离中，最终在其他 BFRB：BFD相互作用保守的病原菌。