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，它威胁着现代医学的重要程序。 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 获得性感染和囊性纤维化患者死亡的主要原因。对抗抗生素 需要新的抗生素和新的靶点。我们建议验证铁稳态作为一种新的 抗生素未来发展目标。细菌铁稳态提供了一个显著的脆弱性， 必需的铁必须从宿主体内获取，这使得入侵病原体的营养物质稀缺 （营养免疫）。病原体已经进化出了从宿主那里“偷”铁的机制，但这些机制取决于 对铁平衡的调节作用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）及其相关的铁氧还蛋白（Bfd），它们是调节胞浆铁所必需的 浓度的重要的是，BfrB和Bfd只存在于细菌中。这项计划是从我们的水晶 BfrB：Bfd复合物的结构，并从已经表明，阻断BfrB：Bfd相互作用破坏铁 体内平衡，导致铜绿假单胞菌细胞变得缺铁，并且在C. elegans 感染的模式。我们的方法是多学科的，涉及具有细菌铁专业知识的研究人员 代谢和结构生物学（里维拉），有机合成/药物发现（邦斯），化学生物学和 药物化学（彼得森）和微生物学（钱德勒）。其目的是： 1)开发用于在体外和铜绿假单胞菌中阻断BfrB：Bfd相互作用的小分子探针。(A)我们将 利用基于结构的设计原理开发有前途的分子， 库中的BfrB：Bfd相互作用的有效抑制剂。(B)筛选一个大的文库以寻找额外的分子 与BfrB结合这些将经受与BfrB的共结晶试验，并且新兴的结构 用于指导其合成加工成能够阻断BfrB：Bfd相互作用的探针的信息。 2)研究干扰铜绿假单胞菌中BfrB：Bfd相互作用的后果， 干预我们将使用遗传技术来询问阻断BfrB：Bfd相互作用对 铜绿假单胞菌铁稳态当我们了解到这些影响时， 染色体，我们将使用新出现的信息作为基准，以评估化学品的功效 目标1中开发的探测器。 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， 模式生物如铜绿假单胞菌PAO 1，然后在铜绿假单胞菌的临床分离株中，并最终在其他 BfrB：Bfd相互作用是保守的。