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: 9674978
• 负责人: Mario Rivera
• 金额: $ 29.57万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 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/9674978
• 关键词: Chemical; tools; perturbing; iron; homeostasis

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.

抽象的 抗生素耐药性是一个全球问题，它有可能在现代中解除重要程序 医学，例如癌症治疗，器官移植等。P。铜绿疾病是医院的重要来源 囊性纤维化患者的感染和死亡率的主要原因。对抗抗生素 耐药需要新的抗生素和新靶标。我们建议将铁稳态验证为新的 未来开发抗生素的目标。细菌铁稳态具有重要的脆弱性，因为 必须从宿主那里获得必需的铁，这使得营养稀缺到入侵病原体 （营养免疫）。病原体已经发展了从宿主那里“窃取”铁的机制，但是这些取决于 关于良好调节的铁稳态。我们针对铁储存之间的蛋白质/蛋白质相互作用 蛋白质细菌毒素（BFRB）及其相关的铁蛋白（BFD），这对于调节胞质铁是必不可少的 浓度。重要的是，BFRB和BFD仅存在于细菌中。提议的工作是由我们的水晶建立的 BFRB：BFD复合物的结构，并从表明阻断BFRB的情况下：BFD相互作用破坏了铁 稳态，导致铜绿假单胞菌细胞变得不足，在秀丽隐杆线虫中的毒气明显降低 感染模型。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（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）。目的是： 1）出现针对BFRB的小分子问题：体外和铜绿假单胞菌中的BFD相互作用。 （a）我们会的 利用基于结构的设计原理来开发从筛选小的分子中获得的承诺分子 文库成BFRB的潜在抑制剂：BFD相互作用。 （b）屏幕一个大库以找到其他分子 结合BFRB。这些将与BFRB进行共结晶试验和新兴的结构。 用于指导其合成阐述到能够阻止BFRB的问题的信息：BFD相互作用。 2）研究使用遗传和化学的铜绿假单胞菌中BFRB：BFD相互作用的后果 干涉。我们将使用遗传技术来询问阻断BFRB的影响：BFD相互作用对 铜绿假单胞菌的铁稳态。当我们使用基因缺失和突变对这些效果了解这些效果时 染色体，我们将使用新兴信息作为基准来评估化学物质的效率 AIM 1出现了问题。 新问题将成为有史以来询问细菌铁稳态的第一个分子工具，首先是 模型有机体，例如铜绿假单胞菌PAO1，然后在铜绿假单胞菌的临床分离株中，最终在其他 bfrb：BFD相互作用的致病细菌是保守的。