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世界性问题，它有可能解除现代重要程序的武装。 医学，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。对抗抗生素 耐药性需要新的抗生素和新的靶标。我们建议验证铁稳态作为一种新的 抗生素未来发展的目标。细菌铁稳态具有显着的脆弱性，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，但这取决于 良好调节的铁稳态。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) 及其相关铁氧还蛋白 (Bfd)，它们是调节胞质铁所必需的 浓度。重要的是，BfrB 和 Bfd 仅存在于细菌中。拟议的工作以我们的水晶为基础 BfrB:Bfd 复合物的结构，并表明阻断 BfrB:Bfd 相互作用会破坏铁 体内平衡，导致铜绿假单胞菌细胞缺铁，并且秀丽隐杆线虫的毒性显着降低 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）。目标是： 1) 开发小分子探针，用于在体外和铜绿假单胞菌中阻断 BfrB:Bfd 相互作用。 （一）我们会 利用基于结构的设计原理来开发通过筛选小分子获得的有前途的分子 文库转化为 BfrB:Bfd 相互作用的有效抑制剂。 (B) 筛选大型文库以寻找其他分子 结合 BfrB。这些将与 BfrB 进行共结晶试验，并且新兴的结构 用于指导他们合成能够阻断 BfrB:Bfd 相互作用的探针的信息。 2) 使用遗传和化学方法研究干扰铜绿假单胞菌中 BfrB:Bfd 相互作用的后果 干涉。我们将使用遗传技术来探究阻断 BfrB:Bfd 相互作用对 铜绿假单胞菌中的铁稳态。当我们通过基因删除和突变了解这些影响时 染色体，我们将使用新出现的信息作为基准来评估化学物质的功效 目标 1 中开发的探针。 新探针将成为有史以来第一个用于研究细菌铁稳态的分子工具，首先是在 模型生物，如铜绿假单胞菌 PAO1，然后是铜绿假单胞菌的临床分离株，最终是其他 BfrB:Bfd 相互作用是保守的病原菌。