Small molecules for perturbing iron homeostasis in bacterial biofilms

摘要 慢性伤口护理特别复杂的细菌生物膜的形成，通常由假单胞菌 铜绿假单胞菌、鲍曼不动杆菌和金黄色葡萄球菌。生物膜对抗生素治疗是无效的， 这是慢性伤口感染如此难以根除的重要原因。存在显著 我们需要找到对抗生物膜的新策略。细菌铁稳态是一个合适的新靶点， 开发抗感染药物，因为宿主使必需的铁缺乏入侵病原体。细菌已经 它们进化出了从宿主体内“窃取”铁的机制，但这些机制依赖于调节良好的铁体内平衡。到 破坏细菌铁稳态，我们针对铁储存蛋白BfrB及其生理伴侣 蛋白质，Bfd，这是细菌特有的。我们的工作表明，BfrB调节胞浆铁浓度 通过（a）氧化Fe 2+并在其内腔中储存高达约3，000个Fe 3+离子，以及（B）与Bfd形成络合物 还原BfrB内腔的Fe ~（3+），并将Fe ~（2+）释放到胞质溶胶中，使其参与代谢。 通过缺失bfd基因阻断铜绿假单胞菌细胞中的BfrB-Bfd复合物， 引发BfrB中Fe 3+的不可逆积累和同时的胞质铁耗竭，这导致 受损的生物膜维持和生物膜细胞死亡。我们的工作还导致了概念验证的发现 BfrB-Bfd复合物的抑制剂，其在Bfd结合位点结合BfrB，抑制铁动员，并引起铁结合。 生物膜细胞死亡。本申请的目的是将概念验证分子改进为铅 分子，评估它们的抗菌和抗菌膜的有效性，并评估它们潜在的药物适用性。 我们的跨学科团队包括细菌铁稳态的化学和结构生物学专业知识 （里维拉）、有机合成和反应机理（邦斯）、制药工业研究和 抗感染药物的开发（雷茨），药物发现中的X射线晶体学（洛弗尔），微生物遗传学 （钱德勒）和感染动物模型（Morici）。具体目标是：1）发展概念验证类似物 BfrB-Bfd复合物的有效抑制剂。这项工作将遵循一个系统的，迭代的战略， 化学合成优化依赖于抑制剂结合BfrB的晶体结构来设计每个新的 抑制剂的产生。2)评估BfrB-Bfd复合物抑制剂的抗菌效果， 评估其潜在的药物适用性。

ABSTRACT Chronic wound care is especially complicated by the formation of bacterial biofilms, commonly by Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus. Biofilms are recalcitrant to antibiotic therapy, which is an important reason why chronic wound infections are so difficult to eradicate. There is a significant need to discover new strategies to combat biofilms. Bacterial iron homeostasis is a suitable novel target for developing anti-infectives because the host makes essential iron scarce to invading pathogens. Bacteria have evolved mechanisms to “steal” iron from their host, but these depend on well-regulated iron homeostasis. To disrupt bacterial iron homeostasis, we are targeting the iron storage protein BfrB and its physiological partner protein, Bfd, which are unique to bacteria. Our work has shown that BfrB regulates cytosolic iron concentrations by (a) oxidizing Fe2+ and storing up to ~3,000 Fe3+ ions in its internal cavity, and (b) forming a complex with Bfd to reduce Fe3+ in the internal cavity of BfrB and release Fe2+ to the cytosol for its incorporation in metabolism. Blocking the BfrB-Bfd complex in P. aeruginosa cells by deletion of the bfd gene perturbs iron homeostasis by triggering an irreversible accumulation of Fe3+ in BfrB and simultaneous cytosolic iron depletion, which leads to impaired biofilm maintenance and biofilm cell death. Our work also led to the discovery of proof-of-concept inhibitors of the BfrB-Bfd complex, which bind BfrB at the Bfd binding site, inhibit iron mobilization, and elicit biofilm cell death. The objectives of this application are to improve the proof-of-concept molecules into lead molecules, evaluate their antibacterial and antibiofilm effectiveness, and evaluate their potential drug suitability. Our interdisciplinary team includes expertise in chemical and structural biology of bacterial iron homeostasis (Rivera), organic synthesis and reaction mechanisms (Bunce), pharmaceutical industry research and development of anti-infectives (Reitz), X-ray crystallography in drug discovery (Lovell), microbial genetics (Chandler), and animal model of infection (Morici). The specific aims are: 1) Evolve proof-of-concept analogs into potent inhibitors of the BfrB-Bfd complex. This work will follow a systematic, iterative strategy of medicinal chemistry synthesis optimization that relies on crystal structures of inhibitor bound BfrB to design each new generation of inhibitors. 2) Asses the antibacterial effectiveness of inhibitors of the BfrB-Bfd complex and evaluate their potential drug suitability.