Heme Oxygenase: Structure, function and pathogenesis

病原细菌需要铁才能生存和引起感染。亚铁血红素占90%。 铁在宿主体内有效，在许多细菌的定植和毒力中起着重要作用 病原体。铜绿假单胞菌是引起医院感染的一种越来越常见的致病菌。 医院环境中的感染。此外，与假单胞菌感染相关的高发病率 囊性纤维性疾病，再加上这种微生物表现出的天然抗生素耐药性，突出了 需要替代的抗菌策略。此前的生化和生物物理研究表明， 细胞内血红素转运蛋白PHU和铁调节的血红素加氧酶pa-HO在 利用血红素作为铁源。该提案的具体目的是：一)确定关系 血红素利用、铁稳态和毒力之间的关系 生化研究。二)通过计算机辅助药物设计(CADD)和在电子计算机数据库中筛选低- 高概率与胞质血红素结合蛋白结合的分子量化合物 (PHU)或脱氢血红素加氧酶(HO)；iii)确定推定的体外和体内性质 抑制剂。与目标蛋白的结合亲和力将使用生物物理和 光谱学方法(荧光，ITC和核磁共振)和体内分析缓蚀剂的有效性 细菌感染的最低抑菌浓度MIC50筛选和秀丽线虫宿主-病原体模型 通过先导化合物验证和蛋白质抑制剂的结构表征优化抑制剂 通过X-射线结晶学和核磁共振方法，为今后基于CADD的研究奠定了基础 领导配方优化。在拟议的研究完成后，收集化学上不同的低- 分子化合物将被确认和验证为HO和PHU的抑制剂。这些 化合物将通过领先的优化努力进一步开发，目标是开发一种新的类别 抗生素。

Pathogenic bacteria require iron for their survival and ability to cause infection. Heme comprises 90% of the iron available within the host and plays a significant role in the colonization and virulence of many bacterial pathogens. The opportunistic pathogen P. aeruginosa is an increasingly common cause of nosocomial infections in the hospital setting. Furthermore, the high morbidity associated with Pseudomonas infection in cystic fibrosis disease, combined with the natural antibiotic resistance shown by this organism, highlights the need for alternate antimicrobial strategies. Previous biochemical and biophysical studies have shown that the intracellular heme trafficking protein PhuS and the iron-regulated heme oxygenase pa-HO are critical in the utilization of heme as an iron source. The specific aims of the proposal are to i) determine the relationship between heme utilization, iron homeostasis and virulence through a combination of genetic, microarray and biochemical studies. ii) identify by Computer-aided drug design (CADD) and in silico database screening low- molecular weight compounds with a high probability of binding to either the cytoplasmic heme binding protein (PhuS) or the apo-heme oxygenase (HO); iii) Determine the in vitro and in vivo properties of the putative inhibitors. Binding affinity to the target protein will be tested using a combination of biophysical and spectroscopic approaches (fluorescence, ITC and NMR) and in vivo analysis of inhibitor efficacy by high- throughput MIC50 screening and the Caenorhabditis elegans host-pathogen model of bacterial infection; iv) Optimize the inhibitors by lead compound validation and structural characterization of the protein-inhibitor complexes by X-ray crystallographic an NMR methods, thereby providing a foundation for future CADD-based lead compound optimization. Upon completion of the proposed study a collection of chemically diverse low- molecular compounds will have been identified and validated as inhibitors of HO and PhuS. These compounds will be further developed through lead optimization efforts with the goal of developing a novel class of antibiotics.