Pseudomonas aeruginosa heme sensing inhibitors targeting HasAp

项目总结 耐多药铜绿假单胞菌(PA)约占医院感染的10%， 强调开发新的治疗方法的迫切需要。威尔克斯实验室已经证明 囊性纤维化患者慢性肺炎肺部感染可减少对铁载体的依赖 随着时间的推移，对血红素的吸收，同时增加对血红素的依赖。我们的遗传和生化分析的特点是 PA系统和Phu系统分别在血红素的运输和传感中起着非多余的作用。 转录组学显示细胞外血细胞hasAp及其外膜受体的mRNA水平 HASR是急性小鼠肺部感染模型中最显著的上调基因。在相同的模型中， 一株∆HASR菌株的生长和毒力显著降低。此外，氧化还原失活的配方 通过靶向铁摄取系统，金属镓(如Ganite)已被用作临床上的抗菌剂。我们的 初步研究表明，稳定的镓-沙洛芬络合物GaSal与HasAp和嵌段结合 血红素信号的级联，降低了PA感知和利用血红素的能力。与此同时，GaSal 作为外源铁载体对PA的铁载体摄取系统的作用，导致细胞内失调 铁稳态的影响。我们的中心假设是，通过靶向同时抑制PA血红素的感觉 细胞外血细胞HasAp在优化外源铁载体受体摄取的同时是一种新的策略 肺炎链球菌感染的治疗。我们的目标是合成一系列GaSal类似物，并使用已建立的 检测，以识别、验证和表征有效的血红素信号和铁稳态抑制物。至 实现我们的目标，我们将合成新的GaSal类似物，这些类似物是使用一种新型的计算机辅助设计的 药物设计(CADD)方法SILCS(目标1)。合成的化合物将进行荧光定量分析。 以确定它们与HasAp的亲和力。将进一步评估选定的抑制剂对血红素信号的抑制作用。 分别采用转录报告法和13C-血红素LC-MS/MS法进行摄取。GaSal摄取 铁载体受体将通过使用电感耦合等离子体质谱(ICMS)测量细胞内的镓含量来定量。在目标2中，我们 将测定选定化合物对一组PA菌株的MIC50和生物膜抑制作用。对于顶层来说 对于候选人，我们将进一步测试它们在线虫体内的效果。TOP化合物的HasAp结合表位 将通过STD-、HSQC-核磁共振和HDX-MS确定。我们的合作研究团队有一个强大的轨道 化合物的CADD、Hit-to-Lead优化以及体外和体内评估的记录。 总体而言，我们的方法使我们处于独特的地位，可以识别、验证和表征小班第一名 具有抑制血红素信号级联和模拟铁载体底物双重活性的分子 并确定这一新的作用机制是否是一种可行的选择 抗菌剂的开发。

PROJECT SUMMARY Multidrug-resistant (MDR) Pseudomonas aeruginosa (Pa) is responsible for ~10% of nosocomial infections, highlighting the critical need for the development of novel therapeutic approaches. The Wilks Lab has shown that chronic Pa lung infection isolates from cystic fibrosis patients decrease the reliance on iron-siderophore uptake over time, while increasing the reliance on heme. Our genetic and biochemical analysis characterized the Pa Has and Phu systems as having non-redundant roles in heme transport and sensing, respectively. Transcriptomics showed mRNA levels of the extracellular hemophore hasAp and its outer membrane receptor hasR are the most significantly upregulated genes in an acute murine lung infection model. In the same model, a ∆hasR strain showed significantly reduced growth and virulence. Moreover, formulations of the redox inactive metal gallium (e.g., Ganite) have been clinically used as antimicrobials by targeting iron uptake systems. Our preliminary studies have shown that the stable gallium-salophen complex, GaSal, binds to HasAp and blocks the heme-signaling cascade, decreasing the ability of Pa to sense and utilize heme. At the same time, GaSal functions as a xenosiderophore for the siderophore uptake systems of Pa, leading to intracellular dysregulation of iron homeostasis. Our central hypothesis is that simultaneous inhibition of Pa heme sensing by targeting the extracellular hemophore HasAp while optimizing xenosiderophore receptor uptake is a novel strategy for the treatment of Pa infections. Our goal is to synthesize a series of GaSal analogs and test them using established assays, to identify, validate, and characterize potent inhibitors of heme signaling and iron homeostasis. To achieve our goal, we will synthesize new GaSal analogs that have been designed using a novel computer-aided drug design (CADD) methodology SILCS (Aim 1). The synthesized compounds will be subjected to the FQ assay to determine their affinities to HasAp. Selected inhibitors will be further assessed for inhibition of heme signaling and uptake using transcriptional reporter assay and 13C-heme LC-MS/MS assay, respectively. GaSal uptake by siderophore receptors will be quantified by measuring the intracellular Ga levels using ICP-MS. In Aim 2, we will determine MIC50 and biofilm inhibition of selected compounds on a panel of Pa strains. For the top candidates, we will further test their in vivo efficacy in C. elegans. The HasAp binding epitope of top compounds will be determined by STD-, HSQC-NMR and HDX-MS. Our collaborative research team has a strong track record of performing CADD, hit-to-lead optimization, and in vitro and in vivo evaluation of compounds. Collectively, our approach puts us in a unique position to identify, validate, and characterize first in class small molecule with dual activity of inhibiting heme signaling cascade and mimicking the substrate of siderophore receptor of MDR Pa, and to determine whether this novel mechanism of action is a viable option for the development of antimicrobials.