Pseudomonas aeruginosa heme sensing inhibitors targeting HasAp

针对 HasAp 的铜绿假单胞菌血红素传感抑制剂

• 批准号: 10331888
• 负责人: Angela Wilks
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-22 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331888
• 关键词: Acute; Affinity; Anabolism; Animal Model; Antibiotics; Assimilations; Biliverdine; Binding; Binding Sites; Biochemical; Biological Assay; Caenorhabditis elegans; Cells; Chronic; Clinical; Complex; Computer Assisted; Computer-Aided Design; Coupling; Crystallization; Cystic Fibrosis sputum; Deuterium; Development; Drug Design; Epitopes; Feedback; Fluorescence; Formulation; Gallium; Gallium Nitrate; Gases; Genes; Genetic; Genetic Transcription; Goals; Growth; Heme; Heme Iron; Hemorrhage; Homeostasis; Hydrogen; Immune; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Infection; Iron; Isotope Labeling; Ligands; Lung infections; Maps; Mass Spectrum Analysis; Measures; Membrane; Membrane Proteins; Messenger RNA; Metals; Methodology; Methods; Microbial Biofilms; Minimum Inhibitory Concentration measurement; Modeling; Multi-Drug Resistance; Mus; Nosocomial Infections; Oxidation-Reduction; Patients; Peptide Hydrolases; Periodicity; Pharmacology; Positioning Attribute; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Reporter; Research; Role; Series; Siderophores; Signal Transduction; Site; Spottings; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Time; Transcriptional Activation; Type III Secretion System Pathway; Virulence; World Health Organization; acute infection; analog; antimicrobial; base; chronic infection; cystic fibrosis patients; design; extracellular; in vivo; in vivo evaluation; inhibitor; lead optimization; multidrug-resistant Pseudomonas aeruginosa; novel; novel strategies; novel therapeutic intervention; opportunistic pathogen; pathogen; pharmacophore; pulmonary function decline; pyochelin; pyoverdin; receptor; resistant strain; salophen; siderophore receptors; small molecule; trait; transcriptomics; treatment strategy; uptake

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.

项目摘要 多药耐药（MDR）铜绿假单胞菌（Pa）导致约10%的医院感染， 突出了开发新的治疗方法的迫切需要。威尔克斯实验室已经证明 从囊性纤维化患者分离的慢性Pa肺部感染降低了对铁-铁载体的依赖， 随着时间的推移吸收，同时增加对血红素的依赖。我们的基因和生化分析显示 Pa Has和Phu系统分别在血红素运输和传感中具有非冗余作用。 转录组学显示细胞外血细胞hasAp及其外膜受体的mRNA水平 hasR是急性鼠肺感染模型中最显著上调的基因。在同一模型中， 一株HisR菌株显示出显著降低的生长和毒力。此外，氧化还原非活性化合物的制剂 金属镓（例如，Ganite）已通过靶向铁摄取系统而在临床上用作抗微生物剂。我们 初步研究表明，稳定的镓-水杨酸复合物GaSal与HasAp结合， 血红素信号级联，降低Pa感知和利用血红素的能力。与此同时， 作为Pa的铁载体摄取系统的异种铁载体，导致细胞内失调 铁的体内平衡。我们的中心假设是同时抑制Pa血红素感应通过靶向 细胞外血红蛋白HasAp，同时优化异种铁载体受体摄取是一种新的策略， 治疗Pa感染。我们的目标是合成一系列GaSal类似物，并使用已建立的 分析，以鉴定、验证和表征血红素信号传导和铁稳态的有效抑制剂。到 为了实现我们的目标，我们将合成新的砷化镓类似物，这些类似物是使用一种新的计算机辅助设计的。 药物设计（CADD）方法学SILCS（目标1）。将对合成的化合物进行FQ测定 来确定它们与HasAp的亲和力将进一步评估选定的抑制剂对血红素信号传导的抑制 和摄取分别使用转录报告基因测定和13 C-血红素LC-MS/MS测定。砷化镓吸收， 铁载体受体将通过使用ICP-MS测量细胞内Ga水平来定量。 将确定选定化合物对一组Pa菌株的MIC 50和生物膜抑制。中国最高 候选物，我们将进一步测试它们在C.优雅的顶级化合物的HasAp结合表位 将通过STD-，HSQC-NMR和HDX-MS确定。我们的合作研究团队具有强大的跟踪能力， 进行CADD、靶向优化以及化合物的体外和体内评价的记录。 总的来说，我们的方法使我们处于一个独特的位置，可以首先识别、验证和表征小型 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 受体的MDR Pa，并确定这种新的作用机制是否是一个可行的选择， 抗菌剂的发展。