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

基本信息

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
项目类别：
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

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

展开

项目摘要

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.

项目摘要抗多药（MDR）铜绿假单胞菌（PA）负责约10％的医院感染，强调对新型治疗方法发展的批判性需求。威尔克斯实验室已显示从囊性纤维化患者中分离出的慢性PA肺感染剂量降低了对铁螺旋体的依赖随着时间的流逝，吸收了对血红素的依赖。我们的遗传和生化分析表征了 PA具有分别在血红素传输和传感中具有非冗余作用的PHU系统。转录组学显示了细胞外血液hap的mRNA水平及其外膜接收器 HASR是急性鼠肺感染模型中最显着上调的基因。在同一模型中 ∆HASR菌株显示出显着降低的生长和病毒。此外，氧化还原不活动的公式通过靶向铁摄取系统，金属着姜（例如甘酸盐）已被临床用作抗菌剂。我们的初步研究表明，稳定的甲藻素复合物（Gasal）与HASAP和块结合血红素信号的级联反应，降低了PA感知和使用血红素的能力。同时，加油作为PA的铁载体摄取系统的异种载体，导致细胞内失调铁稳态。我们的中心假设是通过靶向同时抑制PA血红素感应优化异种载体受体摄取的同时，细胞外的血果哈同是是一种新颖的策略 PA感染的治疗。我们的目标是合成一系列的加油型类似物，并使用已建立的测定，识别，验证和表征血红素信号传导和铁稳态的有效抑制剂。到实现我们的目标，我们将合成使用新型计算机AID设计的新的Gasal类似物药物设计（CADD）方法学SILCS（AIM 1）。合成化合物将经过FQ分析确定他们的亲密关系。将进一步评估选定的抑制剂以抑制血红素信号传导并分别使用转录报告基因测定法和13C-血红素LC-MS/MS分析的吸收。加油的吸收通过使用ICP-MS测量细胞内GA水平，将量化型基团受体。在AIM 2中，我们将确定MIC50和生物膜在PA菌株面板上所选化合物的抑制。对于顶部候选人，我们将进一步测试其在秀丽隐杆线虫中的体内效率。顶级化合物的哈希绑定插件将由STD-，HSQC-NMR和HDX-MS确定。我们的协作研究团队有很强的轨道执行CADD的记录，命中率优化以及化合物的体外和体内评估。总的来说，我们的方法使我们处于独特的位置，以识别，验证和特征在小班级中首先具有抑制血红素信号级联的双重活性的分子，并模仿铁载体的底物 MDR PA的受体，并确定这种新颖的作用机理是否是可行的选择抗菌素的发展。