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：
10231736
负责人：
Angela Wilks
金额：
$ 23.18万
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 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10231736
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/antagonist 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

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.

项目概要多重耐药 (MDR) 铜绿假单胞菌 (Pa) 导致约 10% 的医院感染，威尔克斯实验室强调了开发新型治疗方法的迫切需要。从囊性纤维化患者中分离出的慢性巴肺感染降低了对铁铁载体的依赖我们的遗传和生化分析表明，随着时间的推移，对血红素的依赖增加。 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 是急性小鼠肺部感染模型中最显着上调的基因。 a ΔhasR 菌株显示出生长和毒力显着降低。此外，氧化还原失活的制剂。金属镓（例如 Ganite）已在临床上通过针对铁吸收系统用作抗菌剂。初步研究表明，稳定的镓-salophen 复合物 GaSal 与 HasAp 结合并阻断血红素信号级联反应，降低 Pa 感知和利用血红素的能力。作为 Pa 铁载体摄取系统的异铁载体，导致细胞内失调我们的中心假设是通过靶向同时抑制 Pa 血红素感应。细胞外血细胞 HasAp 同时优化异铁载体受体的摄取是一种新的策略我们的目标是合成一系列 GaSal 类似物并使用已建立的方法对其进行测试。测定，以识别、验证和表征血红素信号传导和铁稳态的有效抑制剂。为了实现我们的目标，我们将合成新的 GaSal 类似物，这些类似物是使用新型计算机辅助设计的药物设计 (CADD) 方法 SILCS（目标 1）合成的化合物将接受 FQ 测定。以确定它们与 HasAp 的亲和力，将进一步评估所选抑制剂对血红素信号传导的抑制作用。分别使用转录报告测定和 13C-血红素 LC-MS/MS 测定进行摄取。在目标 2 中，我们将通过使用 ICP-MS 测量细胞内 Ga 水平来量化铁载体受体。将确定所选化合物对一组 Pa 菌株的 MIC50 和生物膜抑制作用。候选者，我们将进一步测试它们在秀丽隐杆线虫中的体内功效。顶级化合物的 HasAp 结合表位。将通过 STD-、HSQC-NMR 和 HDX-MS 确定。我们的协作研究团队拥有强大的追踪能力。执行 CADD、先导化合物优化以及化合物的体外和体内评估的记录。总的来说，我们的方法使我们处于独特的地位，可以在同类小型产品中率先识别、验证和表征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 MDR Pa 受体，并确定这种新的作用机制是否是一个可行的选择抗菌药物的开发。