Aminoglycoside potentiators for P. aeruginosa therapy

用于治疗铜绿假单胞菌的氨基糖苷类增效剂

基本信息

批准号：
8585819
负责人：
Donald T Moir
金额：
$ 30万
依托单位：
MICROBIOTIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8585819
关键词：
Affect Amikacin Aminoglycoside Antibiotics Aminoglycoside resistance Aminoglycosides Animal Model Antibiotic Therapy Antibiotics Bacteria Bacterial Infections Biological Assay Biological Factors Burn injury Cells Cephalosporinase Chemicals Chemosensitization Chronic Clinical Clinical Treatment Cystic Fibrosis Development Disease Dose Drug Efflux Drug Tolerance Drug resistance Effectiveness Environment Enzymes Exhibits Gene Deletion Genes Goals HIV Human Immune In Vitro Infection Lead Libraries Luciferases Malignant Neoplasms Mechanical ventilation Mediating Membrane Microbial Biofilms Molecular Target Monobactams Mus Operon Pathway interactions Patients Peptide Hydrolases Pharmaceutical Preparations Phase Phosphotransferases Pneumonia Population Prevalence Property Proteins Pseudomonas aeruginosa Relative (related person)Reporter Research Resistance Ribosomal RNA Series Signal Transduction Specificity Stress Structure Structure-Activity Relationship System Testing Therapeutic Tobramycin Toxic effect Toxicology Treatment outcome Ventilator Xenorhabdus luminescens analog base biological adaptation to stress cellular targeting cystic fibrosis patients cytotoxicity efflux pump improved in vivo inhibitor/antagonist innovation killings luminescence member mutant nephrotoxicity novel ototoxicity pathogen preclinical study promoter public health relevance rRNA methylase resistance allele resistant strain response screening sensor small molecule

项目摘要

DESCRIPTION: Pseudomonas aeruginosa is a common cause of serious infections in patients with immune deficiency (e.g., HIV and cancer), cystic fibrosis (CF), on mechanical ventilation or with burn wounds. While P. aeruginosa exhibits an alarming variety of intrinsic drug resistances, aminoglycosides (AMGs) such as tobramycin and amikacin, are still effective in treating diseases caused by this pathogen, including CF and ventilator- associated pneumonia. Nevertheless, the long-term utility of even these agents is in jeopardy due to the increasing prevalence of strains with intrinsic or acquired resistances. Recently, we discovered two members of a membrane stress reduction network, a two-component system (AmgRS) and a membrane protease (FtsH), that protect P. aeruginosa from AMGs in vitro and in vivo. Deletion of these genes yielded P. aeruginosa strains that are up to 16-fold more sensitive to inhibition by AMGs, even if they carry the rmtD 16S rRNA methylase AMG-resistance allele or are grown as biofilms. In addition, the AMG tobramycin was more effective in rescuing mice infected with these P. aeruginosa mutants from lethality. The goal of this project is to discover and develop new drugs that increase the efficacy of AMGs against P. aeruginosa. Our strategy is to identify small molecules that inhibit FtsH and/or AmgRS activity and to develop them into innovative adjunctive therapies that potentiate AMGs. Such therapies will significantly improve clinical treatment outcomes because they are expected to (a) increase the AMG-mediated killing of P. aeruginosa biofilms and planktonic cells, (b) sensitize clinical isolates exhibiting AMG resistance, (c) decrease the rate of development of AMG resistance, (d) reduce AMG-associated oto- and nephrotoxicities by enabling the use of lower doses, and (e) extend the spectrum of more AMGs to include P. aeruginosa. In preliminary studies, we constructed and optimized a cell- based reporter strain to identify FtsH or AmgRS inhibitors. This strain carries a transcriptional fusion of the amgRS- and ftsH-responsive P. aeruginosa gene PA2549 promoter to a luciferase operon, and exhibits increased signal in response to deletion of ftsH and decreased signal in response to deletion of amgRS. This novel reporter strain was validated in a pilot screen of known bioactive compounds, exhibiting a Z'-Factor >0.5 and successfully identifying compounds that increased or decreased RLU values. In Phase I, we will apply the PA2549-lux reporter strain to screen libraries of >300,000 discrete chemical compounds and natural products, confirm the hits, and validate them as potent, selective potentiators of AMG antibiotics vs. P. aeruginosa. The strongest potentiators acting on multiple AMGs and clinical P. aeruginosa isolates will be profiled to exclude those exhibiting cytotoxicity or membrane effects and will be re-synthesized to confirm structure. Analogs will be synthesized to demonstrate responsive SAR and favorable predicted ADME properties. In Phase II, we will develop the most promising validated hits into lead compounds by optimizing their activity and specificity and evaluate them for efficacy and toxicity in animal models of infection.

描述：铜绿假单胞菌是免疫缺乏症（例如HIV和癌症），囊性纤维化（CF），机械通气或烧伤伤口的常见原因。尽管铜绿假单胞菌表现出令人震惊的固有耐药性，但氨基糖苷（AMGS）如二霉素和amikacin仍然有效地治疗由这种病原体引起的疾病，包括CF和呼吸机 - 相关的肺炎。然而，由于固有或获得性抗性的菌株患病率的增加，这些药物的长期效用也受到危害。最近，我们发现了膜应力减少网络的两个成员，两个组件系统（AMGRS）和一个膜蛋白酶（FTSH），它们可以保护铜绿假单胞菌在体外和体内免受AMG的影响。这些基因的缺失产生的铜绿假单胞菌菌株对AMG的抑制更敏感，即使它们携带RMTD 16S rRNA甲基甲基酶AMG耐药等位基因或作为生物膜生长。此外，AMG毒素在挽救感染了这些铜绿假单胞菌突变体的小鼠免于致死性方面更有效。该项目的目的是发现和开发新药，以提高对铜绿假单胞菌的疗效。我们的策略是确定抑制FTSH和/或AMGRS活性的小分子，并将其发展为增强AMG的创新辅助疗法。这种疗法将显着改善临床治疗结果，因为预计（a）增加了AMG介导的铜绿假单胞菌生物膜和浮游细胞的杀害，（b）敏感性临床分离株表现出AMG耐药性，（c）降低AMG抗性的发育速率，（D）降低AMG伴随的OTO型和NEPHOX的依次（d），并降低了OTOX的依次（dy）。扩展更多AMG的光谱以包括铜绿假单胞菌。在初步研究中，我们构建并优化了基于细胞的报告菌株，以鉴定FTSH或AMGRS抑制剂。该菌株带有 AMGRS和FTSH响应性铜绿假单胞菌基因PA2549启动子与荧光素酶操纵子的转录融合，并响应于FTSH的缺失而表现出增加的信号，并响应于AMGR的缺失而降低信号。在已知生物活性化合物的试验屏幕中验证了这种新型的记者菌株，该菌株表现出Z'FACTOR> 0.5，并成功鉴定出增加或降低RLU值的化合物。在第一阶段，我们将对> 300,000个离散化合物和天然产物的屏幕库应用PA2549-LUX报告菌株，确认命中并将其验证为AMG抗生素与P. P. P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p。a中。作用于多个AMG和临床铜绿菌分离株的最强的电位器将被构造，以排除那些表现出细胞毒性或膜效应的人，并将重新合成以确认结构。类似物将合成以证明响应式SAR和有利的预测ADME特性。在第二阶段，我们将通过优化其活性和特异性，并评估它们在感染动物模型中的功效和毒性，从而将最有希望的验证效果开发为铅化合物。