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陷（例如 HIV 和癌症）、囊性纤维化 (CF)、机械通气或烧伤患者严重感染的常见原因。虽然铜绿假单胞菌表现出令人震惊的多种内在耐药性，但氨基糖苷类 (AMG)，如妥布霉素和阿米卡星，仍然可以有效治疗由这种病原体引起的疾病，包括囊性纤维化和呼吸机相关性肺炎。然而，由于具有内在或获得性耐药性的菌株日益流行，即使这些药物的长期效用也受到威胁。最近，我们发现了膜应力降低网络的两个成员，即双组分系统 (AmgRS) 和膜蛋白酶 (FtsH)，它们可以在体外和体内保护铜绿假单胞菌免受 AMG 的侵害。删除这些基因产生的铜绿假单胞菌菌株对 AMG 的抑制敏感度高出 16 倍，即使它们携带 rmtD 16S rRNA 甲基化酶 AMG 抗性等位基因或生长为生物膜。此外，AMG 妥布霉素在拯救感染这些铜绿假单胞菌突变体的小鼠免于致死方面更有效。该项目的目标是发现和开发新药，以提高 AMG 对抗铜绿假单胞菌的功效。我们的策略是识别抑制 FtsH 和/或 AmgRS 活性的小分子，并将其开发为增强 AMG 的创新辅助疗法。此类疗法将显着改善临床治疗结果，因为它们预计会（a）增加 AMG 介导的对铜绿假单胞菌生物膜和浮游细胞的杀伤，（b）使表现出 AMG 耐药性的临床分离株变得敏感，（c）降低铜绿假单胞菌的发展速度。 AMG 耐药性，(d) 通过使用较低剂量来减少 AMG 相关的耳毒性和肾毒性，以及 (e) 扩展更多 AMG 的使用范围以包括 P.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来鉴定 FtsH 或 AmgRS 抑制剂。该菌株携带 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，并表现出响应 ftsH 删除的信号增加和响应 amgRS 删除的信号减少。这种新型报告菌株在已知生物活性化合物的中试筛选中得到了验证，表现出 Z' 因子 >0.5，并成功识别了增加或减少 RLU 值的化合物。在第一阶段，我们将应用 PA2549-lux 报告菌株来筛选超过 300,000 种离散化合物和天然产物的文库，确认命中，并验证它们作为 AMG 抗生素与铜绿假单胞菌的有效、选择性增强剂。对多种 AMG 和临床铜绿假单胞菌分离株起作用的最强增效剂将进行分析，以排除那些表现出细胞毒性或膜效应的增效剂，并将重新合成以确认结构。将合成类似物以证明响应性 SAR 和有利的预测 ADME 特性。在第二阶段，我们将通过优化先导化合物的活性和特异性来开发最有希望的经过验证的化合物，并评估它们在感染动物模型中的功效和毒性。