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

• 批准号: 8454071
• 负责人: Donald T Moir
• 金额: $ 30万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454071
• 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

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(如艾滋病毒和癌症)、囊性纤维化(CF)、机械通风或烧伤创面患者严重感染的常见原因。虽然铜绿假单胞菌表现出惊人的内在耐药性，但氨基糖苷类药物(AMG)，如妥布霉素和阿米卡星，仍然有效地治疗由这种病原体引起的疾病，包括CF和呼吸机相关性肺炎。然而，由于具有内在或获得性耐药性的菌株的日益流行，即使是这些药物的长期用途也处于危险之中。最近，我们发现了膜应激网络的两个成员，一个双组分系统(AmgRS)和一个膜蛋白水解酶(FtsH)，它们在体外和体内都能保护铜绿假单胞菌免受AMGs的影响。这些基因的缺失使铜绿假单胞菌对AMG的抑制敏感性提高了16倍，即使它们携带rmtD 16S rRNA甲基酶AMG抗性等位基因或作为生物膜生长也是如此。此外，AMG妥布霉素在拯救感染这些铜绿假单胞菌突变株的小鼠方面更有效。该项目的目标是发现和开发新药，以提高AMGs对铜绿假单胞菌的疗效。我们的策略是识别抑制FtsH和/或AmgRs活性的小分子，并将它们开发成增强AMG的创新辅助疗法。此类疗法将显著改善临床治疗结果，因为它们有望：(A)增加AMG介导的对铜绿假单胞菌生物被膜和浮游细胞的杀灭作用，(B)使表现出AMG耐药性的临床菌株变得敏感，(C)降低AMG耐药性的发展速度，(D)通过使用较低剂量减少AMG相关的耳肾毒性，以及(E)扩大更多AMG的谱以包括铜绿假单胞菌。在初步研究中，我们构建并优化了一种基于细胞的报告菌株来鉴定FtsH或AmgRS抑制剂。这种菌株携带一种 将AmgRS和FtsH应答的铜绿假单胞菌PA2549基因启动子转录融合到荧光素酶操纵子上，并表现出对FtsH缺失的信号增强和amgRS缺失的信号减弱。这种新的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，显示出Z‘-因子&gt；0.5，并成功地识别了增加或降低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&gt；30万种离散化合物和天然产物的文库，确认命中结果，并验证它们是AMG抗生素与铜绿假单胞菌的有效、选择性增强剂。作用于多个AMG和临床铜绿假单胞菌的最强增效剂将被剖析，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。将合成类似物来展示响应性的SAR和良好的预测ADME特性。在第二阶段，我们将通过优化它们的活性和特异性，将最有希望的验证HITS开发成先导化合物，并在动物感染模型中评估它们的有效性和毒性。