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患者严重感染的常见原因（例如，HIV和癌症），囊性纤维化（CF），机械通气或烧伤。虽然铜绿假单胞菌表现出惊人的多种内在耐药性，但氨基糖苷类（AMG）如妥布霉素和阿米卡星仍然有效治疗由该病原体引起的疾病，包括CF和呼吸机相关性肺炎。然而，由于具有内在或获得性抗性的菌株的日益流行，甚至这些药剂的长期效用也处于危险之中。最近，我们发现了两个成员的膜应力降低网络，双组分系统（AmgRS）和膜蛋白酶（FtsH），保护铜绿假单胞菌从AMG在体外和体内。删除这些基因产生的铜绿假单胞菌菌株对AMG抑制的敏感性高达16倍，即使它们携带rmtD 16 S rRNA甲基化酶AMG抗性等位基因或作为生物膜生长。此外，AMG妥布霉素在挽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的药物，提高AMG对铜绿假单胞菌的疗效。我们的策略是鉴定抑制FtsH和/或AmgRS活性的小分子，并将其开发成增强AMG的创新治疗方法。这种疗法将显著改善临床治疗结果，因为预期它们（a）增加AMG介导的对铜绿假单胞菌生物膜和增殖细胞的杀伤，（B）使表现出AMG抗性的临床分离株敏感，（c）降低AMG抗性的发展速率，（d）通过能够使用较低剂量来降低AMG相关的耳毒性和肾毒性，和（e）扩展更多AMG的谱以包括铜绿假单胞菌。在初步研究中，我们构建并优化了基于细胞的报告菌株以鉴定FtsH或AmgRS抑制剂。这种菌株携带一种 在一些实施方案中，本发明涉及amgRS-和ftsH-响应性铜绿假单胞菌基因PA 2549启动子与荧光素酶操纵子的转录融合，并且表现出响应于ftsH缺失的增加的信号和响应于amgRS缺失的减少的信号。该新型报告菌株在已知生物活性化合物的中试筛选中得到验证，表现出Z '-因子>0.5并成功鉴定了增加或降低RLU值的化合物。在第一阶段，我们将应用PA 2549-lux报告菌株筛选> 300，000个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效剂。对作用于多种AMG和临床铜绿假单胞菌分离株的最强增效剂进行分析，以排除表现出细胞毒性或膜效应的增效剂，并重新合成以确认结构。将合成类似物，以证明响应SAR和有利的预测ADME性能。在第二阶段，我们将通过优化其活性和特异性，将最有希望的经验证的命中物开发成先导化合物，并在动物感染模型中评估其疗效和毒性。