Inhibitors of S. aureus bNOS for adjunctive therapy

用于辅助治疗的金黄色葡萄球菌 bNOS 抑制剂

• 批准号: 8492028
• 负责人: Donald T Moir
• 金额: $ 29.05万
• 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: 2012-07-01 至 2014-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8492028
• 关键词: Animal Model; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacillus (bacterium); Bacillus anthracis; Bacteria; Bacterial Infections; Biochemical; Biological Assay; Blood Circulation; Cell Death; Cells; Cessation of life; Chemicals; Chemosensitization; Communicable Diseases; Community Hospitals; Development; Dose; Drug Design; Drug resistance; Effectiveness; Enzymes; Fluorescent Probes; Fluoroquinolones; Gene Deletion; Genes; Genus staphylococcus; Glycopeptide Antibiotics; Glycopeptides; Goals; Growth; Hospitals; Host Defense; Hydrogen Peroxide; Hydroxyl Radical; Immune; In Vitro; Infection; Inhibitory Concentration 50; Isoenzymes; Lactams; Lead; Libraries; Mediating; Membrane; Monobactams; Nitric Oxide; Nitric Oxide Synthase; Nosocomial Infections; Oxidative Stress; Parents; Patients; Pharmaceutical Preparations; Phase; Production; Reaction; Reactive Oxygen Species; Reporter; Research; Resistance; Resistance development; Respiratory Burst; Site; Skin; Skin Tissue; Sodium Chloride; Soft Tissue Infections; Specificity; Staphylococcus aureus; Superoxide Dismutase; System; Therapeutic; Toxic effect; Toxicology; Urban Hospitals; bactericide; base; beta-Lactam Resistance; catalase; clinically relevant; community setting; cytotoxicity; improved; in vivo; inhibitor/antagonist; innovation; killings; methicillin resistant Staphylococcus aureus; neutrophil; novel; pathogen; preclinical study; pressure; public health relevance; resistant strain; screening; small molecule; soft tissue

DESCRIPTION (provided by applicant): Staphylococcus aureus is a major cause of community and hospital-acquired infections of the skin, soft tissue, and bloodstream. The recent dramatic increase in occurrence of strains resistant to beta-lactam antibiotics (MRSA) has reduced therapeutic options significantly, but most MRSA strains remain sensitive to other bactericidal antibiotics in the fluoroquinolone (FQ) and glycopeptide (GP) classes. Recent research has demonstrated that bactericidal antibiotics stimulate the production of reactive oxygen species (ROS) in bacteria, which contribute to cell death in a manner similar to the host immune oxidative burst. Alleviation of this oxidative stress by the production of nitric oxide (NO) by bacterial NO synthase (bNOS) enhances the survival of several bacterial species, including staphylococci, to antibacterial therapy and to neutrophil killing. Consequently, small molecule bNOS inhibitors will provide an adjunctive therapeutic approach to bolster the effectiveness of antibiotics and neutrophils against MRSA and potentially reduce the selective pressure for development of drug-resistance by increasing the duration of effective circulating levels of antibiotic. Substantial differences between mammalian and bacterial NOS enzymes indicate that selective inhibition of bNOS is feasible. The overall goal of this project is to discover and develop drugs that increase the efficacy of clinically relevant bactericidal antibiotics against pathogenic staphylococci such as MRSA by specifically inhibiting bacterial NO production. Our strategy is to identify small molecule bNOS inhibitors and to develop them into innovative adjunctive therapies that increase the bactericidal activity of FQ, GP, and beta-lactam antibiotics. In preliminary studies, we established proof of concept for bNOS as a novel target for adjunctive therapy by demonstrating that the growth and viability of strains of S. aureus, B. subtilis, and B. anthracis carrying deletions of the bNOS gene were more sensitive to several bactericidal antibiotics than were their wild-type parents. NO in bacterial cells was shown to activate catalase, induce sodA (superoxide dismutase), suppress the Fenton reaction (production of reactive hydroxyl radicals from H2O2), and rescue cells from ROS generated by bactericidal drugs and by the immune oxidative burst. These results indicate that bactericidal drugs can be potentiated by targeting bacterial systems that reduce ROS damage, such as bNOS. In Phase I, we will construct and optimize luminescent, fluorescent, and biochemical primary and secondary screening assays for bNOS inhibitors, apply them to libraries representing >300,000 discrete chemical compounds, confirm the hits, and validate them as potent, selective potentiators of several antibiotics and neutrophils vs. MRSA and other drug- resistant species. The most potent, broadest acting bNOS inhibitors will be characterized to eliminate compounds with off-target activity vs. the 3 mammalian NOS isozymes and cytotoxicity. In Phase II, we will develop the most promising validated hits into lead compounds by optimizing their activity and specificity using rational drug design and evaluate them for efficacy and toxicity in animal models of infection.

描述（由申请人提供）：金黄色葡萄球菌是社区和医院获得性皮肤、软组织和血流感染的主要原因。最近，对β-内酰胺类抗生素（MRSA）耐药的菌株的发生率急剧增加，显著减少了治疗选择，但大多数MRSA菌株对氟喹诺酮类（FQ）和糖肽类（GP）中的其他杀菌抗生素仍然敏感。最近的研究表明，杀菌抗生素刺激细菌中活性氧（ROS）的产生，这有助于以类似于宿主免疫氧化爆发的方式导致细胞死亡。通过产生一氧化氮（NO）缓解这种氧化应激 通过细菌NO合酶（bNOS）的作用增强了几种细菌物种（包括葡萄球菌）对抗菌治疗和嗜中性粒细胞杀伤的存活。因此，小分子bNOS抑制剂将提供一种连续的治疗方法，以加强抗生素和中性粒细胞对MRSA的有效性，并通过增加抗生素有效循环水平的持续时间，潜在地降低耐药性发展的选择压力。哺乳动物和细菌NOS酶之间的实质性差异表明选择性抑制bNOS是可行的。该项目的总体目标是发现和开发药物，通过特异性抑制细菌NO的产生来提高临床相关杀菌抗生素对致病性葡萄球菌（如MRSA）的疗效。我们的策略是确定小分子bNOS抑制剂，并将其开发成创新的预防性疗法，增加FQ，GP和β-内酰胺类抗生素的杀菌活性。在初步的研究中，我们通过证明链球菌菌株的生长和活力，建立了bNOS作为连续治疗新靶点的概念证据。aureus，B. B. subtilis和B. B.携带bNOS基因缺失的炭疽菌比它们的野生型亲本对几种杀菌抗生素更敏感。细菌细胞中的NO显示出激活过氧化氢酶，诱导sodA（超氧化物歧化酶），抑制芬顿反应（从H2 O2产生活性羟基自由基），并从由杀菌药物和免疫氧化爆发产生的ROS中拯救细胞。这些结果表明，可以通过靶向减少ROS损伤的细菌系统（如bNOS）来增强杀菌药物。在第一阶段，我们将构建和优化bNOS抑制剂的发光、荧光和生物化学初级和二级筛选测定，将它们应用于代表> 300，000种离散化合物的文库，确认命中，并验证它们作为几种抗生素和中性粒细胞与MRSA和其他耐药物种的有效选择性增效剂。最有效、作用最广泛的bNOS抑制剂将被表征为消除具有脱靶活性（相对于3种哺乳动物NOS同工酶）和细胞毒性的化合物。在第二阶段，我们将通过使用合理的药物设计优化其活性和特异性，并在动物感染模型中评估其有效性和毒性，将最有希望的经验证的命中物开发为先导化合物。