Development of HTP Assay for Inhibitors of Aerobactin Production

Aerobactin 生产抑制剂的 HTP 检测方法的开发

• 批准号: 9101161
• 负责人: ANDREW M GULICK
• 金额: $ 48.26万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9101161
• 关键词: Address; Adopted; Amikacin; Aminoglycoside resistance; Anabolism; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Appearance; Area; Asia; Asians; Bacillus (bacterium); Bacteria; Biochemical; Biochemistry; Biological Assay; Biosynthetic Proteins; Carbapenems; Cells; China; Citrates; Clinical; Clinical Microbiology; Community-Acquired Infections; Data; Development; Disease Outbreaks; Drug Targeting; Drug resistance; Endophthalmitis; Enzymes; Europe; Exhibits; Extended-spectrum β-lactamase; FDA approved; Frequencies; Gram-Negative Bacteria; Growth; Healthcare; Hospitals; Human; Hydroxylysine; Incidence; Individual; Infection; Iron; Klebsiella pneumonia bacterium; Left; Libraries; Life; Ligase; Liver Abscess; Long-Term Care; Meningitis; Modeling; Multi-Drug Resistance; Mus; Natural Products; Necrotizing fasciitis; North America; Nosocomial Infections; Organ; Patients; Pharmaceutical Preparations; Phenotype; Physical condensation; Pilot Projects; Plasmids; Play; Pneumonia; Production; Pyogenic Liver Abscess; Quinolones; Recording of previous events; Reporting; Resistance; Retrospective Studies; Role; Series; Siderophores; Specificity; Testing; Tissues; Travel; United States National Institutes of Health; Virulence; Virulent; aerobactin; antimicrobial; beta-Lactams; capsule; carbapenemase; combat; high risk; high throughput screening; hydroxamate; in vivo; inhibitor/antagonist; miniaturize; mortality; mutant; novel therapeutics; pathogen; public health relevance; resistance frequency; screening; single molecule; small molecule; small molecule inhibitor; small molecule libraries

 DESCRIPTION (provided by applicant): A hypervirulent strain of Klebsiella pneumonia (hvKP) has recently been identified that causes life-threatening community-acquired infections in otherwise healthy individuals. Infections with this strain often begin with liver abscess and have the ability to spread metastatically to other organs. Originally identified in the Pacific Rim, incidence in the US is rising. The transfer of antibiotic resistance from classical strains of K. pneumonia to this hypervirulent strain, which has been experimentally demonstrated, raises the possibility of a resistant, hypervirulent strain and motivates our efforts to identify alternate antibiotic strategies. The phenotype of this hvKP strain includes higher capsule expression, resulting in a hypermucoviscous appearance, and increased production of iron acquisition factors. Specifically, the hvKP strains produce aerobactin, a hydroxamate siderophore that allows the bacteria to grow in low-iron conditions. We have demonstrated in mouse infection models that this aerobactin expression is responsible for virulence. We therefore propose to develop a high-throughput screen to identify small molecule inhibitors of aerobactin production. The collaborative team of Drs. Gulick and Russo brings expertise in all areas of clinical microbiology, biochemistry of natural product biosynthesis, and inhibitor development. We have adopted a two- pronged approach in which we will simultaneously develop a high-throughput biochemical screen for inhibitors of one of the aerobactin biosynthetic proteins as well as a phenotypic screen with live cells to identify in vivo-active compounds. Both assays have been demonstrated in low-throughput format and we will optimize conditions for high throughput screening. We will additionally perform pilot studies to statistically validate the assays with thre commercially available libraries totaling 4400 compounds, which are available at the neighboring Roswell Park Small Molecule Screening Center. Finally, we will develop orthogonal biochemical and whole cell secondary assays that will be used to confirm specificity and remove false positives.

 描述（由申请人提供）：最近已鉴定出肺炎克雷伯菌（hvKP）的一种高毒力菌株，它会导致其他健康个体出现危及生命的社区获得性感染。这种菌株的感染通常始于肝脓肿，并有能力转移至其他器官。最初在环太平洋地区发现，但美国的发病率正在上升。抗生素耐药性从肺炎克雷伯菌的经典菌株转移到这种高毒力菌株（已通过实验证明）增加了耐药性高毒力菌株的可能性，并激励我们努力寻找替代抗生素策略。该 hvKP 菌株的表型包括较高的荚膜表达，导致粘液粘稠的外观，并增加铁获取因子的产生。具体来说，hvKP 菌株产生需氧菌素（aerobactin），这是一种异羟肟酸铁载体，可使细菌在低铁条件下生长。我们在小鼠感染模型中证明，需氧菌素的表达是造成毒力的原因。因此，我们建议开发一种高通量筛选来鉴定需氧菌素产生的小分子抑制剂。博士的协作团队。 Gulick 和 Russo 带来了临床微生物学、天然产物生物合成的生物化学和抑制剂开发各个领域的专业知识。我们采用了双管齐下的方法，其中我们将同时开发一种针对需氧菌素生物合成蛋白抑制剂的高通量生化筛选，以及利用活细胞进行表型筛选以鉴定体内活性化合物。两种测定均已以低通量形式进行了验证，我们将优化高通量筛选的条件。我们还将进行试点研究，以统计验证使用 3 个市售文库（总计 4400 种化合物）的测定方法，这些文库可在邻近的罗斯威尔公园小分子筛选中心获得。最后，我们将开发正交生化和全细胞二次测定，用于确认特异性并消除假阳性。