Development of HTP Assay for Inhibitors of Aerobactin Production

Aerobactin 生产抑制剂的 HTP 检测方法的开发

基本信息

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
项目类别：
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

项目摘要

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，并具有转移性传播的能力。最初在环太平洋地区发现，美国的发病率正在上升，抗生素耐药性从典型的肺炎克雷伯菌菌株转移到了这种高毒力菌株，这一点已得到实验证明。这种 hvKP 菌株的表型包括更高的荚膜表达，导致高粘液外观，并增加铁获取因子的产生。具体来说，hvKP 菌株产生需氧菌素。，一种允许细菌在低铁条件下生长的异羟肟酸铁载体，我们已经在小鼠感染模型中证明了这种需氧菌素的表达是导致毒力的原因。鉴定 aerobactin 生产的小分子抑制剂的高通量筛选 Gulick 和 Russo 博士的合作团队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的所有领域的专业知识。其中，我们将同时开发一种高通量生化筛选，用于筛选需氧菌素生物合成蛋白之一的抑制剂，以及用活细胞进行表型筛选，以识别两种检测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，我们将另外进行试点研究，以使用总共 4400 种化合物的市售文库仔细验证检测。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。