Development of HTP Assay for Inhibitors of Aerobactin Production

 描述(由申请人提供)：最近发现了一种超强毒力肺炎克雷伯氏菌株(HvKP)，它会在其他健康的个人中引起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，并有能力转移到其他器官。最初发现于环太平洋地区，美国的发病率正在上升。抗生素耐药性从经典肺炎克雷伯菌菌株转移到这种已被实验证明的超强毒力菌株，增加了产生耐药超强毒力菌株的可能性，并激励我们努力寻找替代抗生素策略。这种hvKP菌株的表型包括更高的被膜表达，导致粘液高度粘连的外观，以及增加铁获取因子的产生。具体地说，hvKP菌株产生好氧蛋白，这是一种异羟甲酸铁载体，允许细菌在低铁条件下生长。我们已经在小鼠感染模型中证明了这种需氧肌动蛋白的表达与毒力有关。因此，我们建议开发一种高通量筛选来识别好氧肌动蛋白产生的小分子抑制物。古力克博士和鲁索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。我们采取了双管齐下的方法，同时开发了高通量的生化筛选，用于筛选其中一种需氧肌动蛋白生物合成蛋白的抑制剂，以及与活细胞的表型筛选，以鉴定体内活性化合物。这两种分析都是低通量格式的，我们将优化高通量筛选的条件。此外，我们还将进行试点研究，以对三个商业可用库进行统计验证，这些库总计4400种化合物，可在邻近的罗斯威尔公园小分子筛选中心获得。最后，我们将开发用于确认特异性和消除假阳性的正交生化和全细胞二次检测。

 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.