Development of HTP Assay for Inhibitors of Aerobactin Production

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 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.