Development of New Therapeutics Against Bacillus Anthracis

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Petrobactin, a siderophore required for Bacillus anthracis infection and pathogenesis is a member of a new family of iron-chelating agents derived by a non-ribosomal peptide synthetase (NRPS) independent pathway. Initial studies in the Hanna laboratory demonstrated that the genetic locus (as/?) responsible for petrobactin biosynthesis is required for growth of B. anthracis in macrophages and virulence in mice. More recently, we have revealed the biosynthetic basis for siderophore assembly, and have pursued detailed biochemical studies on the three "early" enzymes comprising the Asb pathway. For this competing continuation GLRCE project, we propose studies to confirm the catalytic function and mechanism of two remaining Asb enzymes (AsbB, AsbF), and develop robust functional assays to identify inhibitors of petrobactin biosynthesis. In this work, high throughput screening, chemical diversity, and chemoinformatic capabilities of the University of Michigan Center for Chemical Genomics will be harnessed in order to discover and develop new anti-infective agents targeting these NRPS-independent siderophore synthesis pathways. The specific objectives include: 1. Continue to identify highly potent chemical and natural product inhibitors of AsbF, AsbA, AsbB from hits against in vitro high throughput screens (Collaboration with the Center for Chemical Genomics (CCG), U-M) and characterize their efficacy through in vitro biochemical and in vivo growth inhibition studies [Collaboration with Dr. Phil Hanna, U-M]. 2. Characterize the biochemical mechanism of AsbF, shown to function as a 3-dehydroshikimate dehydratase for 3,4-dihydroxybenzoic acid precursor biosynthesis. Continue to assess the biochemical role of AsbB, including substrate tolerance and kinetic parameters. Pursue crystallographic analysis on each of the six Asb proteins to provide the basis for future inhibitor design studies [Collaboration with Dr. Andrzej Joachimiak, ANL].