Small molecules for perturbing iron homeostasis in bacterial biofilms

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ABSTRACT Chronic wound care is especially complicated by the formation of bacterial biofilms, commonly by Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus. Biofilms are recalcitrant to antibiotic therapy, which is an important reason why chronic wound infections are so difficult to eradicate. There is a significant need to discover new strategies to combat biofilms. Bacterial iron homeostasis is a suitable novel target for developing anti-infectives because the host makes essential iron scarce to invading pathogens. Bacteria have evolved mechanisms to “steal” iron from their host, but these depend on well-regulated iron homeostasis. To disrupt bacterial iron homeostasis, we are targeting the iron storage protein BfrB and its physiological partner protein, Bfd, which are unique to bacteria. Our work has shown that BfrB regulates cytosolic iron concentrations by (a) oxidizing Fe2+ and storing up to ~3,000 Fe3+ ions in its internal cavity, and (b) forming a complex with Bfd to reduce Fe3+ in the internal cavity of BfrB and release Fe2+ to the cytosol for its incorporation in metabolism. Blocking the BfrB-Bfd complex in P. aeruginosa cells by deletion of the bfd gene perturbs iron homeostasis by triggering an irreversible accumulation of Fe3+ in BfrB and simultaneous cytosolic iron depletion, which leads to impaired biofilm maintenance and biofilm cell death. Our work also led to the discovery of proof-of-concept inhibitors of the BfrB-Bfd complex, which bind BfrB at the Bfd binding site, inhibit iron mobilization, and elicit biofilm cell death. The objectives of this application are to improve the proof-of-concept molecules into lead molecules, evaluate their antibacterial and antibiofilm effectiveness, and evaluate their potential drug suitability. Our interdisciplinary team includes expertise in chemical and structural biology of bacterial iron homeostasis (Rivera), organic synthesis and reaction mechanisms (Bunce), pharmaceutical industry research and development of anti-infectives (Reitz), X-ray crystallography in drug discovery (Lovell), microbial genetics (Chandler), and animal model of infection (Morici). The specific aims are: 1) Evolve proof-of-concept analogs into potent inhibitors of the BfrB-Bfd complex. This work will follow a systematic, iterative strategy of medicinal chemistry synthesis optimization that relies on crystal structures of inhibitor bound BfrB to design each new generation of inhibitors. 2) Asses the antibacterial effectiveness of inhibitors of the BfrB-Bfd complex and evaluate their potential drug suitability.