Pathogenic bacteria require iron for their survival and ability to cause infection. Heme comprises 90% of the iron available within the host and plays a significant role in the colonization and virulence of many bacterial pathogens. The opportunistic pathogen P. aeruginosa is an increasingly common cause of nosocomial infections in the hospital setting. Furthermore, the high morbidity associated with Pseudomonas infection in cystic fibrosis disease, combined with the natural antibiotic resistance shown by this organism, highlights the need for alternate antimicrobial strategies. Previous biochemical and biophysical studies have shown that the intracellular heme trafficking protein PhuS and the iron-regulated heme oxygenase pa-HO are critical in the utilization of heme as an iron source.
The specific aims of the proposal are to i) determine the relationship between heme utilization, iron homeostasis and virulence through a combination of genetic, microarray and biochemical studies. ii) identify by Computer-aided drug design (CADD) and in silico database screening low- molecular weight compounds with a high probability of binding to either the cytoplasmic heme binding protein (PhuS) or the apo-heme oxygenase (HO);iii) Determine the in vitro and in vivo properties of the putative inhibitors. Binding affinity to the target protein will be tested using a combination of biophysical and spectroscopic approaches (fluorescence, ITC and NMR) and in vivo analysis of inhibitor efficacy by high- throughput MIC50 screening and the Caenorhabditis elegans host-pathogen model of bacterial infection;iv) Optimize the inhibitors by lead compound validation and structural characterization of the protein-inhibitor complexes by X-ray crystallographic an NMR methods, thereby providing a foundation for future CADD-based lead compound optimization. Upon completion of the proposed study a collection of chemically diverse low- molecular compounds will have been identified and validated as inhibitors of HO and PhuS. These compounds will be further developed through lead optimization efforts with the goal of developing a novel class of antibiotics.
The rapid rise in antibiotic resistance in the past two decades increases the need for alternative therapeutic strategies. A current approach to developing new antibiotics is to target non-essential functions that reduce virulence of infecting organisms but decrease the selective pressure on the organism to undergo mutation. Heme utilization provides such a non-essential target as it is directly linked to virulence but is not essential for survival. Therefore the identification and development of novel inhibitors of intracellular trafficking and heme utilization by targeting the cytoplasmic heme binding protein, PhuS, and the iron-regulated heme oxygenase pa-HO will provide a new and novel class of antimicrobials.
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