Pseudomonas aeruginosa heme sensing inhibitors targeting HasAp

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PROJECT SUMMARY Multidrug-resistant (MDR) Pseudomonas aeruginosa (Pa) is responsible for ~10% of nosocomial infections, highlighting the critical need for the development of novel therapeutic approaches. The Wilks Lab has shown that chronic Pa lung infection isolates from cystic fibrosis patients decrease the reliance on iron-siderophore uptake over time, while increasing the reliance on heme. Our genetic and biochemical analysis characterized the Pa Has and Phu systems as having non-redundant roles in heme transport and sensing, respectively. Transcriptomics showed mRNA levels of the extracellular hemophore hasAp and its outer membrane receptor hasR are the most significantly upregulated genes in an acute murine lung infection model. In the same model, a ∆hasR strain showed significantly reduced growth and virulence. Moreover, formulations of the redox inactive metal gallium (e.g., Ganite) have been clinically used as antimicrobials by targeting iron uptake systems. Our preliminary studies have shown that the stable gallium-salophen complex, GaSal, binds to HasAp and blocks the heme-signaling cascade, decreasing the ability of Pa to sense and utilize heme. At the same time, GaSal functions as a xenosiderophore for the siderophore uptake systems of Pa, leading to intracellular dysregulation of iron homeostasis. Our central hypothesis is that simultaneous inhibition of Pa heme sensing by targeting the extracellular hemophore HasAp while optimizing xenosiderophore receptor uptake is a novel strategy for the treatment of Pa infections. Our goal is to synthesize a series of GaSal analogs and test them using established assays, to identify, validate, and characterize potent inhibitors of heme signaling and iron homeostasis. To achieve our goal, we will synthesize new GaSal analogs that have been designed using a novel computer-aided drug design (CADD) methodology SILCS (Aim 1). The synthesized compounds will be subjected to the FQ assay to determine their affinities to HasAp. Selected inhibitors will be further assessed for inhibition of heme signaling and uptake using transcriptional reporter assay and 13C-heme LC-MS/MS assay, respectively. GaSal uptake by siderophore receptors will be quantified by measuring the intracellular Ga levels using ICP-MS. In Aim 2, we will determine MIC50 and biofilm inhibition of selected compounds on a panel of Pa strains. For the top candidates, we will further test their in vivo efficacy in C. elegans. The HasAp binding epitope of top compounds will be determined by STD-, HSQC-NMR and HDX-MS. Our collaborative research team has a strong track record of performing CADD, hit-to-lead optimization, and in vitro and in vivo evaluation of compounds. Collectively, our approach puts us in a unique position to identify, validate, and characterize first in class small molecule with dual activity of inhibiting heme signaling cascade and mimicking the substrate of siderophore receptor of MDR Pa, and to determine whether this novel mechanism of action is a viable option for the development of antimicrobials.