Small Molecule Antivirulents Targeting the Iron-Regulated Heme Oxygenase (HemO) of <i>P. aeruginosa</i>

Bacteria require iron for survival and virulence and employ several mechanisms including utilization of the host heme containing proteins. The final step in releasing iron is the oxidative cleavage of heme by HemO. A recent computer aided drug design (CADD) study identified several inhibitors of the bacterial HemOs. Herein we report the near complete HN, N, CO, Cα, and Cβ chemical shift assignment of the <i>P. aeruginosa</i> HemO in the absence and presence of inhibitors (<i>E</i>)-3-(4-(phenylamino)­phenylcarbamoyl)­acrylic acid (<b>3</b>) and (<i>E</i>)-<i>N</i>′-(4-(dimethylamino)­benzylidene) diazenecarboximidhydrazide (<b>5</b>). The NMR data confirm that the inhibitors bind within the heme pocket of HemO consistent with in silico molecular dynamic simulations. Both inhibitors and the phenoxy derivative of <b>3</b> have activity against <i>P. aeruginosa</i> clinical isolates. Furthermore, <b>5</b> showed antimicrobial activity in the in vivo C. elegans curing assay. Thus, targeting virulence mechanisms required within the host is a viable antimicrobial strategy for the development of novel antivirulants

Iminoguanidines as Allosteric Inhibitors of the Iron-Regulated Heme Oxygenase (HemO) of <i>Pseudomonas aeruginosa</i>

New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of <i>Pseudomonas aeruginosa</i>, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound <b>23</b> showed a binding affinity of 5.7 μM and an MIC₅₀ of 52.3 μg/mL against <i>P. aeruginosa</i> PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound <b>23</b> showed an EC₅₀ of 11.3 μM. The compounds showed increased activity against clinical isolates of <i>P. aeruginosa</i>, further confirming the target pathway. This class of inhibitors acts by binding to an allosteric site; the novel binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange mass spectrometry (HXMS)