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)