Small Molecule Antivirulents Targeting the Iron-Regulated Heme Oxygenase (HemO) ofP. aeruginosa

Abstract

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 P. aeruginosa HemO in the absence and presence of inhibitors (E)-3-(4-(Phenylamino)phenylcarbamoyl) acrylic acid (3) and (E)-N’-(4-(dimethylamino)benzylidene) diazenecarboximidhydrazide (5). The NMR data confirms the inhibitors bind within the heme pocket of HemO consistent with in silico molecular dynamic simulations. Both inhibitors and the phenoxy derivative of 3 have activity against P. aeruginosa clinical isolates. Furthermore 5 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 Pseudomonas aeruginosa

“…30 Selected compounds included those containing hydrophilic or lipophilic substitutions and individually resolved protons ( 9, 12 , and 17 ) as well as the largest substitution, compound 24 . Integration of the difference peaks allows for comparison of relative magnetization transfer, and therefore degree of interaction, of each proton with HemO (Figure 4).…”

“…Additionally, the methoxy protons of compound 17 show much less interaction with HemO than the aromatic protons, much like the N -methyl protons of compound 1 . 30 The largest compound, 24 , contains two sets of aromatic protons with overlapping signals that cannot be resolved. The two unique signals, for the benzylimine proton and the para-position proton, show the largest relative interactions at 100% and 82%, respectively.…”

“…Preparation of samples and collection of STD spectra were conducted as previously reported with slight modifications. 30 Briefly, to a 950 µL solution of 50 mM sodium phosphate (pH = 7.4) in D 2 O was added 50 µL of desired compound dissolved in DMSO- d 6 and 10 µL of purified HemO such that final concentration of HemO was between 3 and 5 µM. After 4 h incubation at room temperature the mixture was centrifuged at 6000g for 3 min to remove residual precipitate.…”

Regulation of Pseudomonas aeruginosa Virulence by Distinct Iron Sources

“…Combined, these studies indicate that the heme acquisition pathway of P. aeruginosa is capable of regulating gene expression by at least two distinct mechanisms. While the impacts of these systems on virulence gene expression are not yet known, earlier studies showed that inhibition of HemO attenuated virulence of P. aeruginosa in a Caenorhabditis elegans model of infection [68]. More studies are clearly needed to understand the full impact of heme-mediated gene regulation during P. aeruginosa pathogenesis.…”

Beyond Traditional Antimicrobials: A Caenorhabditis elegans Model for Discovery of Novel Anti-infectives

“…Through in silico drug design and model simulations, two chemical entities were identified as inhibitors of P. aeruginosa heme oxygenase. One of these compounds prolonged the lifespan of infected nematodes and decreased bacterial colony forming units in the C. elegans intestine, albeit showing poor antimicrobial activity in vitro (Hom et al, 2013). Recently, Zhu et al (2015) used the C. elegans model to characterize an inhibitor of LasB, a vital virulence factor of P. aeruginosa .…”