The effects of novel heme oxygenase inhibitors on the growth of Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.micpath.2019.01.047

Objectives: This study was aimed to develop small molecule inhibitors of the P. aeruginosa heme oxygenase (pa-HemO) as potential treatment of infections caused by P. aeruginosa. Methods: New compounds were designed based on the crystal structure of pa-HemO. The binding affinities (K_D) were determined using intrinsic fluorescence quenching assays. The anti-microbial effects of the new compounds was evaluated by minimal inhibitory concentration 50% (MIC₅₀). Results: Eleven compounds were synthesized as potential pa-HemO inhibitors. New compounds demonstrated K_D values ranging from 1.5 to 180 μM, and MIC₅₀ values ranging from 26 to 260 μg/mL. The compounds had good affinity with HemO and promising anti-microbial effects on P. aeruginosa. Conclusions: The new inhibitors described herein can inhibit the growth of P. aeruginosa via the inhibition of pa-HemO. There may be broad prospects for HemO inhibitors to treat P. aeruginosa related infections.

Full text of DOI:10.1016/j.micpath.2019.01.047

MicrobialPathogenesis129(2019)64–67
Contents lists available at ScienceDirect
Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath
The effects of novel heme oxygenase inhibitors on the growth of
Pseudomonas aeruginosa
Jingming Zhaoa,b,∗,1, Dongdong Liangb,1, Elizabeth Robinsonb, Fengtian Xueb,∗∗
a Department of Respiratory Medicine, The Affiliated Hospital of Qingdao University, 16#, Jiangsu Road, Qingdao, 266003, PR China
b Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Objectives: This study was aimed to develop small molecule inhibitors of the P. aeruginosa heme oxygenase (pa-
HemO) as potential treatment of infections caused by P. aeruginosa.
Methods: New compounds were designed based on the crystal structure of pa-HemO. The binding affinities (KD)
were determined using intrinsic fluorescence quenching assays. The anti-microbial effects of the new compounds
was evaluated by minimal inhibitory concentration 50% (MIC50).
Results: Eleven compounds were synthesized as potential pa-HemO inhibitors. New compounds demonstrated KD
values ranging from 1.5 to 180 μM, and MIC50 values ranging from 26 to 260 μg/mL. The compounds had good
affinity with HemO and promising anti-microbial effects on P. aeruginosa.
Pseudomonas aeruginosa
Heme oxygenase
Inhibitor
Anti-microbial
Iron
Conclusions: The new inhibitors described herein can inhibit the growth of P. aeruginosa via the inhibition of pa-
HemO. There may be broad prospects for HemO inhibitors to treat P. aeruginosa related infections.
1. Introduction
[10]. P. aeruginosa has elegant systems dedicated to the acquisition of
heme from host hemoproteins. P. aeruginosa can take heme from he-
Pseudomonas aeruginosa is a life-threating Gram-negative bacterium
that causes infections in humans [1]. The P. aeruginosa infection can be
controlled by antibiotic including β-lactams and aminoglycosides [2].
However, drug resistance has been observed and seriously increased
over the past decades, which causes severe antibiotic misuse [3,4].
Iron is an essential cofactor for enzymes found within all kingdoms
of life. P. aeruginosa is no exception to this rule, and therefore, it must
acquire iron from their hosts in order to survive the extremely low iron
environment. P. aeruginosa can obtain iron by different strategies: (i)
production of Fe3+ chelating molecules and iron carriers such as pyo-
verdine and pyochelin, and uptake of ferric iron-carrier complexes by
TonB-dependent receptors (TBDR) [5], (ii) ingestion of an exogenous
iron carrier complex [6], (iii) intake of heme molecules from he-
moglobin from the host [7], and (iv) absorption and utilization of fer-
rous iron. Reducing Fe3+ to Fe2+ extracellularly using phenazine and
Fe2+ specific uptake system (Feo system) [8]. In the host, depending on
the type of infection (acute or chronic infection), P. aeruginosa adopts
different iron uptake strategies to meet its own needs [9].
moglobin through both Has and Phu systems [7]. In the periplasm, the
ingested heme binds to the periplasmic binding protein and gets
transported into the cell via the ATP-binding cassette (ABC) transport
system. In the ABC transport system, heme is first combined with the
heme chaperone PhuS and then transported to the HemO that catalyses
the degradation of heme to biliverdin, CO and Fe2+ [11]. Importantly,
the activity of pa-HemO provides critical driving force for the flux of
heme into cells. Therefore, if HemO is inhibited, P. aeruginosa loses its
ability to take up heme as an iron source [12].
The pa-HemO has a unique heme-binding active site. The solvent
accessible surface of pa-HemO (∼7.5 Å3) is much smaller than the
mammalian enzyme HO1 (43.6–59.7 Å3) [13–16]. Moreover, different
than the binding mode in HO1 and other known heme oxygenases,
heme binds in the active site of pa-HemO with a significantly rotated
(∼100°) orientation as a result of the unique amino acid network
present in the active site [13–16]. These structural differences provide
foundations for selective inhibition of the pa-HemO over other heme
oxygenases such as HO1 [17].
The majority of iron within the human body is in the form of heme
In this study, we developed competitive inhibitors of the pa-HemO
∗ Corresponding author. Department of Respiratory Medicine, The Affiliated Hospital of Qingdao University, 16#, Jiangsu Road, Qingdao, 266003, PR China.
∗∗ Corresponding author. Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, 20 Penn Street, Room 637, Baltimore,
MD 21201, United States.
E-mail addresses: wch7650@hotmail.com (J. Zhao), fxue@rx.umaryland.edu (F. Xue).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.micpath.2019.01.047
Received 28 September 2018; Received in revised form 19 November 2018; Accepted 30 January 2019
Availableonline01February2019
0882-4010/©2019ElsevierLtd.Allrightsreserved.

J. Zhao et al.
MicrobialPathogenesis129(2019)64–67
Fig. 1. Synthesis scheme of compounds.
Alkylation of benzo [cd]indol-2(1H)-one (1) with
selected alkyl halides using NaH as a base in DMF
gave compounds
2 in good to excellent yields.
Vilsmeier formylation of compounds 2 with DMF and
POCl3 generated aldehydes 3 with acceptable yields.
NaBH4 mediated reduction of aldehyde 3 yielded
alcohol inhibitors 4. The hydroxyl group of com-
pound 4 was converted into bromides (5) using PBr3
in good yields, which was then converted into azide
6
using NaN3 in DMSO at room temperature.
Staudinger reduction of azide 6 using PPh3 provided
benzylamine with almost quantitative yields.
7
Reagents and conditions: (a) NaH, alkyl iodide or
Benzyl bromide, DMF, 0-rt, 12 h, 66–90%; (b) POCl3,
DMF, 0–110 °C, 3.5 h, 35–75%; (c) NaBH4, EtOH,
65 °C, 2 h, 86–94%; (d) PBr3, DCM, 0-rt, 2 h,
80–88%; (e) NaN3, DMSO, 3 h, 94–98%; (f) PPh3,
MeOH, reflux, 2 h, 93–98%.
Figure automatically generated by CS ChemDraw
Ultra 12.0 software.
to suppress the binding of heme to HemO and inhibit the use of heme by
P. aeruginosa. New compounds were designed based on the x-ray crystal
structure of pa-HemO [18]. The development of these new HemO in-
hibitors will help open up new avenues for the treatment of P. aerugi-
nosa infections.
2.4. Growth inhibition of P. aeruginosa laboratory strains PAO1
Growth inhibition assays were conducted as previously described
[18] with modifications to qualitatively confirm that the inhibitors
specifically targeted the heme uptake pathway. Two media were used
in this study. One is DTSB as an iron-free medium, and another is LB as
a nutrient-rich formulations medium. Briefly, PAO1 monoclonal co-
lonies were picked into 25 mL Luria Broth (LB) or Dialyzed Tryptic Soy
Broth (DTSB) medium shaking at 220 rpm and culturing at 37 °C ther-
mostat bacterial overnight. Then the bacterium concentration was di-
luted back to an OD600 of 0.05. A 96-well cell culture plate was set up
with 200 μL of appropriate cultures in LB media and chelated DTSB
media. The DTSB media were supplemented with either 10 μM heme or
10 μM FeCl3. The test groups were added 9–300 μg/mL of inhibitor and
the control group was added 10 μg/mL gentamicin. OD600 was recorded
on a BioTek Synergy H1 hybrid microplate reader after 4 h. MIC50 va-
lues were calculated as average of triplicate as previously described
[18].
2. Material and methods
2.1. Synthesis of new inhibitors for HemO
To take advantage of the unique active site of pa-HemO, we de-
signed a series of inhibitors. Both 1H NMR and 13C NMR spectra of new
compounds were recorded on spectrometers at 400 MHz and 100 MHz,
respectively. Mass spectra were recorded using electrospray as the io-
nization technique. The syntheses of new compounds are detailed in the
supplementary material. A general synthetic route to prepare the new
inhibitors was shown in Fig. 1.
2.2. KD value determination using the intrinsic fluorescence quenching
assays
2.5. Statistical analysis
The data were expressed as‾X SD. Statistical analysis was tested
with SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). The difference was sta-
tistically significant at P < 0.05.
Fluorescence quenching assays were conducted as previously de-
scribed [18] with modifications made to enable the use of high
throughput liquid handling system Biomek FXP. Binding affinities were
determined in black flat-bottom 96-well plates (Costar 3915) with a
total volume of 200 μL solutions using a BioTek Synergy H1 hybrid
microplate reader. HemO concentration was kept at 1 μM in Tris buffer
(20 mM, pH = 8.0). Inhibitors were spotted into the assay plates in
triplicate as a solution in DMSO with a final concentration ranging of
0.9–500 μM. HemO was excited at 295 nm and the emission spectra was
recorded from 300 to 500 nm. The KD values were calculated from the
plots of total binding vs ligand concentration, where total binding
corresponds to the percentage of binding represented by the decrease in
fluorescence at the maximum emission wavelength (332 nm). Decrease
in fluorescence was corrected using experimentally determined ex-
tinction coefficients for each compound [19].
3. Results and discussion
Eleven compounds were tested as potential pa-HemO inhibitors. The
KD values of the majority of compounds ranged from 8 to 90 μM
(Table 1). Among these compounds, the compound 7c indicated the
best KD value (1.5
value (180
to the active site with modest affinity, suggesting that the compounds
can be suitable developing inhibitors for pa-HemO.
0.8 μM) and compound 7d has the largest KD
37 μM). Overall, the results show that compounds bind
Compared with the control group without DMSO, only the testing
group containing 1% DMSO did not show obvious effects on the growth
of P. aeruginosa (P > 0.05). Other testing groups with higher con-
centrations of DMSO significantly inhibited the growth of the bacteria.
The OD600 measurement curve is shown in Fig. 2. Therefore, in the
subsequent MIC50 assay, we kept the DMSO concentration at 1%. The
research data of DMSO toxicity to P.aeruginosa growth are detailed in
supplementary material.
2.3. The maximum non-toxic concentration of DMSO in cultures
A 96-well plate was added 200 μL of the medium containing the
bacterial liquid. The test groups were treated with various concentra-
tion of DMSO (1–10%) while the control group was not added with
DMSO. The toxicity of DMSO on bacterial growth was determined by
measuring the OD600 values each hour for 14 h.
The inhibitory activities of new compounds against P. aeruginosa
were determined in both DTSB media supplemented with heme or free
iron and LB media (Table 1). The MIC50 values of the compounds
65

J. Zhao et al.
MicrobialPathogenesis129(2019)64–67
Table 1
compounds had good anti-microbial effects against P. aeruginosa in both
DTSB medium supplemented with heme and LB medium. However, the
compounds did not inhibit the growth of P. aeruginosa at different times
in DTSB + FeCl3 media. The OD600 values of 300 μg/ml compounds at
different time in DTSB + FeCl3 media are showed in the session 3 of
supplemental materials. The MIC50 values of the compounds in LB
medium were lower than those obtained from the experiments in DTSB
medium (P < 0.05). The research data of MIC50 values of the com-
pounds on PAO1 are detailed in supplementary material.
We have developed new inhibitors that inhibits pa-HemO, a key
enzyme for iron acquisition of P. aeruginosa. Conventionally the broth
dilution method is commonly applied for measuring the MIC50. It re-
quires the preparation of series dilutions of the compounds. Due to the
relatively low water solubility of many therapeutic candidates, polar
organic solvents such as methanol, ethanol or DMSO are widely used to
enhance the aqueous solubility of compounds. DMSO is the most
commonly preferred since they are miscible with water [20]. However
in various bioassays, DMSO has been reported for their antimicrobial
effect [21]. Thus it becomes essential to ensure that the final con-
centration of DMSO is not interfere with the bioassay (MIC determi-
nation). We determined the highest possible concentration of DMSO
that can be safely used to measure the MIC50 of new compounds
without obvious cytotoxicity. The results showed that 1% was the
highest concentration that DMSO has no significant effects on bacterial
growth. Therefore, the concentration of DMSO used in our MIC50 assay
was set at 1%.
In this experiment, most of the compounds had good KD and MIC50
measurements. This suggests that these compounds inhibited the up-
take of heme by P. aeruginosa and bacteria growth by competitively
binding to pa-HemO. When the concentration of heme reaches a certain
level, it also has a toxic effect on P. aeruginosa [22]. Reducing heme
toxicity can also be accomplished by the heme oxygenase-mediated
degradation of heme. Due to the action of pa-HemO inhibitors, the in-
gested heme cannot be utilized and have a toxic effect on P. aeruginosa,
leading to the inhibition of bacterial growth.
Compound 7d showed relatively weak binding affinities while in-
dicating the inhibition of P.aeruginosa growth by MIC50 test. We rea-
soned that this compound may block the growth of P.aeruginosa by a
different mechanism. The specific mechanism of action will be further
studied in future trials. Compound 7b and 7c demonstrated good KD
values but had relatively poor MIC50 values. We reasoned that the
presence of the special group in these compounds might prohibit the in
vitro effects of inhibitors, as evidenced in a number of previous studies
[23,24].
The KD and MIC50 values of new compounds.
a
a
Compound Structure
KD (X
(μM)
SD)
MIC50 (DTSB)
MIC50 (LB)
(μg/mL)
(μg/mL)
2b
36
2
7
174
100
2c
2d
2e
4c
46
240
230
42
80
14
8.3
67
5
150
26
3
17
10
35
1
250
230
4d
7a
65
90
260
58
120
52
7b
7c
1.9
1.5
180
160
89
0.8
37
160
7d
7e
180
78
230
220
120
180
5
a
The listed result was the average of three independent experiments.
ranged from 42 to 260 μg/mL in DTSB medium and from 26 to 230 μg/
mL in LB medium. Among these new inhibitors, compound 2e has the
smallest MIC50 value in both DTSB and LB medium. The compound 4d
has the largest MIC50 value in DTSB medium and the compound 4c has
the largest MIC50 value in LB medium. The results showed most
DTSB is an iron-depleted medium [25]. In the culture environment
of DTSB plus heme, the compounds mainly inhibited the growth of P.
aeruginosa by competitively binding to pa-HemO and inhibiting the
utilization of heme. LB medium is a nutrient-rich formulations that
provide peptides, peptones, vitamins (including iron) and trace
Fig. 2. The determination of DMSO toxicity on P.
aeruginosa growth by measuring OD600 value.
The toxicity of DMSO on bacterial growth was de-
termined by measuring the OD600 value for 14 h each
hour. The column 1 is non-DMSO control group and
column 12 is non-PAO1 group. From column 2 to
column 11 are 1% DMSO group to 10% DMSO group.
Compared with the control group without DMSO,
only the 1% DMSO test group did not affect the
growth of P. aeruginosa (P > 0.05). The growth of P.
aeruginosa was gradually inhibited in the remaining
groups as the concentration of DMSO increased.
Figure automatically generated by BioTek Synergy
H1 hybrid microplate reader.
66

J. Zhao et al.
MicrobialPathogenesis129(2019)64–67
elements [26]. In LB medium, the compounds may interfere with the
other iron metabolic pathways of P. aeruginosa or inhibit the growth of
P. aeruginosa by other mechanisms than inhibiting the uptake of heme
by bacteria. This may lead that compounds show stronger inhibitory
effects on P. aeruginosa in the LB medium test group than in DTSB
medium test group. The specific mechanism needs further study.
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Declarations of interest
None.
Ethical approval
Not applicable.
Informed consent
Not applicable due to study design.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.micpath.2019.01.047.
Funding
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This work was supported by the [Chinese Medicine Science and
Technology Development Project Fund of Shandong Province] under
Grant [NO.2017-200], the [Postdoctoral Applications Research Project
Fund of Qingdao], under Grant [NO.2016055], and [The Affiliated
Hospital of Qingdao University Youth Research Fund], under Grant
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