Structure-based design and biological evaluation of inhibitors of the pseudomonas aeruginosa heme oxygenase (pa-HemO)

Full text of DOI:10.1016/j.bmcl.2018.02.027

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design and biological evaluation of inhibitors of the
pseudomonas aeruginosa heme oxygenase (pa-HemO)
Dongdong Liang a,1, Elizabeth Robinson a,1, Kellie Hom a, Wenbo Yu a, Nam Nguyen a, Yue Li b,
Qianshou Zong c, Angela Wilks a, , Fengtian Xue a,
⇑
⇑
a Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, United States
b Department of Chemistry and Biochemistry, University of Maryland College Park, College Park 20740, United States
c College of Biological and Chemical Sciences and Engineering, Jiaxing University, Jiaxing City, Zhejiang 314001, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 January 2018
Revised 3 February 2018
Accepted 13 February 2018
Available online xxxx
Ó 2018 Elsevier Ltd. All rights reserved.
Introduction
drive the metabolic flux of heme into the cell.21–23 Furthermore,
the P. aeruginosa
DhemO isogenic mutant, or a strain comple-
Pseudomonas aeruginosa is an opportunistic Gram-negative bac-
terial and one of the most common causes of hospital-acquired
infections worldwide.1,2 It is the major human pathogen that leads
to severe lung deterioration in cystic fibrosis (CF) patients.3 The
treatment of Pseudomonas is further complicated by its resistance
to most available antibiotics.4 For P. aeruginosa infections
polymyxins B and E (colistin) are the only choices of antibiotics
despite their high toxicity.5 Therefore, it is urgent to develop new
therapeutic agents for MDR P. aeruginosa.
Iron is an essential nutrient for survival and virulence of P.
aeruginosa.6 It regulates a number of virulence factors in P. aerugi-
nosa including exotoxins, proteases, and biofilm formation.7–10
Within the host iron is not readily available due to sequestration
by iron binding proteins such as transferrin and ferritin or in the
form of heme.11 However Pseudomonas overcomes this situation
through a variety of mechanisms including the secretion of sidero-
phores (pyoverdine and pyochelin),12,13 ferrous iron uptake
(Feo),14 and its unique heme acquisition systems.15–19
mented with a non-functional pa-HemO, showed significant atten-
uation of infection in a mouse lung infection model, when
compared to the wild type strain (unpublished results). This data
suggests pa-HemO inhibitors, by blocking a key mechanism of
the iron acquisition system, represents a promising therapeutic
target for P. aeruginosa infections.
Small molecule inhibitors of pa-HemO have been reported.24–26
These inhibitors achieved activities by binding either to the heme-
binding active site25 or an allosteric site26 depending on their
structural features. Most of these known inhibitors are close
derivatives of high throughput screen (HTS) hits, which commonly
suffer from major drawbacks such as modest potency (KD 20–50
mM) and poor pharmacological properties. For instance, we
recently reported pa-HemO inhibitor 1 (Fig. 1), with the KD value
of 18.4 mM and limited chemical stability.25
The pa-HemO has a unique heme-binding active site. The sol-
vent accessible surface of pa-HemO (ꢀ7.5 Å3) is much smaller than
the mammalian enzyme HO1 (43.6–59.7 Å3).27–30 More interest-
ingly, different to the binding mode in human HO1 (Fig. 2A) and
other known isozymes, heme binds in the pa-HemO active site
with a dramatically rotated (ꢀ100°) orientation28 as a result of
the unique amino acid network present in the active site (Fig.
2B). These structural differences provide opportunities for selective
inhibition of the pa-HemO over other HemOs such as HO1.31
To take advantage of the unique active site of pa-HemO, we
designed a series of inhibitors (2–31) based on a 3-(4-oxo-2-
thioxothiazolidin-3-yl)propanoic acid scaffold (Fig. 3). New com-
pound design was directed by the computer-aided drug design
(CADD) Site Identification by Ligand Competitive Saturation
(SILCS) method as reported previously.26 The carboxylic acid or
As part of the heme acquisition system, heme degradation by
the enzyme heme oxygenase (HemO) is essential for P. aeruginosa
to acquire iron from the host.20 P. aeruginosa HemO (pa-HemO) cat-
alyzes the reaction to breakdown heme and release iron, along
with CO and b- and d-biliverdin.20 It has also been shown by the
Wilks Lab that the catalytic activity of pa-HemO was pivotal to
⇑
Corresponding authors.
E-mail addresses: awilks@rx.umaryland.edu (A. Wilks), fxue@rx.umaryland.edu
(F. Xue).
1
D.L. and E.R. contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2018.02.027
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
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D. Liang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 1. Chemical structure of the pa-HemO inhibitor 1.
tetrazole groups of compounds 2–31 mimic one of the two acid
groups of heme, to form an electrostatic interaction with the basic
residue Lys132 of pa-HemO. The 4-oxo-2-thioxothiazolidin-3-yl
fragment (shown in brown) is chosen to mimic one of the pyrrole
rings of the porphyrin, to fit into the hydrophobic binding pocket
around Val33 (Fig. 2B). The same moiety could also form potential
interaction with the iron-chelating residue His26 of pa-HemO. We
note the controversial reports on the application of rhodanine in
medicinal chemistry;32,33 studies in our lab to modify the rhoda-
nine are on-going. The substituted A-ring is selected to interact
Fig. 3. Design and general structure of new inhibitors 2–31.
the configuration change of the enzyme upon inhibitor binding.
Overall, the results show that compound 3 binds to the active site
with modest affinity, suggesting that the scaffold can be suitable
developing inhibitors for pa-HemO.
As shown in Scheme 1, Knoevenagel condensation of selected
aromatic aldehydes (32) with 3-(4-oxo-2-thioxothiazolidin-3-yl)
ethylene derivatives (33a-d) in the presence of 10 equivalents of
sodium acetate (NaOAc) in acetic acid (AcOH) gave compounds
2–23 and 28–31 in good to excellent yields upon heating. The syn-
thesis of compounds 24–27 has been achieved using a two-step
procedure. Suzuki coupling of boronic acid 34 with substituted
iodobenzenes in the presence of Pd(0) catalyst generated com-
pound 35 in modest to good yields. Aldehyde 35 was then submit-
ted to Knoevenagel condensation to provide inhibitors 24–27 in
good yields.
Compared to inhibitor 2, the 2-furan analog 4 was slightly more
potent, while the catechol analog 5 indicated similar activity
(Table 1). Similar to the results of the tetrazole inhibitor 3, slightly
enhanced affinities were observed when the carboxylic acid groups
of compounds 4 and 5 were replaced by the aromatic acid bioisos-
tere tetrazole. Introduction of an electrodonating methoxy substi-
tution at either the m- (8) or p-position (9) generated new
inhibitors with ꢀ2-fold improved inhibitory potency. The p-F sub-
stituted analog 10 indicated similar affinity to that of inhibitor 2.
Larger halogen Cl-substitution at the same position yielded com-
pound 11 with >3-fold increased affinity, although the diCl-substi-
tuted analog 12 didn’t further improve the potency. The aliphatic
p-iPr analog 13 showed a 5-fold improvement in potency com-
pared to compound 2. Despite of the decreased potency of the 1-
naphthyl analog 14, the 2-naphthyl analog 15 showed a KD value
of 3.9 mM. Similarly good potency was observed with the indole
analog 16.
with the hydrophobic pocket next to residues Phe189 and Q52
,
while the extended tail B-ring is employed to extend the com-
pound to interact with residues Phe117 and Asn19 of pa-HemO.
To test our hypothesis, we first synthesized two pyridinyl com-
pounds 2 and 3 (Fig. 4A). Compounds 2 and 3 indicated modest
affinities to pa-HemO with the KD values of 51 mM and 27 mM,
respectively, using the previously established fluorescence
quenching assay.26 The potency of the tetrazole analog 3 was
almost 2-fold higher than that of the carboxylic inhibitor 2. We
reasoned that the enhancement in potency might due to the pres-
ence of residue Phe128 next to Lys132 (Fig. 2B), which can form
additional
p–p stacking interaction with the tetrazole fragment
of inhibitor 3. We then performed saturation transfer difference-
nuclear magnetic resonance (STD-NMR) experiments to confirm
the binding of compound 3 to pa-HemO, and found that both the
pyridine moiety and aliphatic tail of the inhibitor show clear bind-
ing to the enzyme (Fig. 4B). To further characterize the interaction
between inhibitor 3 and pa-HemO, we carried out 1H,15N-HSQC
NMR experiments (Fig. 4C). The presence of compound 3 caused
multiple chemical shift perturbations (CSPs) in the heme binding
site of pa-HemO. Interestingly residues with CSP over 3-fold of
standard deviation (r ,
), including Leu32, Ser35, Lys36, Phe39, Arg85
Glu104, Ser119, Glu137, Asn141, Phe189, Gly190 and Arg195, are mostly
located in the vicinity of the heme-binding active site (Fig. 4C). Of
them only Arg85 and Glu104 locate on the back surface of the
enzyme. The strong CSPs of these two residues can be results of
Fig. 2. Heme-binding active sites of A) HO1 (PDB 1 N45) and B) pa-HemO (PDB 1SK7).
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D. Liang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Fig. 4. Characterization of initial compounds 2–3. A) Chemical structures and KD values of compounds 2–3 in the fluorescence quenching assays. B) STD-NMR experiment
results: 1H spectra (black) and the STD spectrum (blue) of compound 3. Note that the 1H NMR of compound 3 indicated a mixture of two rotamers with estimation of 1:1 ratio.
One of the two rotamers binds favorably to apo pa-HemO. C) 1H,15N-HSQC NMR results of compound 3. Black: apo pa-HemO; Red: compound 3 mixed with apo pa-HemO.
Scheme 1. Synthesis of compounds 2–31. Reagents and conditions: (i) 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid, NaOAc:AcOH (1:10),105 °C, 6–14 h, 47–87%; (ii)
iodobenzene, Pd(PPh3)2Cl2, K2CO3, dioxane:H2O (3:1), 100 °C, overnight, 71–87%.
To explore inhibitors with potential interactions to the
hydrophobic pocket formed by Phe117 and Asn19, a collection of
11 new inhibitors (17–27) were synthesized with a B-ring included
in their chemical structures (Table 2). We first tested structurally
rigid bi-Ph compounds 17 and 18. The m-Ph analog 17 indicated
a significant decrease of the inhibitory potency, however, the p-
Ph compound 18 indicated a promising KD value of 3.3 mM. With
the introduction of a flexible linker between the A- and B-rings,
compounds 19–22 were synthesized and tested. The results indi-
cated that the m-PhNH analog 21 gave the best inhibitory potency
with a KD value of 1.2 mM. We have also studied inhibitors with
extended chemical structures based upon the furan analog 4
(23–27). While the p-methylester-Ph substitution (23) signifi-
cantly diminished the inhibitory potency, the same substituent at
the m-position led to compound 25 with a KD value of 7.6 mM.
The CN group (26) at the same position was detrimental to the
binding affinity. The m-OH substituted analog gave the most
potent compound 27 with a KD value of 1.1 mM, which is similar
to the value of the substrate heme.24
1 and 2, this analysis results in a high PI ꢀ 0.70 and R2 ꢀ 0.53 for
the correlation between LGFE and KD as shown in Fig. 5. This indi-
cates FragMaps are of utility with respect to both qualitatively
directing ligand design and yield a reasonable level of quantitative
predictability, indicating the utility of the approach in the further
refinement of the presented compounds.
The binding sites of inhibitors 18, 21, and 27 were studied using
both SILCS calculation (Fig. S1) and 1N,15N-HSQC NMR experiments
(Fig. 6 and Tables S2–S5). With the presence of inhibitor 18, signif-
icant CSPs (> 3r) were observed for residues N19, L32, S35, K36,
Q52, E104, V118, S119, S183, A185, and R195 (Fig. 6A and S2). All
these residues are located in the vicinity to the heme binding
active site of pa-HemO. Perturbation of six residues, L32, S35,
K36, E104, S119 and R195 were also detected for inhibitor 3, sug-
gesting that these inhibitors may bind to the same binding site of
the enzyme. Compared to inhibitor 18, the addition of inhibitor 21
led to significant CSPs for less residues including L32, Q52, E104,
L116, S119, L177, S183, L193 and R195 (Fig. 6B and S3). Most of
these residues overlap with those for compounds 3 and 18, indicat-
ing that compound 21 likely adapts similar binding mode in the
heme binding site of the pa-HemO. The presence of inhibitor 27
caused significant CSP for pa-HemO residues L18, E37, A40, Q56,
L68, S77, L113, G190, and F197 (Fig. 6C and S4). In addition,
Given the availability of the assay results, further analysis of the
utility of the SILCS LGFE scores was undertaken (Fig. 5). Correlation
analysis and calculation of the predictive index (PI)34 with respect
to LGFE scores were performed. Based on all compounds in Tables
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D. Liang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 1
Table 2
Structure features, calculated properties, KD values of compounds 4–16.
Structure features, calculated properties, KD values of compounds 17–27.
.
.
b
b
LGFEa (kcal/mol)
LEa
KD (mM)
Cmpd
A-B
LGFEa (kcal/mol)
À31.1
LEa
KD (mM)
20
Cmpd
A
R
17
À1.24
2
4
5
6
7
–CO2H
tetrazole
–CO2H
–22.7
À20.8
–23.9
–23.7
À1.26
À1.04
À1.14
À1.03
38 10
26
57
45
6
8
6
18
19
20
À29.8
À31.8
À30.5
À1.19
À1.22
À1.17
3.3 0.9
7.0 2.7
6.5 1.3
tetrazole
8
–CO2H
À26.1
À26.7
À1.24
25
8
9
–CO2H
À1.27
23
4
10
11
12
13
–CO2H
–CO2H
–CO2H
–CO2H
–23.9
À26.6
À27.7
À26.9
À1.19
À1.33
À1.32
À1.22
44
15
14
10
9
2
4
4
21
22
À31.4
À1.21
À1.13
1.2 0.2
7.4 0.6
–32.6
23
24
À30.9
À28.9
À1.10
À1.00
28
6
6.5 1.0
7.6 1.1
14
–CO2H
À29.6
À1.28
13
2
25
À30.7
À1.10
15
16
–CO2H
–CO2H
À30.0
À26.6
À1.30
À1.21
3.9
1
26
27
À30.1
À28.7
À1.16
À1.15
13 1.4
1.1 0.2
6.4 1.4
a
Ligand Grid Free Energy (LGFE) and Ligand Efficiency (LE) calculated with
SILCS.26
a
Ligand Grid Free Energy (LGFE) and Ligand Efficiency (LE) calculated with
SILCS.26
b
The listed result was the average of three independent experiments.
b
The listed result was the average of three independent experiments.
1N,15 N-HSQC peaks of heme binding site residues L32, Q52, S119
and R195 disappeared due to their fast exchange rate. These results
indicated that upon the binding of inhibitor 27, the heme binding
site of pa-HemO becomes more flexible.
with the results obtained from computational analyses. Further
structure optimization is undergoing for anti-microbial activities
of inhibitors.
The anti-pseudomonas activity of the three most potent com-
pounds, 21, 27, and 18 were evaluated using both the MIC50 by
growing P. aeruginosa in minimal media supplemented with heme
or free iron, and biofilm formation assays (Table 3). Our results
indicated all three compounds showed poor anti-bacterial effects
on pseudomonas in both assays. We reasoned that the presence
of the carboxylic acid group in these compounds might prohibit
the in vivo effects of inhibitors, as evidenced in a number of previ-
ous studies.34,35 It has been well documented that compounds,
with in vivo activities toward pseudomonas, usually employs basic
amino groups.35 Therefore, we have synthesized and tested com-
pounds 30 and 31, which showed KD values of 43 and 18 mM,
respectively. However, these compounds also did not have signifi-
cant antibacterial activity.
In summary, 4-Oxo-2-thioxothiazolidin-3-yl-propanoic acid
based inhibitors of pa-HemO are described to interact with the
unique network of residues in the heme-binding active site of
the enzyme. SAR efforts of the series resulted in analogs 21 and
27, with around 1 mM affinities. NMR studies confirmed the bind-
ing site of selected inhibitors of the family, which is consistent
Fig. 5. Correlation plots between LGFE and KD. Coefficient of determination (R2) and
predictive index (PI) are shown for the quality of correlation.
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D. Liang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Fig. 6. A representation of the pa-HemO based on Protein Data Bank structure (PDB: 1SK7) is shown, indicating residues perturbed upon binding of compounds 18 (A), 21 (B),
and 27 (C). (A) In magenta, residues with > 3 chemical shift perturbations listed as the following in increasing order: A84, Q52, L116, L193, L177, E104, S183, R195, L32, and
S119. (B) In magenta, residues with > 3
E104. (C) In magenta, residues with >3
r
r
chemical shift perturbations listed as the following in increasing order: K36, Q52, A185, V118, R195, L32, S35, S119, N19, S119 and
r
chemical shift perturbations listed as the following in increasing order: A185, E37, S77, G190, Q56, L113, L68, A40, L18 and F197. In
blue, residues L32, Q52, S119 and R195 (shown in purple) disappeared due to the fast exchange rate.
Table 3
Structure features and properties of compounds 28–31.
Cmpd
Structure
KDa(mM)
MIC50 (mg/mL)
Biofilm assay (mg/mL)
21
1.2 0.2
>300
>300
27
1.1 0.2
>300
>300
18
28
29
3.3 0.9
2.3 0.5
5.2 0.7
>300
NAb
NAb
>300
>300
>300
30
31
43 11
>300
NAb
>300
>300
18
3
a
The listed result was the average of three independent experiments.
NA refers to no microbial inhibition activity occurred.
b
Acknowledgements
A. Supplementary data
We thank the National Health Institute grant #AI102883 to A.
W., and Dr. Amanda Oglesby-Sherrouse for advice on the biofilm
formation assays.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.02.027.
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D. Liang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
19. O’Neill MJ, Bhakta MN, Fleming KG, Wilks A. Proc Natl Acad Sci USA.
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