Novel 2-Substituted 3-Hydroxy-1,6-dimethylpyridin-4(1 H)-ones as Dual-Acting Biofilm Inhibitors of Pseudomonas aeruginosa

Article abstract of DOI:10.1021/acs.jmedchem.0c00763

2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS), a compound from P. aeruginosa, functions as both a quorum sensing (QS) regulator and a potent iron chelator to induce expression of pyoverdine and pyochelin which are involved in high-affinity iron transport syste

Full text of DOI:10.1021/acs.jmedchem.0c00763

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Article
Novel 2-Substituted 3-Hydroxy-1,6-dimethylpyridin-4(1H)-ones
as Dual-Acting Biofilm Inhibitors of Pseudomonas aeruginosa
Jun Liu, Jin-Song Hou, Yi-Bin Li, Zhi-Ying Miao, Ping-Hua Sun, Jing Lin, and Wei-Min Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00763 • Publication Date (Web): 31 Aug 2020
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Novel 2-Substituted 3-Hydroxy-1,6-dimethylpyridin-4(1H)-ones
as Dual-Acting Biofilm Inhibitors of Pseudomonas aeruginosa
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Jun Liu, Jin-Song Hou, Yi-Bin Li, Zhi-Ying Miao, Ping-Hua Sun*, Jing Lin*,
Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
*
Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M.
Chen). Fax: +86 20 8522 4766.
E-mail address: pinghuasunny@163.com(P.-H. Sun), linjing_jnu@163.com (J. Lin),
twmchen@jnu.edu.cn (W.-M. Chen).
ABSTRACT
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-Heptyl-3-hydroxy-4(1H)-quinolone (PQS), a compound from P. aeruginosa,
functions as both a quorum sensing (QS) regulator and a potent iron chelator to induce
expression of pyoverdine and pyochelin which are involved in high-affinity iron
transport systems. A potential dual-acting anti-biofilm strategy requires molecules
designed to interfere with iron uptake and the QS system of P. aeruginosa. A series of
2
-substituted 3-hydroxy-1,6-dimethylpyridin-4-ones have been designed, synthesized
and tested as biofilm inhibitors of P. aeruginosa. One compound,
N-((1,3,6-trimethyl-4-oxo-1,4-dihydropyridin-2-yl)methyl)hexanamide (10d) exhibits
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8.67% biofilm inhibitory activity at 20 μM. Further mechanistic studies have
confirmed that this compound not only inhibits the QS systems of P. aeruginosa, but
also acts as an iron chelator to compete strongly with pyoverdine, causing iron
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deficiency in bacteria. The pyoverdine receptor FpvA was revealed as the target of
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INTRODUCTION
Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen that by the
formation of biofilm often causes drug resistance in clinically acquired infections1.
The quorum sensing (QS) systems mediated by chemical signal molecules are among
the most common regulatory mechanisms in the biofilm formation of P. aeruginosa
2
and the release of bacterial virulence factors . P. aeruginosa possesses three main QS
systems, las, rhl and pqs, which are mediated by three chemically distinct classes of
signal molecules, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL)3,
4,5
N-butanoyl-L-homoserine lactone (C4-HSL) , and 2-heptyl-3-hydroxy-4(1H)
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,7
quinolone (PQS) . In the las system for example, when the concentration of
3
-oxo-C12-HSL exceeds a certain threshold, it binds to the transcriptional receptor
LasR and activates the expression of genes that produce virulence factors such as
elastase. Similar to 3-oxo-C12-HSL, C4-HSL and PQS can bind to their respective
receptors RhlR and PqsR and activate certain target genes, including those responsible
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for the production of rhamnolipids and pyocyanin respectively . Recent reports show
that fourth inter-cellular communication signal molecule, IQS
2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde), which is capable of sensing
a
(
phosphate depletion stress, modulates bacteria pathogenesis by interacting with the
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quorum sensing network . In the past decade, QS inhibitors (QSIs) have been
considered to be agents that potentially can reduce pathogenicity and overcome
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bacterial antibiotic tolerance. Many anti-biofilm compounds that act as specific QSIs
have been described in recent years, but none of them have been successfully used
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clinically . Consequently, new strategies for the design and development of biofilm
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inhibitors are necessary.
Iron is an essential element in P. aeruginosa and plays a crucial role in bacterial
biofilm formation12,13. Microorganisms acquire iron from their hosts. The ferrous
cation is the form involved in bacterial biochemical pathways, but under physiological
conditions it is relatively unavailable and P. aeruginosa indirectly exploits the more
readily available ferric cation as a source14,15. Bacteria have evolved to have a series
of iron acquisition systems, including iron transport systems based on pyoverdine
(Pvd) and pyochelin (Pch) which uptake iron16,17. Published results have shown that
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iron chelators can interfere with bacterial biofilm formation and more interesting, as
presented in Figure 1, PQS not only acts as a QS signal molecule in regulating the
formation of bacterial biofilm but also functions as a potent iron chelator which
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induces expression of the Pvd and Pch iron transport systems . This suggests that
PQS analogs could be developed as inhibitors that interfere with both iron uptake and
QS systems, thus performing as dual-acting inhibitors of P. aeruginosa biofilm
formation.
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Figure 1. Diagrammatic representation of the role of PQS, and regulation of biofilm formation
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between PqsR and iron uptake systems in P. aeruginosa . 2-Alkyl-4-quinolone (AQ) biosynthesis
depends on the actions of PqsA, B, C and D on anthranilate (provided via anthranilate synthase,
PhnAB) to generate HHQ. PQS is produced from HHQ via the pqsH gene product. Both PQS and
HHQ are co-inducers of PqsR which drives the expression of the pqsABCDE operon. Unlike
HHQ, PQS induces microvesicle formation and is a potent iron chelator which induces expression
of the pyoverdin and pyochelin high affinity iron transport systems. Gray arrows represent PQS
biosynthesis. Black arrows indicate gene expression. Blue arrows represent the combination of
3
+
signal molecules to pqsR or Fe and red arrows indicate up-regulation.
In this study, a class of 2-substituted 3-hydroxy-1,6-dimethylpyridin-4-one
derivatives with the structural characteristics of both QS inhibitors and iron chelators
were designed and evaluated as dual-acting biofilm inhibitors of P. aeruginosa. The
structure–activity relationships (SAR) of 3-hydroxy-1,6-dimethylpyridine-4-one
derivatives were investigated. N-((1,3,6-Trimethyl-4-oxo-1,4-dihydropyridin-2-yl)
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methyl)hexanamide (10d) was found to exhibit 68.67% biofilm inhibitory activity at
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0 μM. There was also a reduction in the cytotoxicity for Vero and LO2 cells caused
by the acute infectivity of P. aeruginosa. Further experiments demonstrated that 10d
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affects both iron acquisition systems and QS systems.
RESULTS AND DISCUSSION
Chemistry.
A working hypothesis is that the structures derived from the QS signal molecules
and iron chelators could be combined to produce novel biofilm inhibitors which
interfere with both iron chelation and bacterial QS systems. Since the PQS molecule
contains a 3-hydroxypyridin-4-one fragment found in known iron chelators,
3
-hydroxypyridin-4-one was selected as the core skeleton of a series of new
compounds. Some of the QS signal molecules have a hydrophobic alkyl side chain
and a series of side chains were therefore used to conjugate with hydroxypyridinone
(Figure 2). To explore the effects of different side chains, including the hydrophobic
alkyl group (10a–10f) and a cyclic or phenyl ring group (11a–11u), the biofilm
inhibitory activity of the compounds against P. aeruginosa were examined.
Compounds with a sulfonamide bond replacing an amide bond (13a–13g) and those
with an amino group replacing an amide bond (16a–16j) were designed and
synthesized (Figure 2).
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Core skeleton
O
OH
O
N
OH
N
H
N
R
Iron chelators
O
O
1
0a-10f, 11a-11u
OH
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N
H
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O
Hybrid scaffold
Study of the linker
OH
OH
O
PQS
H
N
R
N
S
R
N
N
O
Side chain
Linker
13a-13g
O
H
N
O
O
O
OH
H
C -HSL
4
N
alkyl chain, phenyl ring
heterocyclic ring, aliphatic ring
O
R =
N
R
H
N
O
1
6a-16j
O
O
3
-oxo-C -HSL
1
2
Figure
2.
The
design
of
a
drug
scaffold,
and
the
structures
of
3
-hydroxy-1,6-dimethylpyridine-4-one derivatives with various 2-substituted side chains.
Synthesis of target compounds 10a–10f, 11a–11u, and 13a–13g. The precursor,
allomaltol was prepared from the readily available kojic acid according to a published
2
0
method . Compound 1 was synthesized by condensation of allomaltol with
formaldehyde in the presence of sodium hydroxide. The 3-hydroxyl group was then
protected by a benzyl group to obtain 2. Next, the 2-(1-hydroxyl) group of 2 was
protected using 3,4-dihydro-2H-pyran to obtain 3. Compound 3 reacted with
methylamine, resulting in the formation of 4, and the protecting group was removed
by heating with 2 mol/L HCl to yield 5. The transformation of the 2-(1-hydroxyl)
group in 5 to a 2-(1-amino) group was achieved by the Mitsunobu reaction of 5 with
phthalimide, triphenylphosphine and diethyl azodicarboxylate, leading to 6 which was
subsequently hydrolyzed by hydrazine hydrate, resulting in the formation of
2-(aminomethyl)-3-(benzyloxy)-1,6-dimethylpyridin-4(1H)-one (7) (Scheme 1).
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Scheme 1. Synthetic route to compound 7.
O
O
O
O
OBn
OTHP
OH
OH
OH
OBn
OH
b
c
a
1
1
1
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O
O
O
O
allomaltol
d
1
2
3
O
O
O
OBn
OTHP
OBn
OBn
O
N
e
f
OH
N
N
N
O
4
5
6
O
g
OBn
NH2
N
7
Reagents and conditions：(a) Formaldehyde, NaOH, H O, rt, overnight; (b) NaOH, BnCl, MeOH,
2
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0 ℃, overnight; (c) THP, PTS, dry DCM, rt, 3 h; (d) 33-35% MeNH , EtOH, 75 °C, overnight;
2
(e) 2 mol/L HCl, EtOH, 80 ℃, 4 h; (f) Phthalimide, Ph P, diethyl azodicarboxylate (DEAD), dry
3
THF, 0 ℃-rt., overnight; (g) NH NH , EtOH, 80 ℃, 4 h.
2
2
Acylation or sulfonylation of the amine (7) with different acid chlorides or
sulfuryl chlorides in the presence of the catalyst pyridine afforded the derivatives 8a–
8
f, 9a–9u and 12a–12g. Subsequent removal of the benzyl protecting group by
hydrogenolysis with Pd/C gave compounds 10a–10f, 11a–11u, 13a–13g (Scheme 2,
Scheme 3).
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Scheme 2. Synthetic route to 10a–10f, 11a–11u derivatives.
O
O
O
N
OBn
NH2
OBn
OH
a
H
N
b
H
R1
N
R1
N
N
O
O
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8a-8f
Series : 10a-10f
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a-9s
1
1a-11u
Compound
a, 10a
b, 10b
c, 10c
R
Compound
R
Compound
R
8
9
f, 11f
9
n, 11n
8
OMe
8
9g, 11g
9h, 11h
9o, 11o
8d, 10d
8e, 10e
N
9
p, 11p
CF3
N
8
f
O
O
Cl
F
9
i, 11i
9
q, 11q
n=7
O
1
0f
9
j, 11j
F
9r, 11r
9s, 11s
n-7
OMe
OMe
9
a, 11a
9
k, 11k
CF3
9
b, 11b
9
c, 11c
9
t, 11t
9
l, 11l
OMe
OMe
9
d, 11d
9
m, 11m
9u, 11u
O
O
OMe
9e, 11e
,
Reagents and conditions: (a) R -CO Cl, dry DMF, pyridine, 0 ℃-rt, 4 h; or (8f, 9m) RCOOH,
1
2
EDCI，DMAP, rt., overnight; (b) H , 10% Pd/C, MeOH, overnight or (10f) ATF: H O (1:1), rt., 4
2
2
h and H , 10% Pd/C, MeOH, overnight.
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Scheme 3. Synthetic route to 13a–13g.
O
O
O
N
OBn
NH2
OBn
OH
a
b
H
N
H
O
O
N
N
N
S
S
R
R
1
1
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1
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O
O
7
12a-12g
Series : 13a-13g
Compound
R
Compound
R
Compound
12f, 13f
R
1
2a
Br
12c, 13c
12d, 13d
12e, 13e
R
N
12g
13g
1
3a
H
N
OMe
12b, 13b
F
Reagents and conditions: (a) R-SO Cl, dry DMF, pyridine, 0 ℃-rt, overnight; (b) H , 10% Pd/C,
2
2
MeOH, overnight.
Synthesis of the target compounds 16a–16j. Compounds 16a–16j were
synthesized in three steps from compound 5 as shown in Scheme 4. The
2
-(1-hydroxyl) group of 5 was chlorinated using thionyl chloride to obtain 14. The
chlorine in 14 was replaced by various primary amines to give 15a–15j. Finally, the
benzyl protecting group was hydrogenolyzed using Pd/C to obtain 16a–16j (Scheme
4
).
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Scheme 4. Synthetic route to 16a–16g derivatives.
O
O
O
O
N
OBn
OH
OBn
Cl
OBn
H
N
OH
a
b
c
0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
N
N
N
R
R
5
14
15a-15j
Series : 16a-16j
Compound
R
Compound
15e, 16e
R
Compound
15i, 16i
R
,
F
O
O
1
5a, 16a
N
O
15b, 16b
15f, 16f
15j, 16j
N
CF3
1
5c, 16c
15g, 16g
15h, 16h
N
1
5d, 16d
N
Reagents and conditions: (a) SOCl ; (b) R-NH , dry DMF, TEA, 75 °C, 5-6 h (c) H , 10% Pd/C,
2
2
2
MeOH, overnight.
Biological Evaluation.
Determination of minimum inhibitory concentrations (MIC). We tested the
effect of target compounds on bacterial growth (Table 1) and our results showed that
at a concentration of 512 M, these synthetic derivatives exhibited no influence on
the growth of P. aeruginosa PAO1.
Anti-biofilm screening and SAR study. The inhibitory effect of compounds on
biofilm was determined by a crystal violet staining assay. Azithromycin and our
previously reported active pyranone derivative (6a)50 were utilized as positive
controls. Azithromycin and 6a displayed IC values in the staining assay of 6.76 ±
5
0
2.28 μM and 20.31 ± 0.50 μM, respectively. As detailed in Table 1, some target
1
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compounds with an amide linker exhibited biofilm inhibitory activity (IC < 100 μM)
5
0
and 10d, a target compound with an alkyl chain of six carbons, displayed the best
anti-biofilm effect with an IC value of 6.57 ± 0.74 μM. The results showed that
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
both extending (10e, 10f) and shortening (10a, 10b, 10c) the length of the alkyl chain
sharply reduced the biofilm inhibitory activity. To study the effect of the R group on
biofilm formation, compounds with various aliphatic and aromatic rings introduced
into the R group (11a–11u) were synthesized. Compounds 11a–11u with aliphatic
and aromatic rings displayed lower bioactivities (IC > 26.49 ± 1.62 μM) than 10d
5
0
(IC50 = 6.57 ± 0.74 μM). Interestingly, we found that compound 11e with a
tetrahydrofuran in the side chain had the highest activity to inhibit biofilm with an
IC value of 26.49 ± 1.62 μM, while compounds substituted with cycloalkanes (11a–
5
0
1
1d) revealed much decreased effect on biofilm inhibition. We investigated if the
substituents on the phenyl ring and the number of carbon atoms between the cyclic
group and the amide bond have any influence on anti-biofilm activity and found that
target compounds (11j–11n) containing various groups on the phenyl ring exhibited
weak biofilm inhibitory activity with IC values > 38.42 ± 10.18 μM. In contrast,
5
0
p-methoxyphenyl derivative (11l) showed a better anti-biofilm effect than the
m-methoxyphenyl derivative 11m or the o-methoxyphenyl derivative 11n.
Compounds 11s, 11t, and 11u with 1, 2, and 3 carbons between the cyclic group and
the amido bond of the side chain, respectively and electron donating groups on the
benzene ring were synthesized in a manner similar to that used for 11l and it was
found that the increases in the chain length drastically reduced the biofilm inhibitory
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
3
3
3
4
4
4
4
4
4
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4
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5
5
5
5
5
5
5
6
activity of these compounds.
To explore the influence of the linker on anti-biofilm activity, the amide linker
was replaced by a sulfonamide, providing compounds 13a–13g. Compounds with a
sulfonamide bond as a linker retained some anti-biofilm activity, but most of them
exhibited a biofilm inhibitory activity (IC > 51.63 ± 8.61 μM) which is lower than
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
that of 10d. We explored the amino bond as a linker in compounds 16a–16j. The
anti-biofilm activities of compounds 16a–16j which contain the amino bond as a
linker were still weaker than that of 10d. On the basis of these results, some
preliminary SARs of the 3-hydroxy-1,6-dimethylpyridin-4(1H)-one derivatives can be
summarized as follows: (1) compounds in the amide-linked series with six carbon
alkyl chains (10d) exhibited efficient biofilm inhibitory activity; (2) compounds in the
amide-linked series with various aliphatic and aromatic rings introduced into the
chain exhibited reduced activity, and the length of the chain between the cyclic group
and the linker had a dramatic negative influence on the biofilm inhibitory activity; (3)
replacement of the amide bond with a sulfonamide or amino bond failed to increase
the biofilm inhibitory activity.
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Table 1. Biological evaluation of derivatives against the P. aeruginosa PAO1.
IC50a.b( μM)
IC50a.b( μM)
Compound
Structure
Compound
11g
Structure
O
N
O
N
OH
H
N
OH
H
N
10a
45.14 ± 5.22
41.67 ± 1.37
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
OH
H
N
OH
H
N
N
10b
56.88 ± 0.44
57.94 ± 2.13
6.57 ± 0.74
11h
11i
11j
> 100
> 100
> 100
N
O
N
O
O
O
CF3
OH
H
N
OH
H
N
N
10c
N
O
N
O
O
O
OH
H
N
OH
H
N
F
1
0d
0e
N
O
N
O
O
O
OH
H
N
OH
H
N
CF3
1
> 100
> 100
> 100
11k
11l
61.05 ± 10.29
38.42 ± 10.18
> 100
N
O
N
O
O
O
OH
H
N
OH
H
N
OMe
10f
N
O
N
O
n = 7
O
O
O
OH
H
N
OH
H
N
11a
11m
N
O
N
OMe
O
O
O
N
OH
H
N
OH
11b
64.13 ± 0.62
65.07 ± 4.57
11n
11o
H
N
69.69 ± 5.79
46.78 ± 9.37
N
O
O
O
OMe
O
OH
H
N
OH
11c
H
N
N
O
N
O
O
O
OH
OH
11d
H
N
63.43 ± 7.62
11p
H
N
28.12 ± 6.74
N
N
O
O
O
N
O
N
OH
H
N
OH
O
1
1e
H
26.49 ± 1.62
75.56 ± 2.87
11q
11r
> 100
N
O
O
O
Cl
O
N
O
N
OH
H
N
OH
11f
H
54.95 ± 8.92
N
O
F
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
IC50a.b( μM)
IC50a.b( μM)
Compound
Structure
Compound
16b
Structure
O
O
OH
H
N
OH
11s
65.45 ± 7.29
H
52.45 ± 7.84
N
N
O
N
O
OMe
OMe
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
OH
H
N
OH
11t
59.07 ± 8.87
16c
16d
H
> 100
N
N
O
N
O
O
N
OH
H
N
OH
1
1u
3a
> 100
H
14.92 ± 1.49
N
N
O
OMe
O
N
O
OH
H
OH
O
N
H
1
> 100
16e
> 100
S
N
OMe
N
O
O
OH
H
O
N
OH
OMe
O
N
13b
N
O
N
S
> 100
16f
H
> 100
> 100
O
N
F
OH
H
O
OH
CF3
O
N
13c
59.31 ± 2.19
16g
H
S
N
O
N
O
OH
H
O
N
O
OH
N
N
S
N
13d
51.63 ± 8.61
16h
16i
H
16.89 ± 0.43
O
N
N
O
N
OH
O
N
H
OH
F
O
N
O
13e
S
> 100
H
> 100
O
N
N
O
OMe
OH
O
H
O
N
OH
N
S
1
1
3f
O
> 100
16j
H
34.96 ± 8.78
N
N
N
O
O
OH
OH
H
O
3g
N
HN
35.31 ± 8.49
DFP
> 100
N
S
O
N
O
N
OH
16a
H
24.53 ± 6.53
N
aIC50 = concentration of inhibitor needed to inhibit biofilm formation by 50%. b For most
compounds, the screening concentration of IC was 20-100 μM.
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Compound 10d prevents P. aeruginosa biofilm formation without interfering
with bacterial growth. Preliminary screening of biofilm inhibitory activity in the P.
aeruginosa PAO1 strain indicated that compound 10d has the best activity. To
evaluate the potential anti-biofilm activity of 10d comprehensively, its effects on the
biofilm formation in P. aeruginosa PAO1 strains at concentrations of 1.25, 2.5, 5, 10
and 20 μM were measured. The results showed that 10d reduced P. aeruginosa PAO1
biofilm biomass development in a dose-dependent manner and that it also has biofilm
inhibitory activity with an inhibition rate >25% at concentrations as low as 1.25 μM
(Figure 3A). The results of crystal violet staining assay in a glass tube confirmed that
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0d inhibits the formation of biofilm in a concentration-dependent manner (Figure
3B). Confocal laser scanning microcopy was used to observe the effect of 10d on
biofilms and, as shown in Figure 3C, after treatment with 10d at concentrations of
1
.25, 2.5, 5, 10 and 20 μM, the biofilm (green fluorescence) formation was reduced in
a dose-dependent manner. We confirmed that 10d (Figure S311 A) has no inhibitory
effect on the growth of bacteria at 1.25, 2.5, 5, 10, 20 μM concentrations. These
results show that 10d could act as a biofilm inhibitor by acting on specific targets
related to biofilm regulation rather than by inhibiting bacterial growth. The potential
targets and inhibitory mechanisms of the compounds were explored systematically.
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Effects of 10d on P. aeruginosa biofilm formation. (A) Biofilm formation at different
concentrations of 10d (1.25, 2.5, 5, 10, 20 μM) for 24 h in a microtiter plate. Bacterial counts
were quantified in triplicate. (B) Biofilm formation at different concentrations of 10d (1.25, 2.5, 5,
1
0, 20 μM) for 24 h in a glass tube. Bacterial counts were quantified in triplicate. (C) 3D image of
P.aeruginosa PAO1 biofilm after incubation with 10d (1.25, 2.5, 5, 10, 20 μM) for 24 h. Control
was untreated. The experiments were done in triplicate.
Compound 10d functions as an iron chelator, inducing P. aeruginosa–related
iron deficiency. Considering that iron ions have a regulatory effect on bacterial
biofilm formation, we first verified the iron-chelating ability of 10d and the influence
of iron content on bacteria. The pFe(III) value, defined as the negative logarithm of
free iron concentration in a solution, was selected as the parameter with which to
2
1
assess the ligand affinity for the metal under biological conditions and pFe(III) of
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10d was 20.6. The pFe(III) values of 10d were determined using a fluorescence
2
2
method with deferiprone (DFP) as the reference compound . The results showed that
1
0d exhibits satisfactory chelating ability with a pFe(III) value of 22.00 ± 0.14, which
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
is stronger than the DFP pFe(III) value of 20.81 ± 0.44. The ability of 10d to cause
bacterial iron deficiency and inhibit bacterial biofilm formation was investigated. We
analyzed the effect of various concentrations (1.25, 2.5, 5, 10, 20 μM) of 10d (Table
2
) on the iron content in P. aeruginosa PAO1 through inductively coupled plasma
mass spectrometry. We found that the iron concentration in the bacteria was 0.88 ±
0.30 μM and 1.23 ± 0.24 μM after the addition of compound 10d (20 μM) and
positive control DFP (20 μM), which is significantly lower than that in the control
group (1.91 ± 0.80 μM). Additionally, these data revealed that 10d reduced the iron
concentration in bacteria in a concentration-dependent manner, exhibiting a strong
iron-chelating ability which could lead to iron deficiency in bacteria. We also found
that the effect of compound 10d on iron chelation was as strong as the positive control
DFP, and the effects of compound 10d on the induction of iron deficiency and the
inhibition of biofilm formation in bacteria are considerably superior to those of DFP.
The biofilm inhibition rate of DFP is >100 µM. These differences suggest that
compound 10d could exert an efficient anti-biofilm activity dependent on its iron ion
chelation ability, but also could do so by chelating irons and supporting specific
interactions with some targets in the iron acquisition system. One possibility is that
the chelated iron ion is possibly used as a Trojan horse molecule to locate targets, and
the lower activity of DFP could be attributed to its inability to bind to specific targets.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Iron content in P. aeruginosa PAO1.
Concentration
a
Compound
Total Fe (µM)
(µM)
0
1.91 ± 0.80
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
.25
1.55 ± 0.69
1.56 ± 0.71
1.17 ± 0.50
0.97 ± 0.31
0.88 ± 0.30
1.23 ± 0.24
2
.5
1
0d
5
10
2
0
DFP
20
aAll dates represent mean ± S.D. from different experiments performed in triplicate.
Compound 10d competes strongly with Pvd. To investigate the validity of the
aforementioned hypothesis, the effects of 10d on the iron acquisition system were
analyzed. Production of siderophores and utilization is one of the major strategies
2
3
deployed by bacteria to access iron under iron-limiting conditions . P. aeruginosa
excretes two principal siderophores, namely pyoverdine24 (Pvd) and pyochelin25
(Pch). Pvd and Pch can naturally produce fluorescence in P. aeruginosa under
iron-limiting conditions. Consequently, the effects of 10d on the intercellular levels of
Pvd and Pch were first evaluated by fluorescence detection in ABTGC medium
(
B-medium (0.1% MgCl , 0.1% CaCl , 0.1% FeCl ) supplemented with 10% A10,
2 2 3
0
.2% glucose and 0.2% casamino acids) containing 10 μ M (iron-normal medium)
and 50 μM (iron-sufficient medium) of FeCl . We found that 10d (Figure 4A)
3
strongly increases the level of Pvd in a concentration-dependent manner to counteract
iron deficiency in an ABTGC medium containing 10 μM FeCl . However,
3
iron-chelating agents such as DFP show negligible effects on the production of Pvd at
all tested concentrations (1.25, 2.5, 5, 10, 20 μM). Furthermore, 10d (Figure 4B) and
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DFP have only a slight influence on the production of Pch. These results indicated
that 10d may have a high-affinity for the Pvd iron acquisition system that regulates
the iron acquisition pathway, thus causing iron deficiency and inhibition of biofilm
formation. The secretions of Pvd and Pch in P. aeruginosa were nearly undetectable
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
under the condition of sufficient supplementation with FeCl (50 μM in the ABTGC
3
medium), where 10d exhibited less effect on the levels of Pvd and Pch (Figure S312).
Figure 4. (A) Effects of 10d on the production of iron-free pyoverdine at different concentrations
(
1.25, 2.5, 5, 10, 20 μM) in iron supplemented ABTGC (10 μM of FeCl ). (B) Effects of 10d on
3
the production of iron-free pyochelin at different concentrations (1.25, 2.5, 5, 10, 20 μM) in iron
supplemented ABTGC (10 μM of FeCl ). The experiments were done in triplicate. Error bars are
3
means ± SDs. * = p < 0.05, ** = p < 0.01, *** = p < 0.00.
Inactivation of anti-biofilm activity of 10d in Pvds mutant and overexpressed
strains of fpvA. FpvA is a high-affinity outer membrane receptor of Pvd in P.
aeruginosa, and is controlled by two distinct ECF sigma factors, namely σFpvI and
σPvdS. The σPvdS factor is required for activating the synthesis of Pvd and the
2
6
expression of FpvA and is encoded by the Pvds gene and strains with Pvds mutants
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showed negligible ability to acquire iron through the high-affinity pyoverdine iron
acquisition system. To investigate the mechanisms of 10d in the regulation of the
high-affinity Pvd iron acquisition system and to identify its potential target, Pvds
mutant and fpvA-overexpressed strains were selected to evaluate the regulatory role of
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0d in the high-affinity Pvd iron acquisition system. As depicted in Figure 5A, the
inhibitory effects of 10d on biofilm formation in the Pvds mutant strain (the
high-affinity Pvd iron acquisition system deficient strain) was significantly reduced
and the inhibitory effect of 10d at 20 μM was decreased to approximately 20%. These
results confirmed that 10d can act on the high-affinity Pvd iron acquisition system
and indicated that 10d might serve as a biofilm inhibitor. We tested the effect of 10d
on biofilm inhibition in the fpvA-overexpressed strains, and our results showed that
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0d has a weak activity to inhibit biofilm in fpvA-overexpressed P. aeruginosa
(Figure 5B). Taken together, our data suggest that 10d is better able than the normal
iron-chelating agent DFP to induce iron deficiency and biofilm inhibition in P.
aeruginosa and that 10d could compete with Pvd by specifically interacting with the
siderophore receptor FpvA.
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Figure 5. (A) Effects of 10d on biofilm formation in Pvds mutant strain. (B) Effects of 10d on
biofilm formation in fpvA-overexpression strain. The experiments were done in triplicate. Error
bars are means ± SDs. * = p < 0.05, ** = p < 0.01, *** = p < 0.00.
In silico study on 10d with FpvA target. We tried regular free docking between
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FpvA and 10d in the presence or absence of Fe in the binding pocket. Our results
suggested that the 10d is not comparable to Pvd from either the structural or energy
aspects because of its relatively smaller size and weaker interaction. As mentioned
above, 10d competes strongly with Pvd, which raises the question of how the
competition between such different molecules proceeds. After observing the Pvd-Fe
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structure and studying the quantitative reaction of 10d and Fe , we assumed that a
sexadentate chelation of 10d and iron ion could be the key to the competitive binding.
Tripolymers of 10d were built and energy optimized in the opls3 force field in
Maestro, Schrödinger. 529 reasonable conformations in two groups (cis- and trans-
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tripolymers) were obtained by conformational searching . Following a two round
strategy docking, many preferable conformations in the active pocket were obtained,
most of them showing a better score (< -9) than Pvd (-7.37) in the same site and also
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with favorable energy affinities (Figure 6). A 40 ns MD simulation was completed on
the FpvA-10d-Fe complex, to confirm the affinity and potential conformational
change between the FpvA and the test compound. As shown in Figure 6B, the
binding of the 10d-Fe tripolymer was located firmly in the binding site within the beta
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barrel domain of FpvA . Two critical interactions were observed during the
simulation: the hydrogen bonds with ARG69 and TYR661, along with the alkyl tails
inserted into hydrophobic cavities surrounded by PHE660, TYR661, MET608 and
ALA64. An MM-PBSA calculation based on the last 15 ns trajectories gave a
satisfactory △G of -43.56 kcal/mol which is consistent with our hypothesis that 10d
competes with Pvd. Notably, although most of the FpvA structure remained stable,
the conformation of the signal domain was most flexible during the MD simulation.
This non negligible conformation change indicated the potential biological effects
caused by the 10d-Fe chelate.
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Figure 6. Docking conformation of 10d tripolymer in FpvA. (A) Structures of cis- and trans-
tripolymers. (B) tripolymer in the binding pocket of Pvd, (PDB ID 2W6T). (C) cis-(-10.499) and
trans-(-9.182) tripolymers docking results. (D) cis-tripolymer (-10.499) and Pvd (-7.37). cis- and
trans-tripolymers are depicted in green and yellow cylinders, respectively, Pvd is in purple. (E)
key interactions between 10d-Fe and FpvA in MD simulation. (F) RMSD of 10d-Fe and FpvA
during simulation. (G) signal domain conformation change in md simulation.
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0d inhibits QS-controlled P. aeruginosa virulence. On the basis of the
potential of 10d as a dual-acting biofilm inhibitor and the fact that 10d has
approximately 20% biofilm inhibitory activity in the high-affinity Pvd iron acquisition
system deficient strain (Pvds mutant strain), we tested the effect of 10d on the QS
systems, another putative regulatory pathway of compound 10d. Because QS systems
are responsible for the regulation of secretion of the virulence factor in P. aeruginosa
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PAO1 , the effect of 10d on three virulence factors were examined. The virulence
factors include elastase, rhamnolipid, and pyocyanin and they are regulated by the las,
rhl, and pqs systems, respectively. The results are presented in Figure 7. As expected,
10d significantly attenuates the activity of elastase (Figure 7A) and decreases the
levels of rhamnolipid (Figure 7B) and pyocyanin (Figure 7C) in P. aeruginosa
PAO1 in a concentration-dependent manner. Together, these results demonstrate that
the inhibitory effect of 10d on the production of three virulences might be related to
the QS system.
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Figure 7. Effects of 10d on the production of virulence factors at different concentrations (1.25,
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.5, 5, 10, 20 μM). (A) Elastase activity. (B) Rhamnolipid production. (C) Pyocyanin production.
Error bars are means ± SDs. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
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0d Inhibits the fluorescence of QS reporter strains. Three reporter strains,
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PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp were used to further explore
the influence of 10d on the three QS systems. The green fluorescence protein (gfp)
gene was integrated downstream from the promoters of lasB, rhlA, and pqsA,
respectively, and the production of green fluorescent protein (GFP) indicated the
activity of these promoters. As depicted in Figures 8A-8C, compound 10d
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significantly inhibits the fluorescence of PAO1-lasB-gfp, PAO1-rhlA-gfp, and
PAO1-pqsA-gfp strains in a dose-dependent manner (1.25, 2.5, 5, 10, 20 μM). To
verify that 10d targets the corresponding QS genes rather than the GFP protein, the
PAO1-gfp strain was used as a control strain to investigate the influence of 10d on the
fluorescence of the GFP protein. It was shown that 10d has no effect on the
fluorescence of GFP (Figure 8D). These experimental results indicate a direct
inhibitory effect of 10d on the QS pathway. Collectively, our data demonstrated that
the biofilm inhibition of 10d involved both iron uptake and QS systems.
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Figure 8. Dosage-dependent inhibition curves of 10d incubated with the following QS monitors
(A) PAO1-lasB-gfp, (B) PAO1-rhlA-gfp and (C) PAO1-pqsA-gfp. (D) PAO1- gfp. The
experiments were done in triplicate.
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0d Suppresses swimming and swarming motilities of P. aeruginosa. Flagellar
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and pili motility are necessary for P. aeruginosa biofilm development . During the
development of the P. aeruginosa biofilm, swarming and swimming motility are
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mediated by flagella. These motility modes are used for colony expansion along a
semisolid surface, which is correlated with the capacity of P. aeruginosa to colonize
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in vivo . Twitching motility is mediated by pili, a motility mode commonly observed
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in dense aggregates with cell-to-cell contact33,34, and plays a major role in the later
stages of biofilm development. We assessed the effect of 10d on the swimming,
swarming, and twitching motilities of P. aeruginosa PAO1 by inoculating P.
aeruginosa PAO1 after overnight culture onto motility plates. It was found that 10d
(Figure 9A, B) suppressed flagellum-driven swimming and swarming motilities in a
concentration-dependent manner at concentrations of 1.25, 2.5, 5, 10 and 20 μM. This
result was supported by the previously established anti-biofilm activity of 10d.
However, 10d (Figure 9C) had no effect on the twitching motility of P. aeruginosa
PAO1. This phenomenon was inconsistent with the report that indicated twitching
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motility was stimulated by lactoferrin acting as an iron chelator, which can function
as a biofilm inhibitor. This surprising result related to 10d contrasts with the findings
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related to lactoferrin in a published study . An earlier study showed that the twitching
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motility was positively correlated with the presence of lasR, lasI, and rhlI genes , and
the ability of 10d to inhibit the QS pathway was shown in the same study.
Accordingly, the unchanged influence on the twitching motility of 10d could be
attributed to the suppression of twitching motility caused by the inhibition of the QS
pathway which cancels the stimulation caused by 10d as an iron chelator. Therefore,
10d exhibits no effect on the twitching motility of P. aeruginosa PAO1. This result
further confirmed that 10d is a dual-acting biofilm inhibitor rather than a single target
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iron-chelating biofilm inhibitor. Furthermore, according to the characteristics of the
mechanism of the biological effects of 10d, we speculate that 10d may mainly act on
the PVD receptor FpvA to cause iron deficiency at low concentrations, which will
stimulating twitching motility, and thus cause results in d and e (Figure 9C) to likely
exceed that of blank control.
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Figure 9. Motility inhibition assays of P. aeruginosa PAO1. Three motility assays were
conducted on plates containing 10d on agar at different concentrations (1.25, 2.5, 5, 10, 20 μM).
(A) Swimming motility. (B) Swarming motility. (C) Twitching motility. The results shown are
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representative of results from three independent experiments.
Through a series of evaluations of biological characterization, we demonstrated
that 10d can not only selectively act on the Pvd receptors FpvA to cause iron
deficiency but also can interfere with the QS pathway to reduce QS-regulated
virulence factors (elastase, rhamnolipid, and pyocyanin) as well as the bacterial
motility in P. aeruginosa.
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Effect of 10d on cytotoxicity. We evaluated the toxicity of 10d in the African
green monkey kidney cell line Vero and the noncancerous human hepatocyte cell line
LO2 using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The chelating agent DFP can affect the iron content in normal cells and
possesses cytotoxicity within a certain range36-38. We found that 10d (Figure. 10) is
less cytotoxic than DFP to Vero and LO2 cells, and has less effect on the viability of
Vero and LO2 cells at concentrations up to 50 μM. Taken together, these results
indicated that compound 10d has low toxicity in Vero and LO2 cells.
Figure 10. Vero (A) and LO2 (B) cells were treated with 10d at various concentrations (0, 3.125,
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.25, 12.5, 25, 50, 100 μM) for 24 h. Cell viability was determined using an MTT assay. Error
bars are means ± SDs. * = p < 0.05, ** = p < 0.01, *** = p < 0.00.
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10d Reduces P. aeruginosa acute virulence in cells. To assess the potential of
the synthesized compounds for reduction of P. aeruginosa virulence, we evaluated the
ability of 10d in vitro to block P. aeruginosa acute virulence against the African
green monkey kidney cell line Vero and noncancerous human hepatocyte cell line
LO2 using cell viability as a readout. Cell survival was quantified 4 h post-infection
with P. aeruginosa in the presence or absence of 10d. P. aeruginosa was pre-treated
with different concentrations of 10d (0, 1.25, 2.5, 5, 10, and 20 μM) for 24 h. The
Vero and LO2 cells were infected with P. aeruginosa for 4 h. Subsequent MTT assay
showed that the pre-treatment with 10d significantly and dose-dependently promoted
the survival of infected Vero and LO2 cells (Figure 11). Notably, in the presence of
compound 10d (20 μM), the cell survival rate of Vero was increased from 41.5% to
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6.4% (Figure 11A) and that of LO2 was enhanced from 40.7% to 89.9% (Figure
1B). These data indicate that 10d has considerable therapeutic potential as a dual
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biofilm inhibitor in acute P. aeruginosa infections.
Figure 11. 10d rescues P. aeruginosa cell cytotoxicity. (A) anti-virulence efficacy in Vero cell
infection assays of 10d at various concentrations (1.25, 2.5, 5, 10, 20 μM). (B) anti-virulence
efficacy in LO2 cell infection assays of 10d at various concentrations (1.25, 2.5, 5, 10, 20 μM).
Error bars are means ± SDs. * = p < 0.05, ** = p < 0.01, *** = p < 0.00.
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