Design and Synthesis of Novel c-di-GMP G-Quadruplex Inducers as Bacterial Biofilm Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.1c00465

The formation of biofilms by clinical pathogens typically leads to chronic and recurring antibiotic-resistant infections. High cellular levels of cyclic diguanylate (c-di-GMP), a ubiquitous secondary messenger of bacteria, have been proven to be associate

Full text of DOI:10.1021/acs.jmedchem.1c00465

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Article
Design and Synthesis of Novel c‑di-GMP G‑Quadruplex Inducers as
Bacterial Biofilm Inhibitors
Teng-Fei Xuan,† Zi-Qiang Wang,† Jun Liu, Hai-Tao Yu, Qian-Wen Lin, Wei-Min Chen,* and Jing Lin*
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ABSTRACT: The formation of biofilms by clinical pathogens
typically leads to chronic and recurring antibiotic-resistant
infections. High cellular levels of cyclic diguanylate (c-di-GMP),
a ubiquitous secondary messenger of bacteria, have been proven to
be associated with a sessile biofilm lifestyle of pathogens. A
promising antibiofilm strategy involving the induction of c-di-GMP
to form dysfunctional G-quadruplexes, thereby blocking the c-di-
GMP-mediated biofilm regulatory pathway, was proposed in this
study. In this new strategy, a series of novel c-di-GMP G-
quadruplex inducers were designed and synthesized for develop-
ment of therapeutic biofilm inhibitors. Compound 5h exhibited
favorable c-di-GMP G-quadruplex-inducing activity and 62.18
6.76% biofilm inhibitory activity at 1.25 μM without any DNA
intercalation effect. Moreover, the favorable performance of 5h in interfering with c-di-GMP-related biological functions, including
bacterial motility and bacterial extracellular polysaccharide secretion, combined with the reporter strain and transcriptome analysis
results confirmed the c-di-GMP signaling-related action mechanism of 5h.
c-di-GMP is synthesized and degraded by diguanylate
cyclases (DGCs) and phosphodiesterases (PDEs), respec-
tively.4−6 Therefore, interference of c-di-GMP signal molecule
production has been proposed as an attractive strategy for the
treatment of bacterial biofilm-related diseases.15−17 Since 2000,
with the establishment of various rapid sensitive screening
methods,18,19 many DGC-specific inhibitors and PDE-specific
activators have been produced from synthetic c-di-GMP
derivatives.20−22 However, many gene copies encode c-di-
GMP catalysts in bacteria. For example, 17 proteins encode the
DGC domain (GGDEF), 5 proteins encode the PDE domain
(EAL or HD-GYP), and 16 proteins encode both domains
(GGDEF-EAL) in P. aeruginosa.23,24 The diversity and
redundancy of c-di-GMP regulatory enzymes lead to
substantial difficulties in their targeting.25,26 Moreover, recent
studies have demonstrated that the c-di-GMP concentration in
bacteria is not regulated globally, and some DGCs and PDEs
can only affect the local c-di-GMP concentration.27 Although
studies related to c-di-GMP receptor inhibitors have been
reported, because of a lack of in-depth understanding of the
INTRODUCTION
■
Biofilms are bacterial populations or communities attaching in
organized structures to a surface, and they play a considerable
role in the persistence of bacterial infections.1,2 Bacteria within
biofilms are several orders of magnitude more resistant to
antibiotics than are planktonic bacteria, and more than 80% of
clinically malignant infections are associated with bacterial
biofilm resistance.3,4 Standard antibacterial treatments typically
fail to eradicate biofilms. None of the current drugs specifically
targeting bacterial biofilms have proven to be successful in
clinical practice.5,6 Developing effective biofilm inhibitors is
one of the most pressing challenges in antibacterial drug
development.
Second messengers are small signal molecules that have
critical regulatory functions in bacteria in response to external
stimuli or major internal signals.3,7 Among them, the cyclic
diguanylate (c-di-GMP) signal molecule is ubiquitous in
bacteria and is widely found in Gram-positive and Gram-
negative bacteria, including many major medical pathogens
related to multidrug resistance, such as the common bacteria
Pseudomonas aeruginosa, Klebsiella pneumoniae, or Escherichia
coli.8,9 More importantly, c-di-GMP, as the second messenger
in bacteria, is involved in regulating the formation of bacterial
biofilms.10,11 Studies have revealed that a high concentration of
c-di-GMP in bacteria can promote the formation of bacterial
biofilms, whereas a low concentration can inhibit such
formation.12 The c-di-GMP pathway is thus an attractive
drug target for controlling bacterial infection.13,14
Received: March 15, 2021
https://doi.org/10.1021/acs.jmedchem.1c00465
© XXXX American Chemical Society
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Figure 1. Structural designs of new compounds based on TO and positive molecules.
a
Scheme 1. Synthesis of Compounds 2a−n and 3a−c
a
Reagents and conditions: (a) MeI, sulfolane, 60 °C, 24 h. (b) Aldehyde, CH3OH/EtOH, reflux.
relevant mechanisms, this area of research requires further
discussion.28 Considering this finding, directly targeting the c-
di-GMP molecule may be the most feasible approach to
targeting the c-di-GMP pathway. In recent years, researchers
have determined that some proline-rich short peptides with
cationic and aromatic groups, or arginine-rich short peptides
isolated from Caulobacter crescentus, can combine with c-di-
GMP with nanomolar affinity, thus blocking the c-di-GMP
signal pathway and inhibiting bacterial biofilm formation.29,30
Although these studies have greatly expanded the research and
development of new types of biofilm inhibition, because of the
stability of polypeptide structures, no inhibitor suitable for
clinical use has been reported.
Studies have indicated that c-di-GMP can form G-
quadruplex structures in the presence of univalent cations
such as K+ or some planar aromatic molecules such as
proflavine and thiazole orange (TO).31,32 More importantly, c-
di-GMP loses its functions after being aggregated into the G-
quadruplex.33,34 Although the abovementioned small mole-
cules have adverse effects (e.g., considerable biological toxicity
B
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a
Scheme 2. Synthesis of Compounds 5a−n and 6a−c
a
Reagents and conditions: (a) MeI, CH3CN, reflux, 24 h. (b) Piperidine, CH3OH/EtOH, reflux.
possibly caused by its DNA intercalation effect) and thus
cannot be developed into a useful therapeutic, it undoubtedly
has specific effects on inducing c-di-GMP to form polymers
with no biological activity.35 Therefore, acting directly on the
signal molecule c-di-GMP and reducing its effective concen-
tration by inducing it to form a functionally deleted G-
quadruplex is likely to be a promising strategy for the
development of antibiofilm agents.
Here, for the development of therapeutic biofilm inhibitors
through this novel strategy, we designed and synthesized a
series of benzothiazole and quinoline derivatives based on the
structure of the reported G-quadruplex inducers. The
structure−activity relationships in inducing c-di-GMP G-
quadruplex formation and in antibiofilm activity were
investigated, and compound 5h was found to exhibit high
inducing activity and 62.18 6.76% biofilm inhibitory activity
at 1.25 μM. Furthermore, motility, exopolysaccharide (EPS)
production, reporter strain, and transcriptome analyses were
performed to reveal the c-di-GMP signaling-related action
mechanism of 5h. Compound 5h exhibited no DNA
intercalation effect and low biological toxicity, indicating its
potential in the development of therapeutic antibiofilm agents.
or quinoline part of TO to reduce its planar system and
introducing the phenyl ring or heterocyclic group through the
double bond to retain its planar aromaticity. We anticipated
obtaining inducers that have no DNA intercalation effect but
retain strong induction activity (Figure 1). The facile synthetic
route and all series of compounds from S-I to S-IV (2a−n, 3a−
c and 5a−n, 6a−c) are illustrated in Schemes 1 and 2. The
preparation of key intermediate 1 was performed with
commercially available 2-methylquinoline and iodomethane
as the starting material through methylation. Intermediate 4
was obtained through the reaction of 2-methylbenzothiazole
and iodomethane. The prepared intermediates 1 and 4 were
reacted with various commercially available aromatic aldehydes
through a nucleophilic reaction, which provided the target
compounds 2a−n, 3a−c and 5a−n, 6a−c. We confirmed the
structures and purities of the compounds by using 1H and 13
C
nuclear magnetic resonance (NMR) spectrometry, high-
resolution mass spectrometry (HRMS), and high-performance
liquid chromatography (HPLC) (see details in the Exper-
imental Section and Supporting Information).
Evaluation of the Compounds’ Ability to Induce c-di-
GMP to Form G-Quadruplex. The c-di-GMP G-quadruplex-
inducing activity of the compounds was first determined using
circular dichroism (CD), which is a powerful tool for
identifying the aggregation state of nucleic acids.32,36 CD can
be used to qualitatively determine the presence of G-
quadruplex formation in solution, although it cannot provide
the detailed molecular structure of the G-quadruplex
(tetramolecular or octamolecular complexes).36 A positive
CD peak at approximately 300 nm indicates the presence of
the c-di-GMP G-quadruplex, and the height of the peak
RESULTS AND DISCUSSION
■
Chemistry. To overcome the toxicity of conventional G-
quadruplex inducers while retaining their selectivity, by
drawing on lessons from the interaction model between
small molecules and c-di-GMP, the design strategy of DNA G-
quadruplex inducers, and the characteristics of reported
inducers, we used TO as the template compound and designed
a series of novel inducers by retaining only the benzothiazole
C
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Figure 2. Actual CD measurement was performed on the sample of c-di-GMP mixed with compounds of various inducers. Compound TO was
used as a positive control, and no compound served as a blank control. (A) CD data of compounds 2a−n and 3a−c. (B) CD data of compounds
5a−n and 6a−c. (C) CD peak intensities of various samples at 300 nm.
indicates the strength of the inducing ability. In this
experiment, the previously reported inducer TO was used as
a positive control, and the induced peak intensity of each
compound at 300 nm was analyzed using a histogram to
identify compounds with clear c-di-GMP G-quadruplex-
inducing activity. As shown in Figure 2, several of the new
synthetic compounds, namely, 2a, 2e, 2f, 2h, 3a, 3b, 5f, 5h,
and 6a, induced strong positive absorption peaks at
approximately 300 nm, which indicated that these compounds
can induce c-di-GMP to form G-quadruplexes.
The derivatives had varying effects in inducing c-di-GMP G-
quadruplex formation. A comparison of the CD assay results
revealed that the conversion of quinoline and benzothiazole
moieties had little effect on the inducing activity, but the type
of aromatic group (Ar) had a considerable effect. For
compounds in series A and B, when Ar was an unsubstituted
phenyl ring (2k and 5k), the compounds exhibited weak
inducing ability. Replacing the phenyl ring with other aromatic
rings, including the pyridyl ring and pyrazinyl ring (3b, 3c and
6b, 6c), did not significantly improve the inducing activity.
However, various substitutions on the phenyl ring had
considerable influence on the inducing ability. When a
hydrogen bond donor or acceptor was introduced into the
phenyl ring, it tended to exhibit enhanced inducing activity.
Compounds 2a, 2e, 3a, 5a, 5e, and 6a exhibited higher
inducing activity than did the corresponding compounds in
which the hydrogen atom on the amino or hydroxy
substituents had been replaced (2b−d, 2g, 5b−d, and 5g) or
the compounds in which the amino or hydroxy substituent had
been replaced by another group (2l−n and 5l−n). Moreover,
compared with compounds 2e and 5e, when introducing an
electron donating group at the ortho position of the hydroxyl
group, the inducing activity was maintained (2e vs 2h) or
improved (5e vs 5h), but if an electron-withdrawing group was
introduced at the ortho position (2i−j and 5i−j), the activity
decreased or even disappeared. These structure−activity
relationships are consistent with our theoretical speculation
that the compounds would stabilize the G-quadruplex through
π−π interaction in the form of intercalation or terminal
stacking, and the formation of hydrogen bonds played a key
role in this binding pattern; thus, substituents containing
hydrogen bond donors and receptors had higher inducible
activity.
Antibiofilm Screening. To explore whether our com-
pound can effectively induce c-di-GMP molecules to form G-
quadruplexes, thus reducing their relative concentration in
bacteria and ultimately inhibiting bacterial biofilms, the
inhibitory effect of the compounds on the biofilm was
subsequently determined using a crystal violet (CV) staining
assay. According to related reports,37−40 bacteria perform
selective gene mutation to develop greater resistance in
unfavorable growth environments, for example, both the
rugose small colony variants of P. aeruginosa and the bacteria
that cause chronic cystic fibrosis airway infections are caused
by the wspF mutation. To enhance the relevance of the current
study for solving the clinical infection problem, we used the
bacterial model P. aeruginosa PAO1-ΔwspF, which has a wspF
gene deletion mutation and is characterized by the high
expression level of the c-di-GMP monomer.41 The CV staining
assay was conducted after we tested the effect of our
compounds on bacterial growth and determined that our
compounds do not affect the normal growth process of
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Table 1. Biofilm Inhibition Rates of the Derivatives
ab
,
a
ab
,
a
compound
inhibition rate (%)
IC50 (μM)
compound
inhibition rate (%)
IC50 (μM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
3a
3b
3c
2.11 3.62
−15.93 7.61
−2.34 4.09
1.18 2.73
13.38 5.55
29.36 4.71
−9.62 4.21
21.04 3.97
4.21 9.01
−12.72 6.12
18.77 2.94
21.34 3.88
−10.56 7.73
−17.27 3.46
22.18 4.13
8.36 6.33
>100
>100
>100
>100
>100
6.81 1.08
>100
5.4 3.48
>100
>100
>100
>100
>100
>100
9.61 3.49
>100
>100
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
6a
6b
14.53 7.88
−2.67 3.07
−20.73 4.07
2.71 6.74
10.53 4.44
21.72 3.73
−4.78 4.07
62.18 6.76
−7.67 4.84
−21.44 3.78
19.53 6.49
7.72 4.92
−4.77 6.72
−14.92 4.88
17.68 5.43
11.39 4.78
−16.71 7.01
>100
>100
>100
>100
>100
11.54 4.17
>100
0.88 0.28
>100
>100
>100
>100
>100
>100
7.84 2.75
>100
>100
13.03 5.91
6c
a
b
All rates represent mean standard deviation from experiments performed in triplicate. The concentration of the compounds was 1.25 μM.
Figure 3. (A) Representative three-dimensional images of PAO1-ΔwspF biofilms. Biofilms formed in the presence of various concentrations of 5h
after 24 h of incubation. Images were acquired using a confocal microscope with a 63× objective lens. Scale bars represent 40 μm. (B) Biofilm
formation and cell viability in PAO1-ΔwspF in various concentrations of 5h, followed by quantification of planktonic cells by measuring the OD at
600 nm. Biofilm cells attached to the well surface were assayed using CV staining. Error bars are means standard deviations. *p < 0.05, **p <
0.01, and ***p < 0.001.
bacteria even at 512 μM. At a concentration of 1.25 μM,
compound 5h exhibited the highest bacterial biofilm inhibition
rate. Thus, to effectively compare 5h with other compounds,
the biofilm formation inhibition rate of all compounds was
investigated at a concentration of 1.25 μM. All compounds
with no ability to induce c-di-GMP to form G-quadruplexes
according to the CD experiment (i.e., 2b, 2c, 2j, 2g, 2m, 2n,
5b, 5c, 5g, 5i, 5j, 5m, 5n, and 6c) had no ability to inhibit
bacterial biofilms (Table 1). Other compounds with the ability
to induce c-di-GMP to form polymers exhibited the ability to
inhibit bacterial biofilms to a certain extent, among which
compound 5h had a remarkable bacterial biofilm inhibitory
rate of 62.18 6.76% at a concentration of 1.25 μM. Confocal
laser scanning microscope imaging analysis was further
performed to more intuitively observe the biofilm inhibitory
effect of 5h (Figure 3A). Staining with the fluorescent dye
SYTO9 was performed, and the reconstructed three-dimen-
sional images revealed significantly lower biofilm formation in
the glass plate treated with compound 5h than in the control
group. Moreover, 5h reduced PAO1-ΔwspF biofilm biomass
formation in a dose-dependent manner without affecting
bacterial growth (Figure 3B). The inhibitory effect of
compound 5h on the biofilm formation of wild-type strain
PAO1 (with low c-di-GMP levels) and another c-di-GMP
high-expression strain, ATCC 27853, was simultaneously
evaluated. The content levels of c-di-GMP in these two
bacteria were determined using HPLC (Figure S1). The results
indicated that compound 5h had a negligible effect on the
biofilm formation of the PAO1 strain (Figure S2), but it had an
inhibitory effect on the biofilm formation of the ATCC 27853
strain similar to that on the PAO1-ΔwspF strain (Figure S3).
These results indicate that a threshold intracellular concen-
tration of c-di-GMP is required for 5h treatment to be
effective, illustrating that the target of 5h is most likely the c-di-
GMP signaling pathway.
Exploring the Targeting Effect and Stoichiometric
Ratio of Compound 5h and c-di-GMP. We investigated
whether compound 5h targeted c-di-GMP in a series of
experiments. First, the targeting effect of compound 5h and c-
di-GMP was determined by simulating the bacterial environ-
ment in vitro, and the stoichiometric ratio between the two
was calculated. Compound 5h had functions similar to those of
the fluorescent probes we reported previously.42 When
compound 5h interacted with c-di-GMP molecules, its
fluorescence intensity changed significantly. The fluorescence
of compound 5h was very low, but with the increasing
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Figure 4. (A) Dose response for 5h−c-di-GMP interaction. [5h] = 5 μM, buffer: 10 mM Tris-HCl (pH 7.5) containing 1 M KCl. Ex. 520 nm, Em.
540−620 nm. Reaction temperature: −20 °C. (B) Evaluation of the selectivity of 5h. (C) Job plot. Total [5h] + [c-di-GMP] was fixed at 10 μM.
The experiment was performed in triplicate, and all of the triplicate data points are plotted on the graphs.
concentration of c-di-GMP, its fluorescence increased
considerably, which is consistent with the results of previously
reported c-di-GMP inducers;31,32 this finding indicates that our
compound can induce c-di-GMP to form G-quadruplexes
(Figure 4A). We also replaced the c-di-GMP molecules in the
solution with various nucleotides (rNTP, GMP, and dNTP) to
test the specificity of our compound (Figure 4B). The results
revealed that the fluorescence intensity was enhanced only
when c-di-GMP was present in the solution, and almost no
change was observed when it was replaced with other
nucleotides, indicating that the compound only targets c-di-
Figure 5. (A) Effects of various concentrations of compound 5h on
GMP molecules. Finally, to determine the stoichiometry
the motility of PAO1-ΔwspF. (B) Comparison of the range of motion
between our compound and c-di-GMP, we performed a Job
of bacterial swarming in the control group and 5 μM compound
plot (Figure 4C). The Job plot data indicated that the
stoichiometry of 5h to c-di-GMP in the presence of potassium
was 1:4.5. This result is consistent with our expectations. Each
molecule of compound 5h induced four molecules of c-di-
GMP to combine with each other to form a functionally
deleted G-quadruplex.
concentration. Scale bars represent 1 cm. Error bars are means
standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001.
concentration that had the greatest impact on bacterial
motility in this experiment was 5 μM, which is 4 times the
optimal concentration for inhibiting biofilm formation. The
experimental results confirmed the effect of compound 5h on
the swarming capacity of the bacteria regulated by c-di-GMP.
Compound 5h Inhibits Bacterial EPS Production.
Bacteria typically synthesize many polysaccharides, including
structural polysaccharides, intracellular polysaccharides, and
extracellular polysaccharides or EPSs.47 EPSs are water-soluble,
high-molecular-weight biopolymers secreted into the extrac-
ellular environment by some special microorganisms during
growth and metabolism.48 EPSs enhance resistance to
desiccants, oxidative stress, and host defense factors by
protecting nonspecific and specific host immunity, thus
providing microorganisms with additional survival advantages.
EPSs are also the main structural component of the
extracellular polymer matrix, which participates in cell surface
adhesion, cell interaction, and bacterial biofilm formation.49 c-
di-GMP regulates EPSs at the transcription or posttranscrip-
tion level, and this becomes a highly conservative regulatory
mechanism. Studies have revealed that several DGCs (SadC,
WspR, and YfiN/TpbB) and PDEs (BifA and RocR) are
involved in the production of bacterial EPSs. c-di-GMP, as an
Effect of Compound 5h on Bacterial Motility. c-di-
GMP plays a critical role in bacterial motility.43 Generally, low
levels of c-di-GMP are associated with the motility of
individual cells, whereas increased concentrations of c-di-
GMP promote the surface attachment of bacteria; this is also a
key reason for the formation of bacterial biofilms.44,45 On the
basis of the assumption that mobility is one of the primary
processes regulated by c-di-GMP and that the assembly and
activity of the bacterial flagellar motor are also highly regulated
by this second messenger, we evaluated the effect of compound
5h on the swarming capacity of PAO1-ΔwspF to preliminarily
determine whether compound 5h targets c-di-GMP in bacteria.
The results indicated that the motility of bacteria was evidently
improved after the addition of compound 5h (Figure 5).
Compared with the bacteria cultured without the compound,
the motility halo of the bacteria cultured with compound 5h at
concentrations of 5, 3.75, and 2.5 μM increased by 77, 34, and
15%, respectively. Because the expression level of c-di-GMP in
bacteria is 3−5 times higher than that in liquid culture medium
for bacteria cultured on agar plates,46 the compound
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essential signaling molecule, also participates in this complex
regulation process.50,51 In sum, when high c-di-GMP levels are
present in bacteria, EPS synthesis also increases in bacteria,
and when the c-di-GMP level in bacteria decreases, the
amounts of EPSs secreted into the surrounding environment
decreases correspondingly. On the basis of the aforementioned
theory, we initially assessed the effect of various concentrations
of compound 5h on EPS synthesis in the PAO1-ΔwspF strain.
We used the standard Congo red binding assay to quantitate
total EPSs.52 As the compound concentration increased, the
amounts of EPSs produced by bacteria decreased linearly
(Figure 6). When the concentration of compound 5h was 1.25
aeruginosa biofilm formation.54 These genes include pel and psl
operons, which are responsible for guiding the synthesis of Pel
and Psl EPSs as well as the cdrA genes encoding the adhesins
necessary for the structure of Psl EPSs.55,56 On the basis of gel
displacement data, Hickman and Harwood46 proposed that
FleQ inhibits Pel expression through a simple blocking
mechanism in which FleQ is located in the PelA promoter to
prevent RNA polymerase from binding. These researchers
further highlighted how the combination of c-di-GMP and
FleQ induces the dissociation of the FleQ and the PelA
promoter, which enables RNA polymerase to enter DNA and
form an active transcription complex.57,58 In short, when
bacteria have high c-di-GMP levels, the PelA expression
increases correspondingly. By contrast, when bacteria have low
c-di-GMP levels, the PelA expression decreases. Here, we used
the reporter strain PAO1-ΔwspF-pelA-lacZ to evaluate the
effect of compound 5h on the PelA expression in bacteria. The
strain was constructed by inserting lacZ downstream from the
Pel promoter by using the mini-CTX vector. In this strain, lacZ
is controlled by the Pel promoter; thus, lacZ expression could
reflect the activity of the Pel promoter. When treated with our
compound, c-di-GMP levels in bacteria are theoretically
reduced, and the PelA expression in bacteria is reduced in a
corresponding manner. When compound 5h was incubated
with bacteria at a concentration of 1.25 μM for 4 h, the
absorbance at 490 nm, which reflects the activity of lacZ, was
lower than that of the control group (Figure 7). Moreover, the
degree of color change in the experimental group was
significantly smaller than that in the blank control group.
This result indicates that under the action of compound 5h,
the expression of PelA is reduced, supporting our previous
prediction that our compound would reduce active c-di-GMP
levels in bacteria.
Figure 6. Congo red was used to stain and quantitate the secreted
EPS after treatment with various concentrations of compound 5h.
Data are from three parallel experiments. Error bars are means
standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001.
μM, the production of EPS was approximately 30% lower than
that in the control group. The experimental results of each
group are consistent with our previous experimental results,
which confirm that the compound is likely to affect the c-di-
GMP pathway in bacteria and thus exert an effect on the
bacterial biological phenotype regulated by it.
Compound 5h Inhibits lacZ Expression in Reporter
Strains. Several transcriptional regulators that respond to c-di-
GMP have been described.53 Among these regulators, FleQ is
involved in regulating the expression of genes that regulate P.
Noninterference of 5h with the c-di-GMP Network
through DGCs and PDEs. Under physiological conditions,
the intracellular concentration of c-di-GMP is fine tuned by its
metabolism enzymes DGCs and PDEs. Therefore, we
examined whether the c-di-GMP reduction effect of 5h is
caused by its influence on DGC activity and PDE activity or by
it directly inducing c-di-GMP aggregation, thus reducing its
Figure 7. (A) Expression of pelA. (B) Color change of the solution was determined after bacteria were incubated with compound 5h at various
concentrations for 4 h through the constructed lacZ reporting system. (C) Data reflect the effect of various concentrations of compounds on the
expression of bacterial pelA. All experiments were three independent replicates. Error bars are means standard deviations. *p < 0.05, **p < 0.01,
and ***p < 0.001.
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effective concentration. We selected the enzyme WspR, a well
characterized DGC, and RocR, an EAL-type PDE, as
representative enzymes to examine the effect of 5h on DGC
activity and PDE activity. HPLC was employed to measure the
enzymatic activity of WspR and RocR under various
concentrations of 5h.42 Samples were collected every 10 min,
and the enzyme kinetic curves of WspR and RocR were
calculated and drawn. With compound 5h, whether at a
concentration with favorable biofilm inhibitory activity or at
the highest concentration of 5 μM, the rate of synthesis of c-di-
GMP by WspR remained basically unchanged, and compound
5h had no effect on the degradation rate of c-di-GMP by RocR
(Figure 8). This result revealed that the compound did not
reduce the relative content of c-di-GMP in bacteria by affecting
the activities of WspR and RocR.
control. All other treatments were the same. The transcriptome
data indicated that the addition of compound 5h affected the
expression of related genes of P. aeruginosa PAO1-ΔwspF.
After filtering the gene expression data (p < 0.05 and |log2
(fold change)| > 1), we identified 1988 differentially expressed
genes. The volcano plot (Figure 9B) revealed 919 upregulated
genes and 1069 downregulated genes. To elucidate the
functions of these differentially expressed genes, we focused
on the role of c-di-GMP in the two-component system for the
quorum sensing and biofilm formation of P. aeruginosa, and we
conducted enrichment analysis of these data. After screening
the relevant data (p < 0.05 and |log2 (fold change)| > 1.5), we
selected several representative genes closely related to the
biofilm formation and motility and drew a heat map (Figure
9C). The expression of cdrA, which regulates the formation of
bacterial biofilm, was evidently downregulated (Figure 9A). In
the absence of c-di-GMP, the transcriptional regulator FleQ
represses cdrA transcription. In the presence of c-di-GMP, the
second messenger binds to FleQ and induces the release of
FleQ from the cdrA promoter.29 Therefore, the change in cdrA
expression can indirectly reflect the c-di-GMP concentration in
bacteria. Furthermore, Pel and Psl polysaccharides are the
targets of c-di-GMP. When the c-di-GMP concentration in
bacteria decreases, the expression of these genes that regulate
the main components of bacterial biofilm also decreases
accordingly.59 Alg44 is a PilZ domain c-di-GMP receptor
required for alginate polymerization or transport. Alginate is
also a main component in bacterial biofilms.60,61 When we
used compound 5h to reduce the c-di-GMP concentration in
bacteria, Alg44 expression was also significantly downregulated.
Notably, we found that the expression of tpbA, a tyrosine
phosphatase reportedly related to biofilm formation, was
upregulated; this enzyme can not only dephosphorylate tbpB
but also inhibit the biological function of tbpB, thus reducing c-
di-GMP levels in bacteria and inhibiting the formation of
bacterial biofilms.62 In terms of bacterial motility, the
expression of many flagella genes that are positively regulated
Figure 8. Time course of the reaction catalyzed by the DGC WspR
(A) and by the PDE RocR (B) in the presence of various
concentrations of 5h (0, 1.25, and 5 μM). All measurements were
performed in triplicate. Error bars are means standard deviations.
Transcriptome Analysis Revealed the Effect of
Compound 5h on Biofilm Formation and the c-di-
GMP Pathway. Transcriptome analysis can more clearly
reveal the effect of compound 5h on bacterial biofilm at the
gene level. Bacterial samples were divided into two groups:
control group and group B (2 groups × 2 biological replicates).
In group B, P. aeruginosa PAO1-ΔwspF was incubated with
1.25 μM compound 5h for 12 h, whereas group A was a blank
Figure 9. (A) Schematic indicating that 5h sequesters c-di-GMP from the repressor FleQ in the reporter system, resulting in the suppression of
downstream genes such as cdrA. (B) Volcano plot of the number of different genes between group A and group B. (C) Differential gene
thermogram of bacterial biofilm formation and motility in the control group and in group B.
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by FleQ is upregulated to varying degrees because of the
decrease in the c-di-GMP concentration in bacteria; examples
of these genes include flhA, which is involved in flagellar
export, flhF, which is involved in the localization of the flagellar
apparatus, fliD, which encodes the flagellar cap and export
proteins, flgA, which is involved in P-ring formation of the
flagellar basal body, and fleN, which regulates the flagellar
number.63 The transcriptome results clearly indicate that our
compound targeted c-di-GMP and that the compound
regulated bacterial biofilm formation by inducing c-di-GMP
to form dysfunctional G-quadruplexes and reducing the relative
concentration of c-di-GMP in bacteria.
Verification of Transcriptome Results. To verify the
aforementioned results, reverse transcription polymerase chain
reaction (PCR) was applied to analyze the effect of 5h on the
expression of fleN, flhA, pelB, pslE, and cdrA, which are closely
related to the c-di-GMP pathway. Moreover, we selected the
housekeeping gene RpsL, which is not regulated by the c-di-
GMP signal molecule, as the internal reference gene. As
illustrated in Figure 10, a significant repression of pelB, pslE,
Figure 11. (A) Comparison of DNA intercalation between 5h and
TO at the same concentration. Polyacrylamide gel electrophoresis
experiments were performed at a working voltage of 100 V for 40 min.
(B) Cytotoxicity of various concentrations of 5h was detected using
MTT assay in AC16 cells; the data revealed that cell viability was
higher than 90% over the range of effective biofilm inhibitory
concentrations of the compounds. The data were derived from three
independent replicates.
myocardial AC16 cells were used in the analysis. After
repeated experiments, we obtained the data as shown in
Figure 11B. At concentrations of 0.15625−10 μM, the tested
compounds resulted in a cell survival rate above 90%, and at
the optimal concentration for antibacterial biofilm formation
(1.25 μM), the cell survival rate was as high as 96%. Thus,
considering the influence of environment and experimental
conditions, our compound exhibits considerable potential for
application in patent medicine.
CONCLUSIONS
■
On the basis of the crucial role of c-di-GMP in regulating the
formation of bacterial biofilms, we designed and synthesized a
series of benzothiazole and quinoline derivatives as potent c-di-
GMP G-quadruplex inducers. The purpose was to investigate
the possibility of inducing c-di-GMP to form stable but
biologically inactive higher-order aggregates, such as G-
quadruplexes, by using small molecules to inactivate cellular
c-di-GMP and thus control biofilm formation. Through various
experiments, we identified compound 5h as an effective c-di-
GMP G-quadruplex inducer and a promising biofilm inhibitor.
Moreover, for the first time, a systematic structure−activity
relationship analysis of c-di-GMP G-quadruplex inducers was
performed, the results of which may inform the design of new
inducers in the future. The results of fluorescence experiments
confirmed that compound 5h can specifically induce c-di-GMP
to form G-quadruplexes at a stoichiometric ratio of 1:4.5. More
importantly, compound 5h exhibits strong biofilm inhibition at
1.25 μM, and it exhibits excellent performance for many
biofilm-associated phenotypes of bacteria, including motility
and EPS secretion. Furthermore, 5h does not have the
biological toxicity of conventional G-quadruplex inducers such
as TO. The reporter strain and transcriptional-level analysis
confirmed the c-di-GMP signaling-associated action mecha-
nism of compound 5h. In summary, this study suggests the
feasibility of using 5h as a therapeutic candidate for developing
antibacterial biofilm agents that induce c-di-GMP to form
dysfunctional G-quadruplexes. A comparison of the present
strategy with other available methods for regulating biofilm
formation, such as regulating the quorum sensing system,
blocking intrinsic DGC activity, or enhancing intrinsic PDE
activity, reveals that compound 5h does not act on complex
regulatory systems but acts directly on the signaling molecule
Figure 10. Effect of compound 5h on the expression of genes
regulated by the c-di-GMP signaling molecule. The experimental
results were homogenized by the housekeeping gene RpsL. All
measurements were performed in triplicate. Error bars are means
standard deviations.
and cdrA was observed when the strains were treated with 1.25
μM 5h; at the same time, the expression of fleN and flhA,
which are responsible for regulating bacterial motility, has been
promoted correspondingly. This result is consistent with the
transcriptome experimental results and is strong evidence that
the target of compound 5h for inhibiting bacterial biofilm
formation is the c-di-GMP signaling pathway.
Determination of Cytotoxicity of 5h. Most previously
reported c-di-GMP G-quadruplex inducers are aromatic
intercalators (e.g., acridines such as proflavine and acriflavine),
diamidinium, or iminium aromatics, all of which have nucleic
acid intercalation effects.34,64 This toxicity must be avoided for
therapeutic agents inhibiting bacterial biofilms. Thus, we tested
whether compound 5h also has a nucleic acid intercalation
effect by using TO as a positive control. Positive results were
achieved (Figure 11A). No tailing was observed in the lane
where the compound was added, which indicated that our
compound did not have a DNA intercalation effect. By
contrast, several blurred strips appeared in the lane where TO
was added, indicating that TO destroyed the original structure
of the DNA after acting on it. We also used the MTT method
to evaluate the cytotoxicity of compound 5h. Human
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131.98, 130.33, 128.78, 127.54, 125.14, 120.90, 119.46, 114.23,
114.18, 66.30, 47.13, 39.88. Purity: 97.8% by HPLC. HRMS (m/z):
331.1803 [M−I]+, calcd for C22H23N2O, 331.1805.
itself. This strategy of targeting the signaling molecule c-di-
GMP itself, the key factor of the entire network, may facilitate
comprehensive and effective regulation of biofilm formation.
(E)-2-(4-Hydroxystyryl)-1-methylquinolin-1-ium Iodide (2e). The
reaction was performed according to the general procedure A to
1
EXPERIMENTAL SECTION
afford the purple solid 2e in 72% yields. mp 211.7−212.4 °C. H
■
1
NMR (300 MHz, DMSO-d6): δ 8.52 (d, J = 9.3 Hz, 1H), 8.28 (m,
2H), 8.17−8.05 (m, 2H), 8.01−7.91 (m, 1H), 7.79−7.66 (m, 3H),
7.23 (d, J = 15.0 Hz, 1H), 6.59 (d, J = 8.8 Hz, 2H), 4.26 (s, 3H); 13C
NMR (75 MHz, DMSO-d6): δ 171.83, 155.60, 148.59, 140.36,
139.85, 134.02, 129.95, 127.41, 126.49, 123.02, 120.53, 119.73,
118.49, 109.26, 38.69. Purity: 100.0% by HPLC. HRMS (m/z):
262.1222 [M−I]+, calcd for C18H16NO, 262.1226.
Chemical Synthesis. Synthesis and Characterization. H NMR
and 13C NMR spectra were recorded on a Bruker DRX400
spectrometer at 400 MHZ or a NT-300 WB spectrometer at 300
MHz. Chemical shifts (δ) are reported in ppm relative to the internal
standard tetramethylsilane. HRMS spectra were obtained on a
Finnigan MAT-90 spectrometer or an Agilent 6210 series LC/MSD
TOF mass spectrometer. Melting points were determined using a
digital melting point apparatus (Briwell/GH60) without correction.
The purities of the synthesized compounds were confirmed to be
>95% by analytical HPLC performed using a dual pump Agilent 1260
Infinity LC system equipped with an YMC-Triart C18 column and
eluted with methanol/water containing 0.05% TFA. All solvents and
reagents used were purchased from commercial sources and used as
received unless otherwise noted.
(E)-2-(3-Hydroxystyryl)-1-methylquinolin-1-ium Iodide (2f). The
reaction was performed according to the general procedure A to
afford the green solid 2f in 72% yields. mp 238.4−239.2 °C. 1H NMR
(300 MHz, DMSO-d6): δ 9.78 (s, 1H), 9.10 (d, J = 8.9 Hz, 1H), 8.60
(m, 2H), 8.39 (d, J = 7.2 Hz, 1H), 8.19 (m, 2H), 7.97 (t, J = 7.5 Hz,
1H), 7.87 (d, J = 15.9 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.34 (t, J =
7.8 Hz, 2H), 6.95 (dd, J = 7.8, 1.8 Hz, 1H), 4.58 (s, 3H); 13C NMR
(75 MHz, DMSO-d6): δ 158.30, 156.63, 147.49, 144.70, 139.65,
136.64, 135.45, 130.58, 129.54, 128.34, 121.79, 120.60, 119.85,
119.04, 116.03, 39.16. Purity: 99.2% by HPLC. HRMS (m/z):
262.1224 [M−I]+, calcd for C18H16NO, 262.1226.
(E)-2-(4-Methoxystyryl)-1-methylquinolin-1-ium Iodide (2g). The
reaction was performed according to the general procedure A to
afford the green solid 2g in 82% yields. mp 236.4−237.3 °C. 1H NMR
(400 MHz, DMSO-d6): δ 9.01 (d, J = 9.0 Hz, 1H), 8.55 (m, 2H),
8.34 (d, J = 7.4 Hz, 1H), 8.24 (d, J = 15.8 Hz, 1H), 8.19−8.12 (m,
1H), 8.00−7.89 (m, 3H), 7.78 (d, J = 15.8 Hz, 1H), 7.08 (d, J = 8.7
Hz, 2H), 4.54 (s, 3H), 3.85 (s, 3H); 13C NMR (101 MHz, DMSO-
d6): δ 162.62, 156.80, 147.61, 144.05, 139.62, 135.19, 131.87, 130.47,
129.24, 128.10, 128.00, 121.31, 119.73, 117.00, 115.14, 56.09, 40.34.
Purity: 99.8% by HPLC. HRMS (m/z): 276.1379 [M−I]+, calcd for
C19H18NO, 276.1383.
Synthesis of Intermediates. Intermediates 1, 4 were prepared
following the process shown in Schemes 1 and 2, and the details of
the synthesis are described in the Supporting Information.
General Procedure A: Preparation of A and B. A mixture of 1 or 4
and the corresponding aldehyde (2 mmol) in methanol (5 mL) was
heated under reflux for 6−15 h in the presence of a basic catalyst
(pyridine or piperidine, 0.2 mmol). The reaction mixture was allowed
to cool slowly to room temperature, and a dark solid was filtered off,
washed with cold methanol and then with diethyl ether, and dried. If
further purification was required, the resulting dark solid was
recrystallized from methanol.
(E)-2-(4-Aminostyryl)-1-methylquinolin-1-ium Iodide (2a). The
reaction was performed according to the general procedure A to
1
afford the brown solid 2a in 82% yields. mp 253.1−253.9 °C. H
NMR (300 MHz, DMSO-d6): δ 8.80 (d, J = 9.1 Hz, 1H), 8.47 (m,
2H), 8.29−8.20 (m, 2H), 8.12−8.04 (m, 1H), 7.88−7.80 (m, 3H),
7.54 (d, J = 15.5 Hz, 1H), 6.81 (d, J = 8.9 Hz, 4H), 4.44 (s, 3H); 13C
NMR (101 MHz, DMSO-d6): δ 156.89, 154.21, 149.80, 142.22,
139.67, 134.55, 133.02, 130.24, 128.41, 127.18, 122.96, 120.63,
119.27, 114.24, 111.57, 39.44. Purity: 98.8% by HPLC. HRMS (m/
z): 261.1387 [M−I]+, calcd for C18H17N2, 261.1386.
(E)-2-(4-Hydroxy-3-methoxystyryl)-1-methylquinolin-1-ium Io-
dide (2h). The reaction was performed according to the general
procedure A to afford the red solid 2h in 82% yields. mp 266.7−267.4
°C. 1H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 1H), 8.98 (d, J = 9.0
Hz, 1H), 8.55 (t, J = 9.7 Hz, 2H), 8.32 (d, J = 7.8 Hz, 1H), 8.21 (d, J
= 15.7 Hz, 1H), 8.18−8.12 (m, 1H), 7.92 (t, J = 7.5 Hz, 1H), 7.73 (d,
J = 15.7 Hz, 1H), 7.62 (d, J = 1.5 Hz, 1H), 7.44 (dd, J = 8.2, 1.5 Hz,
1H), 6.92 (d, J = 8.2 Hz, 1H), 4.55 (s, 3H), 3.92 (s, 3H); 13C NMR
(101 MHz, DMSO-d6): δ 156.92, 151.47, 148.68, 148.66, 143.72,
139.66, 135.05, 130.42, 129.10, 127.85, 127.15, 125.79, 121.20,
119.70, 116.30, 115.91, 112.49, 56.53, 40.45. Purity: 99.4% by HPLC.
HRMS (m/z): 292.1334 [M−I]+, calcd for C19H18NO2, 292.1332.
(E)-2-(3-Fluoro-4-hydroxystyryl)-1-methylquinolin-1-ium Iodide
(2i). The reaction was performed according to the general procedure
(E)-2-(4-(Dimethylamino)styryl)-1-methylquinolin-1-ium Iodide
(2b). The reaction was performed according to the general procedure
A to afford the green solid 2b in 77% yields. mp 269.1−269.6 °C. 1H
NMR (300 MHz, DMSO-d6): δ 8.80 (d, J = 9.1 Hz, 1H), 8.47 (m,
2H), 8.29−8.20 (m, 2H), 8.12−8.04 (m, 1H), 7.88−7.80 (m, 3H),
7.54 (d, J = 15.5 Hz, 1H), 6.81 (d, J = 8.9 Hz, 2H), 4.44 (s, 3H), 3.07
(s, 6H); 13C NMR (75 MHz, DMSO-d6): δ 156.75, 153.28, 149.37,
142.30, 139.65, 134.59, 132.45, 130.25, 128.46, 127.26, 122.85,
120.66, 119.29, 112.28, 40.18. Purity: 98.9% by HPLC. HRMS (m/
z): 289.1699 [M−I]+, calcd for C20H21N2, 289.1699.
1
A to afford the red solid 2i in 80% yields. mp 237.1−237.9 °C. H
NMR (400 MHz, DMSO-d6): δ 8.75 (d, J = 9.1 Hz, 1H), 8.39 (M,
2H), 8.20 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 15.3 Hz, 1H), 8.06 (t, J =
7.9 Hz, 1H), 7.88 (dd, J = 13.0, 1.7 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H),
7.53 (dd, J = 8.6, 1.7 Hz, 1H), 7.47 (d, J = 15.3 Hz, 1H), 6.85 (t, J =
8.8 Hz, 1H), 4.39 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ
155.69, 155.01, 152.59, 148.24, 140.89, 139.84, 134.17, 131.44,
130.03, 127.68, 126.72, 122.37, 120.74, 120.57, 118.68, 115.85,
110.73, 38.90. Purity: 99.7% by HPLC. HRMS (m/z): 280.1145 [M−
I]+, calcd for C18H15FNO, 280.1132.
(E)-1-Methyl-2-(4-(4-methylpiperazin-1-yl)styryl)quinolin-1-ium
Iodide (2c). The reaction was performed according to the general
procedure A to afford the brown solid 2c in 66% yields. mp 254.4−
1
255.1 °C. H NMR (400 MHz, DMSO-d6): δ 8.88 (d, J = 9.0 Hz,
1H), 8.50 (m, 2H), 8.25 (m, 2H), 8.11 (t, J = 8.0 Hz, 1H), 7.87 (m,
3H), 7.64 (d, J = 15.6 Hz, 1H), 7.06 (d, J = 8.1 Hz, 2H), 4.48 (s, 3H),
3.41 (m, 4H), 2.48 (m, 4H), 2.26 (s, 3H); 13C NMR (101 MHz,
DMSO-d6): δ 156.82, 153.49, 148.61, 142.97, 139.65, 134.84, 132.10,
130.34, 128.76, 127.53, 124.79, 120.90, 119.42, 114.43, 114.00, 54.60,
46.75, 45.93. Purity: 96.2% by HPLC. HRMS (m/z): 344.2122 [M−
I]+, calcd for C23H26N3, 344.2121.
(E)-2-(3,5-Dibromo-4-hydroxystyryl)-1-methylquinolin-1-ium Io-
dide (2j). The reaction was performed according to the general
procedure A to afford the brown solid 2j in 89% yields. mp 267.2−
1
267.7 °C. H NMR (400 MHz, DMSO-d6): δ 9.05 (d, J = 8.9 Hz,
(E)-1-Methyl-2-(4-morpholinostyryl)quinolin-1-ium Iodide (2d).
The reaction was performed according to the general procedure A to
1H), 8.55 (d, J = 9.0 Hz, 1H), 8.48 (d, J = 9.0 Hz, 1H), 8.34 (d, J =
8.0 Hz, 1H), 8.25 (s, 2H), 8.18 (t, J = 7.9 Hz, 1H), 8.10 (d, J = 15.8
Hz, 1H), 7.95 (t, J = 7.5 Hz, 1H), 7.86 (d, J = 15.8 Hz, 1H), 4.56 (s,
3H); 13C NMR (101 MHz, DMSO-d6): δ 156.43, 144.76, 144.39,
139.70, 135.35, 133.69, 130.51, 129.42, 128.17, 121.31, 119.78,
118.73, 112.93, 40.23. Purity: 99.7% by HPLC. HRMS (m/z):
417.9435 [M−I]+, calcd for C18H14Br2NO, 417.9437.
1
afford the brown solid 2d in 66% yields. mp 223.4−223.9 °C. H
NMR (300 MHz, DMSO-d6): δ 8.90 (d, J = 9.1 Hz, 1H), 8.51 (dd, J
= 17.4, 9.1 Hz, 2H), 8.33−8.18 (m, 2H), 8.17−8.07 (m, 1H), 7.94−
7.83 (m, 3H), 7.67 (d, J = 15.6 Hz, 1H), 7.08 (d, J = 9.0 Hz, 2H),
4.49 (s, 3H), 3.81−3.69 (m, 4H), 3.38−3.35 (m, 4H); 13C NMR (75
MHz, DMSO-d6): δ 156.72, 153.64, 148.51, 142.99, 139.59, 134.84,
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(E)-1-Methyl-2-styrylquinolin-1-ium Iodide (2k). The reaction was
performed according to the general procedure A to afford the yellow
solid 2k in 78% yields. mp 230.0−230.6 °C. H NMR (400 MHz,
130.00, 128.86, 124.64, 122.89, 122.22, 120.03, 41.06. Purity: 100.0%
by HPLC. HRMS (m/z): 247.1229 [M−I]+, calcd for C17H15N2,
247.1230.
1
DMSO-d6): δ 9.12 (d, J = 8.9 Hz, 1H), 8.61 (m, 2H), 8.39 (dd, J =
8.0, 1.0 Hz, 1H), 8.21 (m, 2H), 8.04−7.91 (m, 4H), 7.58−7.50 (m,
3H), 4.60 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 156.62,
147.21, 144.80, 139.66, 135.48, 135.33, 131.84, 130.57, 129.63,
129.59, 128.38, 121.78, 120.01, 119.89, 40.70. Purity: 98.1% by
HPLC. HRMS (m/z): 246.1274 [M−I]+, calcd for C18H16N,
246.1277.
(E)-2-(4-Aminostyryl)-3-methylbenzo[d]thiazol-3-ium Iodide
(5a). The reaction was performed according to the general procedure
A to afford the purple solid 5a in 87% yields. mp 247.3−248.1 °C. 1H
NMR (400 MHz, DMSO-d6): δ 8.29 (d, J = 8.0 Hz, 1H), 8.08 (d, J =
8.4 Hz, 1H), 7.99 (d, J = 15.3 Hz, 1H), 7.81−7.75 (m, 3H), 7.67 (t, J
= 7.6 Hz, 1H), 7.55 (d, J = 15.3 Hz, 1H), 6.68 (d, J = 8.7 Hz, 4H),
4.21 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 171.85, 155.20,
150.95, 142.39, 133.92, 129.29, 127.84, 127.17, 124.25, 122.03,
116.35, 114.32, 106.04, 36.02. Purity: 98.5% by HPLC. HRMS (m/
z): 267.0958 [M−I]+, calcd for C16H15N2S, 267.0950.
(E)-2-(4-Fluorostyryl)-1-methylquinolin-1-ium Iodide (2l). The
reaction was performed according to the general procedure A to
1
afford the brown solid 2l in 61% yields. mp 234.2−235.6 °C. H
NMR (300 MHz, DMSO-d6): δ 9.12 (d, J = 8.9 Hz, 1H), 8.59 (d, J =
8.9 Hz, 2H), 8.39 (dd, J = 8.1, 1.2 Hz, 1H), 8.28−8.17 (m, 2H), 8.09
(m, 2H), 8.02−7.89 (m, 2H), 7.42 (t, J = 8.8 Hz, 2H), 4.59 (s, 3H);
13C NMR (75 MHz, DMSO-d6): δ 162.61, 156.55, 145.95, 144.77,
139.65, 135.47, 132.17, 132.05, 130.56, 129.58, 128.35, 121.66,
119.89, 116.87, 116.58, 40.67. Purity: 98.5% by HPLC. HRMS (m/
z): 264.1175 [M−I]+, calcd for C18H15FN, 264.1183.
(E)-2-(4-(Dimethylamino)styryl)-3-methylbenzo[d]thiazol-3-ium
Iodide (5b). The reaction was performed according to the general
procedure A to afford the purple solid 5b in 83% yields. mp 246.2−
1
247.7 °C. H NMR (400 MHz, DMSO-d6): δ 8.30 (d, J = 8.0 Hz,
1H), 8.08 (dd, J = 14.5, 12.0 Hz, 2H), 7.91 (d, J = 8.9 Hz, 2H), 7.79
(t, J = 7.8 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 15.3 Hz, 1H),
6.83 (d, J = 8.9 Hz, 2H), 4.23 (s, 3H), 3.11 (s, 6H); 13C NMR (101
MHz, DMSO-d6): δ 171.80, 153.96, 150.57, 142.41, 133.29, 129.32,
127.89, 127.27, 124.27, 121.93, 116.40, 112.42, 106.73, 100.00, 36.07.
Purity: 98.3% by HPLC. HRMS (m/z): 295.1258 [M−I]+, calcd for
C18H19N2S, 295.1263.
(E)-2-(4-Bromostyryl)-1-methylquinolin-1-ium Iodide (2m). The
reaction was performed according to the general procedure A to
1
afford the green solid 2m in 78% yields. mp 241.9−242.4 °C. H
NMR (400 MHz, DMSO-d6): δ 9.13 (d, J = 8.9 Hz, 1H), 8.59 (d, J =
8.9 Hz, 2H), 8.39 (d, J = 8.1 Hz, 1H), 8.25−8.15 (m, 2H), 8.04−7.91
(m, 4H), 7.75 (d, J = 8.5 Hz, 2H), 4.59 (s, 3H); 13C NMR (101
MHz, DMSO-d6): δ 156.41, 145.75, 144.93, 139.68, 135.56, 134.57,
132.60, 131.43, 130.59, 129.67, 128.47, 125.36, 121.77, 120.79,
119.91, 40.73. Purity: 96.4% by HPLC. HRMS (m/z): 324.0381 [M−
I]+, calcd for C18H15BrN, 324.0382.
(E)-3-Methyl-2-(4-(4-methylpiperazin-1-yl)styryl)benzo[d]-
thiazol-3-ium Iodide (5c). The reaction was performed according to
the general procedure A to afford the purple solid 5c in 78% yields.
mp 229.4−229.8 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.33 (d, J =
7.7 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.03 (d, J = 15.4 Hz, 1H), 7.90
(d, J = 8.9 Hz, 2H), 7.68 (m, 2H), 7.02 (d, J = 9.0 Hz, 2H), 4.26 (s,
3H), 3.47−3.37 (m, 4H), 2.48−2.40 (m, 4H), 2.25 (s, 3H); 13C
NMR (75 MHz, DMSO-d6): δ 171.92, 153.96, 149.92, 142.35,
133.02, 129.42, 128.09, 127.46, 124.38, 123.52, 116.62, 114.08,
108.21, 54.67, 46.57, 46.06, 36.42. Purity: 99.3% by HPLC. HRMS
(m/z): 350.1705 [M−I]+, calcd for C21H24N3S, 350.1691.
(E)-1-Methyl-2-(4-nitrostyryl)quinolin-1-ium Iodide (2n). The
reaction was performed according to the general procedure A to
1
afford the brown solid 2n in 76% yields. mp 214.7−215.5 °C. H
NMR (400 MHz, DMSO-d6): δ 9.21 (d, J = 8.8 Hz, 1H), 8.63 (m,
2H), 8.43 (d, J = 8.0 Hz, 1H), 8.38 (d, J = 8.6 Hz, 2H), 8.24 (m, 5H),
8.02 (t, J = 7.5 Hz, 1H), 4.64 (s, 3H); 13C NMR (101 MHz, DMSO-
d6): δ 155.96, 148.79, 145.49, 143.91, 141.45, 139.75, 135.83, 130.66,
130.50, 129.96, 128.80, 124.62, 124.24, 122.11, 120.01, 41.01. Purity:
99.9% by HPLC. HRMS (m/z): 291.1129 [M−I]+, calcd for
C18H15N2O2, 291.1128.
(E)-3-Methyl-2-(4-morpholinostyryl)benzo[d]thiazol-3-ium Io-
dide (5d). The reaction was performed according to the general
procedure A to afford the purple solid 5d in 88% yields. mp 241.6−
1
242.7 °C. H NMR (300 MHz, DMSO-d6): δ 8.34 (dd, J = 8.0, 0.8
Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 15.5 Hz, 1H), 7.93 (d,
J = 9.0 Hz, 2H), 7.84−7.76 (m, 1H), 7.75−7.66 (m, 2H, HAr,
CH), 7.04 (d, J = 9.0 Hz, 2H), 4.27 (s, 3H), 3.78−3.70 (m, 4H),
3.41−3.36 (m, 4H); 13C NMR (75 MHz, DMSO-d6): δ 172.03,
154.17, 149.87, 142.37, 132.88, 129.47, 128.17, 127.54, 124.40,
123.98, 116.69, 113.98, 108.62, 66.28, 46.93, 36.46. Purity: 98.2% by
HPLC. HRMS (m/z):337.1365 [M−I]+, calcd for C20H21N2OS,
337.1369.
(E)-2-(2-(1H-Pyrrol-3-yl)vinyl)-1-methylquinolin-1-ium Iodide
(3a). The reaction was performed according to the general procedure
A to afford the brown solid 3a in 84% yields. mp 198.2−198.7 °C. 1H
NMR (400 MHz, DMSO-d6): δ 11.60 (s, 1H), 8.81 (d, J = 9.1 Hz,
1H), 8.44 (m, 2H), 8.34 (d, J = 15.3 Hz, 1H), 8.24 (d, J = 7.9 Hz,
1H), 8.08 (t, J = 8.0 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.56 (s, 1H),
7.39 (d, J = 15.3 Hz, 1H), 7.00 (s, 1H), 6.87 (s, 1H), 4.43 (s, 3H);
13C NMR (101 MHz, DMSO-d6): δ 157.27, 144.87, 142.49, 139.56,
(E)-2-(4-Hydroxystyryl)-3-methylbenzo[d]thiazol-3-ium Iodide
(5e). The reaction was performed according to the general procedure
A to afford the yellow solid 5e in 72% yields. mp 266.7−268.5 °C. 1H
NMR (300 MHz, DMSO-d6): δ 10.59 (s, 1H), 8.40 (d, J = 7.4 Hz,
1H), 8.20 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 15.7 Hz, 1H), 7.96 (d, J =
8.7 Hz, 2H), 7.88−7.71 (m, 3H), 6.93 (d, J = 8.6 Hz, 2H), 4.32 (s,
3H); 13C NMR (75 MHz, DMSO-d6): δ 172.52, 162.57, 149.71,
142.43, 133.07, 129.65, 128.53, 127.86, 125.87, 124.58, 117.05,
116.74, 110.57, 36.76. Purity: 99.0% by HPLC. HRMS (m/z):
268.0804 [M−I]+, calcd for C16H14NOS, 268.0796.
(E)-2-(3,4-Dihydroxystyryl)-3-methylbenzo[d]thiazol-3-ium Io-
dide (5f). The reaction was performed according to the general
procedure A to afford the brown solid 5f in 76% yields. mp 236.3−
237.2 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.24 (s, 1H), 9.56 (s,
1H), 8.38 (d, J = 7.9 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.06 (d, J =
15.6 Hz, 1H), 7.84 (t, J = 7.6 Hz, 1H), 7.73 (m, 2H), 7.54−7.38 (m,
2H), 6.90 (d, J = 8.1 Hz, 1H), 4.30 (s, 3H); 13C NMR (75 MHz,
DMSO-d6): δ 172.41, 151.58, 150.20, 146.44, 142.43, 129.62, 128.48,
127.82, 126.43, 124.82, 124.53, 116.99, 116.94, 116.42, 110.43,
100.00, 36.59. Purity: 99.3% by HPLC. HRMS (m/z): 284.0744 [M−
I]+, calcd for C16H14NO2S, 284.0745.
134.58, 130.27, 128.48, 127.17, 126.89, 122.60, 122.23, 120.40,
119.36, 112.74, 107.76, 39.50. Purity: 98.2% by HPLC. HRMS (m/
z): 235.1227 [M−I]+, calcd for C16H15N2, 235.1230.
(E)-1-Methyl-2-(2-(pyrazin-2-yl)vinyl)quinolin-1-ium Iodide (3b).
The reaction was performed according to the general procedure A to
1
afford the brown solid 3b in 72% yields. mp 203.7−204.6 °C. H
NMR (300 MHz, DMSO-d6): δ 9.24 (d, J = 8.8 Hz, 1H), 9.13 (d, J =
1.4 Hz, 1H), 8.86 (m, 1H), 8.78 (d, J = 2.4 Hz, 1H), 8.63 (t, J = 9.5
Hz, 2H), 8.45 (dd, J = 8.1, 1.3 Hz, 1H), 8.35 (d, J = 15.8 Hz, 1H),
8.27 (m, 1H), 8.19 (d, J = 15.8 Hz, 1H), 8.04 (t, J = 7.5 Hz, 1H), 4.61
(s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 155.71, 148.52, 146.52,
146.39, 145.81, 145.75, 141.55, 139.69, 136.00, 130.72, 130.05,
128.92, 125.20, 122.35, 119.96, 41.08. Purity: 98.2% by HPLC.
HRMS (m/z): 248.1179 [M−I]+, calcd for C16H14N3, 248.1182.
(E)-1-Methyl-2-(2-(pyridin-4-yl)vinyl)quinolin-1-ium Iodide (3c).
The reaction was performed according to the general procedure A to
1
afford the brown solid 3c in 74% yields. mp 253.6−254.4 °C. H
NMR (400 MHz, DMSO-d6): δ 9.22 (d, J = 8.8 Hz, 1H), 8.76 (d, J =
5.9 Hz, 2H), 8.63 (m, 2H), 8.44 (d, J = 7.7 Hz, 1H), 8.29−8.18 (m,
2H), 8.11 (d, J = 16.1 Hz, 1H), 8.02 (t, J = 7.5 Hz, 1H), 7.92 (d, J =
5.9 Hz, 2H), 4.64 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ
155.94, 151.05, 145.62, 143.78, 142.17, 139.73, 135.87, 130.68,
(E)-2-(4-Methoxystyryl)-3-methylbenzo[d]thiazol-3-ium Iodide
(5g). The reaction was performed according to the general procedure
K
https://doi.org/10.1021/acs.jmedchem.1c00465
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 2. Bacterial Strains and Cells Used in the Study
a
strains
relevant characteristics
source or reference
PAO1-ΔwspF
PAO1-ΔwspF-mini-CTX-pelA-lacZ
PAO1, ΔwspF; markerless
PAO1, ΔwspF, aatB::miniCTX-pelA-lacZ; Tc
this study
this study
a
cell
AC16
human cardiomyocyte
our group
a
Description of strain antibiotic resistance. Tc, ticarcillin resistance.
A to afford the yellow solid 5g in 82% yields. mp 246.5−247.1 °C. 1H
NMR (300 MHz, DMSO-d6): δ 8.45−8.39 (m, 1H), 8.20 (m, 2H),
8.06 (d, J = 8.8 Hz, 2H), 7.94−7.82 (m, 2H), 7.81−7.72 (m, 1H),
7.11 (d, J = 8.8 Hz, 2H), 4.34 (s, 3H), 3.86 (s, 3H); 13C NMR (75
MHz, DMSO-d6): δ 172.47, 163.35, 149.11, 142.43, 132.64, 129.72,
128.65, 128.01, 127.26, 124.65, 117.17, 115.29, 111.70, 56.22, 36.88.
Purity: 98.6% by HPLC. HRMS (m/z): 282.0958 [M−I]+, calcd for
C17H16NOS, 282.0953.
117.03, 116.73, 114.46, 114.43, 37.19. Purity: 97.7% by HPLC.
HRMS (m/z): 270.0752 [M−I]+, calcd for C16H13FNS, 270.0747.
(E)-2-(4-Bromostyryl)-3-methylbenzo[d]thiazol-3-ium Iodide
(5m). The reaction was performed according to the general procedure
A to afford the orange solid 5m in 80% yields. mp 251.1−251.4 °C.
1H NMR (300 MHz, DMSO-d6): δ 8.48 (d, J = 7.4 Hz, 1H), 8.29 (d,
J = 8.3 Hz, 1H), 8.23 (d, J = 16.0 Hz, 1H), 8.11 (d, J = 16.0 Hz, 1H),
8.04 (d, J = 8.6 Hz, 2H), 7.95−7.87 (m, 1H), 7.85−7.79 (m, 3H),
4.39 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 172.22, 147.36,
142.54, 133.75, 132.74, 131.94, 130.00, 129.06, 128.54, 126.43,
124.86, 117.52, 115.35, 37.15. Purity: 96.7% by HPLC. HRMS (m/
z): 329.9942 [M−I]+, calcd for C16H13BrNS, 329.9947.
(E)-2-(4-Hydroxy-3-methoxystyryl)-3-methylbenzo[d]thiazol-3-
ium Iodide (5h). The reaction was performed according to the
general procedure A to afford the yellow solid 5h in 79% yields. mp
1
268.4−269.2 °C. H NMR (400 MHz, DMSO-d6): δ 10.27 (s, 1H),
8.40 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 15.6
Hz, 1H), 7.89−7.79 (m, 2H), 7.76 (t, J = 7.6 Hz, 1H), 7.68 (s, 1H),
7.54 (dd, J = 8.2, 1.3 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 4.34 (s, 3H),
3.92 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 172.53, 152.46,
150.08, 148.75, 142.48, 129.65, 128.53, 127.86, 126.73, 126.31,
124.57, 117.03, 116.38, 112.97, 110.64, 56.58, 36.75. Purity: 98.0% by
HPLC. HRMS (m/z): 298.0899 [M−I]+, calcd for C17H16NO2S,
298.0902.
(E)-3-Methyl-2-(4-nitrostyryl)benzo[d]thiazol-3-ium Iodide (5n).
The reaction was performed according to the general procedure A to
1
afford the red solid 5n in 82% yields. mp 241.9−242.3 °C. H NMR
(300 MHz, DMSO-d6): δ 8.55−8.50 (m, 1H), 8.43−8.31 (m, 6H),
8.28 (d, J = 16.1 Hz, 1H), 7.97−7.90 (m, 1H), 7.89−7.82 (m, 1H),
4.44 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 171.67, 149.16,
145.42, 142.57, 140.50, 131.04, 130.19, 129.33, 128.91, 125.00,
124.62, 118.50, 117.74, 49.08, 37.50. Purity: 98.9% by HPLC. HRMS
(m/z): 297.0682 [M−I]+, calcd for C16H13N2O2S, 297.0692.
(E)-2-(2-(1H-Pyrrol-3-yl)vinyl)-3-methylbenzo[d]thiazol-3-ium Io-
dide (6a). The reaction was performed according to the general
procedure A to afford the yellow solid 6a in 56% yields. mp 242.5−
243.4 °C. 1H NMR (400 MHz, DMSO-d6): δ 11.72 (s, 1H), 8.32 (d,
J = 8.0 Hz, 1H), 8.18 (d, J = 15.2 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H),
7.78 (t, J = 7.8 Hz, 1H), 7.73−7.65 (m, 2H), 7.47 (d, J = 15.2 Hz,
1H), 7.02 (d, J = 1.6 Hz, 1H), 6.91 (s, 1H), 4.22 (s, 3H); 13C NMR
(101 MHz, DMSO-d6): δ 172.63, 146.10, 142.33, 129.32, 128.69,
127.95, 127.15, 124.35, 122.78, 122.07, 116.51, 108.17, 107.55, 36.21.
Purity: 97.6% by HPLC. HRMS (m/z): 241.0793 [M−I]+, calcd for
C14H13N2S, 241.0799.
(E)-2-(3-Fluoro-4-hydroxystyryl)-3-methylbenzo[d]thiazol-3-ium
Iodide (5i). The reaction was performed according to the general
procedure A to afford the yellow solid 5i in 76% yields. mp 241.7−
1
242.6 °C. H NMR (400 MHz, DMSO-d6): δ 8.28 (d, J = 7.9 Hz,
1H), 8.06 (d, J = 8.4 Hz, 1H), 7.94 (m, 2H), 7.76 (t, J = 7.6 Hz, 1H),
7.70−7.52 (m, 3H), 6.85 (t, J = 8.8 Hz, 1H), 4.21 (s, 3H); 13C NMR
(101 MHz, DMSO-d6): δ 170.58, 156.74, 154.12, 151.70, 148.67,
142.43, 130.95, 129.34, 127.78, 127.28, 124.27, 123.61, 123.54,
120.03, 119.98, 116.82, 116.63, 116.24, 107.87, 49.08, 36.08. Purity:
98.9% by HPLC. HRMS (m/z): 286.0702 [M−I]+, calcd for
C16H13FNOS, 286.0702.
(E)-2-(3,5-Dibromo-4-hydroxystyryl)-3-methylbenzo[d]thiazol-3-
ium Iodide (5j). The reaction was performed according to the general
procedure A to afford the brown solid 5j in 74% yields. mp 248.0−
(E)-3-Methyl-2-(2-(pyrazin-2-yl)vinyl)benzo[d]thiazol-3-ium Io-
dide (6b). The reaction was performed according to the general
procedure A to afford the orange solid 6b in 83% yields. mp 222.1−
1
248.5 °C. H NMR (400 MHz, DMSO-d6): δ 8.44 (d, J = 7.9 Hz,
1
1H), 8.36 (s, 2H), 8.24 (d, J = 8.2 Hz, 1H), 8.10 (d, J = 15.8 Hz, 1H),
7.97 (d, J = 15.8 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.79 (t, J = 7.4 Hz,
1H), 4.36 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 172.09,
154.77, 146.05, 142.47, 134.24, 129.87, 128.86, 128.78, 128.29,
124.78, 117.29, 113.68, 112.79, 37.03. Purity: 97.6% by HPLC.
HRMS (m/z): 423.9012 [M−I]+, calcd for C16H12Br2NOS, 423.9001.
(E)-3-Methyl-2-styrylbenzo[d]thiazol-3-ium Iodide (5k). The
reaction was performed according to the general procedure A to
223.4 °C. H NMR (300 MHz, DMSO-d6): δ 9.20 (d, J = 1.4 Hz,
1H), 8.88 (dd, J = 2.3, 1.5 Hz, 1H), 8.81 (d, J = 2.4 Hz, 1H), 8.57−
8.50 (m, 1H), 8.42−8.31 (m, 3H), 7.99−7.83 (m, 2H), 4.41 (s, 3H);
13C NMR (75 MHz, DMSO-d6): δ 171.20, 147.67, 147.09, 145.91,
143.28, 142.60, 130.30, 129.43, 129.03, 125.12, 119.01, 117.85, 37.44.
Purity: 96.1% by HPLC. HRMS (m/z): 254.0751 [M−I]+, calcd for
C14H12N3S, 254.0752.
(E)-3-Methyl-2-(2-(pyridin-4-yl)vinyl)benzo[d]thiazol-3-ium Io-
dide (6c). The reaction was performed according to the general
procedure A to afford the orange solid 6c in 85% yields. mp 219.5−
1
afford the yellow solid 5k in 72% yields. mp 229.2−229.8 °C. H
NMR (400 MHz, DMSO-d6): δ 8.48 (d, J = 8.1 Hz, 1H), 8.29−8.21
(m, 2H), 8.09−8.05 (m, 3H), 7.93−7.86 (m, 1H), 7.83−7.79 (m,
1H), 7.60−7.53 (m, 3H), 4.40 (s, 3H); 13C NMR (101 MHz,
DMSO-d6): δ 172.41, 148.85, 142.52, 134.50, 132.75, 130.20, 129.94,
129.68, 128.98, 128.42, 124.82, 117.48, 114.64, 37.17. Purity: 98.7%
by HPLC. HRMS (m/z): 252.0842 [M−I]+, calcd for C16H14NS,
252.0841.
1
220.1 °C. H NMR (400 MHz, DMSO-d6): δ 8.95 (d, J = 6.3 Hz,
2H), 8.56 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 16.1 Hz, 1H), 8.37 (d, J =
8.4 Hz, 1H), 8.31−8.27 (m, 3H), 7.99−7.92 (m, 1H), 7.88 (t, J = 7.4
Hz, 1H), 4.47 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 171.12,
147.65, 145.26, 143.53, 142.65, 130.38, 129.54, 129.26, 125.13,
124.53, 121.20, 117.93, 37.74. Purity: 98.0% by HPLC. HRMS (m/
z): 253.0796 [M−I]+, calcd for C15H13N2S, 253.0799.
Biological Assays. Measurement of Fluorescence and CD
Experiments. C-di-GMP, 10 mM Tris-HCl buffer solution (pH =
7.5), and salt solutions were mixed, heated to 95 °C, and kept at 95
°C for 5 min and then slowly cooled back to room temperature and
kept at room temperature for 10 min. 5h was then added to the
mixture and incubated in the refrigerator at −20 °C overnight (about
12 h).
(E)-2-(4-Fluorostyryl)-3-methylbenzo[d]thiazol-3-ium Iodide (5l).
The reaction was performed according to the general procedure A to
1
afford the yellow solid 5l in 80% yields. mp 229.8−231.2 °C. H
NMR (300 MHz, DMSO-d6): δ 8.51−8.45 (m, 1H), 8.31−8.14 (m,
4H), 8.03 (d, J = 15.9 Hz, 1H), 7.88 (tt, J = 6.0, 2.9 Hz, 1H), 7.84−
7.77 (m, 1H), 7.43 (t, J = 8.8 Hz, 2H), 4.39 (s, 3H); 13C NMR (75
MHz, DMSO-d6): δ 172.33, 166.43, 163.09, 147.52, 142.48, 132.89,
132.77, 131.25, 131.21, 129.92, 128.96, 128.37, 124.82, 117.46,
L
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Article
The CD instrument settings were chosen as follows: data pitch, 1
nm; scan speed, 50 nm/min; response, 8 s; bandwidth, 1 nm. The
fluorescence instrument settings were chosen as follows: λex = 520 nm
(slit 6 nm); λem = 540−620 nm (slit 6 nm). The measurements were
carried out at 15 °C.
Microbial Strains and Mediums. The strains and cells used in this
experiment are listed in Table 2. The medium we use to culture
bacteria is Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract,
0.5% NaCl). Cells were cultured using Dulbecco’s modified eagle
medium containing 10% fetal calf serum.
Biofilm Quantification Assays. All synthesized compounds have
undergone the purity detection of 1H and 13C NMR spectroscopy. All
compounds were then accurately weighed and dissolved in DMSO to
prepare a 10 mM stock solution, which was then transferred to a 96-
well plate for biofilm inhibition experiments. P. aeruginosa PAO1-
ΔwspF strain was cultured with LB medium at 37 °C for 24 h. It was
diluted with LB medium until the final OD600 value [optical density
(OD) at 600 nm] of 0.01. The bacteria were incubated with different
concentrations of compounds for 20 h. After that, the bacteria were
removed, washed 4 times with phosphate-buffered saline (PBS)
solution, dried, fixed with 120 μL of methanol, removed after 30 min,
and dried. Then, they were added to 150 μL of 0.1% CV, incubated
for 15 min, and then washed with PBS solution until there was no
liquid CV residue; finally, 120 μL of 33% acetic acid solution was
added into each well and shaken for 10 min, and the absorbance value
was measured at 550 nm using a microplate reader (Bio-Tek).
3D Imaging of Biofilm. The preparation process is the same as the
above biofilm quantification assay experiment. After the bacterial
biofilm is fixed, 1 μM SYTO9 is added and incubated for 15 min
under dark conditions. Fluorescent images were acquired with a Zeiss
LSM 800 confocal microscope. 3D images were obtained using an oil
immersion 63× objective lens and Z-stack program.
Transcriptome Analysis. All transcriptome data in this study are
provided by Beijing Nuohe Zhiyuan Technology Co., Ltd. In simple
terms, P. aeruginosa PAO1-ΔwspF strain was cultured with LB
medium at 37 °C for 24 h. It was diluted with LB medium until the
final OD600 value (OD at 600 nm) of 0.01. The bacteria were
incubated with 1.25 μM 5h for 15 h. Then, the total RNA of bacteria
in the blank control group and experimental group was extracted, and
the library was constructed after passing the test, followed by
sequencing analysis.
Experimental Analysis of RT-PCR. All experimental data are
provided by Guangzhou Ruijie Biotechnology Co., Ltd. The culture
and incubation method of bacteria is consistent with the experimental
method of transcriptome analysis mentioned above. Total RNA was
extracted from 3 mL of bacterial culture solution, and reverse
transcription was performed. The primers designed to amplify the
genes are listed in Table S1. RT-qPCR was performed with SYBR
Premix Ex Taq (Takara) and on the Roche LightCycler 480 real-time
system with the following cycle conditions: initial denaturation at 95
°C for 10 s, followed by 40 cycles of denaturation at 95 °C for 10 s
(first cycle of 30 s) and annealing and extension at 60 °C for 30 s.
Fluorescent signal intensity was collected at the end of the extension
step. The 30S ribosomal protein S12 gene RpsL was chosen as an
internal control to normalize the real-time PCR data in each single
run and to calculate the relative fold change in gene expression by
using the 2−ΔΔCt method. The analysis was performed in duplicate on
three technical replicates.
DNA Embedding Experiment. 10 μM compound 5h and TO were
mixed with 0.005 μg/μL of pBR322 DNA, respectively, and the
volume was made up to 10 μL with ddH2O and incubated at room
temperature for 10 min. Then, 2 μL of 6× DNA loading buffer was
added. Finally, the DNA was subjected to 1 wt % PAGE for
separation. PAGE experiments were run at a working voltage of 100 V
for 40 min.
Analysis of Bacterial Motility. The filtered glucose solution is
added to LB store medium to make its final concentration 0.5%; then
the compounds are added to prepare the corresponding concentration
and finally poured into a flat plate for solidification. P. aeruginosa
PAO1-ΔwspF strain was cultured with LB medium at 37 °C for 24 h.
It was diluted with LB medium until the final OD600 value (OD at 600
nm) of 0.01. 1 μL of bacterial liquid was taken and dropped on the
solidified plate. Then, it was incubated overnight at 37 °C for 14 h.
EPS Quantification. EPS was measured in the quantitative Congo
red (CR) binding assay, as previously described.44 Congo red was
added at a final concentration of 40 μg/mL in 2 mL of PBS and
incubated with shaking for 3 h at 37 °C. After this incubation, the cells
were pelleted via centrifugation, and the absorbance (490 nm) of the
supernatant was measured. One percent PBS plus 40 μg/mL of CR
was used as the reference to determine the amount of CR that was
removed from the solution.
Detection of lacZ Reporter Strains. P. aeruginosa PAO1-ΔwspF
strain was cultured with LB medium at 37 °C for 24 h. Dilute with LB
medium until the final OD600 value (OD at 600 nm) of 0.01. Bacteria
solution and compounds with different concentrations of 100 μL are
added to a 96-well plate and incubated together at 37 °C. After 4 h,
OD600 was determined. Then 100 μL of each sample was sucked out
of a 96-well plate and put into an eight-tube tube, and finally lysis
buffer (100 μL Z buffer + 20 μL 0.1% SDS + 40 μL chloroform) was
added. After mixing the culture medium and lysis buffer for 10 to 15
min, 100 μL of the upper layer liquid of each sample was carefully
sucked into the 96-well plate; then 20 μL of ONPG (4 mg/mL) was
added and incubated for about 10 min at room temperature. Finally,
50 μL of Na2CO3 with a concentration of 1 M was added to terminate
the incubation, and the absorbance was measured at 420 nm.
Enzyme Kinetic Detection of WspR and RocR. The classical
HPLC method was used to detect the kinetics of two proteins, as
reported in our previous work.51 To put it simply, compound 5h with
different concentrations was added to the 1 μM RocR or 1 μM WspR
reaction system, and the kinetic activity of related enzymes was
calculated by regularly detecting the content change of c-di-GMP in
the reaction system.
Cell Culture. AC16 cells cryopreserved in a nitrogen canister were
purchased from ATCC. Cells were cultured in EMDM medium mixed
with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL
streptomycin in a humidified incubator at 37 °C with 5% CO2.
Cytotoxicity by MTT Assay. We used the MTT method here to
detect the toxicity of 5h to cells: collecting cells in the logarithmic
phase, adjusting the concentration of cell suspension, adding 100 μL
to each well in 96-well plate to maintain the density of cells to be
tested at 6000 cells per well, and filling the edge wells with sterile PBS.
The cells were incubated at 37 °C for 5% CO2. After half a day, when
the cells adhere to the wall, the suspension in the wells was replaced
by fresh medium containing different concentrations of drugs or
DMSO, and the concentration of DMSO should be maintained below
0.1% in all wells. Incubation was continued at 37 °C for 24 h with 5%
CO2. After that, 20 μL of MTT solution (5 mg/mL) was added to
each well, and the culture was continued for 4 h. When the culture
was terminated, the culture solution in the well was carefully sucked
off, 150 μL of dimethyl sulfoxide was added to each well, and the
mixture was placed on a shaker to oscillate at a low speed for 10 min,
so that the crystals were fully dissolved. Then, the absorbance value of
each well at 570 nm (OD570) was detected by a microplate reader
(Bio-Tek).
Statistical Analysis. All data points are presented as mean and
standard deviation of independently obtained experiments repeated
at least three times. Statistical significance was calculated by applying
one way analysis of variance followed by Tukey’s multiple comparison
test, *0.01 < p-value < 0.05. Data were analyzed using GraphPad
Prism (GraphPad Software, Inc., California, CA, USA).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00465.
■
sı
Additional tables and figures representing NMR spectra
(1H and 13C NMR) and HRMS of compounds,
comprehensive statement of confirming purity and the
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method, HPLC data of final compounds, determination
of c-di-GMP content in different strains, inhibitory effect
of inducer 5h on PAO1 and ATCC 27853 biofilm, and
related primer information used in the experiment
(PDF)
Molecular formula strings and some data (CSV)
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AUTHOR INFORMATION
Corresponding Authors
■
Wei-Min Chen − College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China; orcid.org/0000-0002-
0292-7841; Phone: +86 20 8522 4497; Email: twmchen@
jnu.edu.cn; Fax: +86 20 8522 4766
Jing Lin − College of Pharmacy, Jinan University, Guangzhou
510632, P. R. China; orcid.org/0000-0001-7707-8066;
Phone: +86 20 8522 1367; Email: linjing_jnu@163.com
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Authors
Teng-Fei Xuan − College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China
Zi-Qiang Wang − College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China
Jun Liu − College of Pharmacy, Jinan University, Guangzhou
510632, P. R. China
Hai-Tao Yu − College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China
Qian-Wen Lin − College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China
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W.; Empting, M.; Titz, A. Novel strategies for the treatment of
Pseudomonas aeruginosa infections. J. Med. Chem. 2016, 59, 5929−
5969.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00465
(16) Bjarnsholt, T.; Ciofu, O.; Molin, S.; Givskov, M.; Høiby, N.
Applying insights from biofilm biology to drug developmentCan a
new approach be developed? Nat. Rev. Drug Discovery 2013, 12, 791−
808.
(17) Römling, U.; Gomelsky, M.; Galperin, M. Y. C-di-GMP: the
dawning of a novel bacterial signalling system. Mol. Microbiol. 2005,
57, 629−639.
(18) Cho, K. H.; Tryon, R. G.; Kim, J.-H. Screening for diguanylate
cyclase (DGC) inhibitors mitigating bacterial biofilm formation.
Front. Chem. 2020, 8, 264.
(19) Christen, M.; Kamischke, C.; Kulasekara, H. D.; Olivas, K. C.;
Kulasekara, B. R.; Christen, B.; Kline, T.; Miller, S. I. Identification of
small-molecule modulators of diguanylate cyclase by fret-based high-
throughput screening. ChemBioChem 2019, 20, 394−407.
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Demizu, Y. Amine skeleton-based c-di-GMP derivatives as biofilm
formation inhibitors. Bioorg. Med. Chem. Lett. 2021, 32, 127713.
(21) Fernicola, S.; Torquati, I.; Paiardini, A.; Giardina, G.;
Rampioni, G.; Messina, M.; Leoni, L.; Del Bello, F.; Petrelli, R.;
Author Contributions
†T.-F.X. and Z.-Q.W. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Pearl River S&T Nova
Program of Guangzhou (201806010116) and the Natural
Science Foundation of Guangdong Province
(2019A1515011801).
ABBREVIATIONS
■
C-di-GMP, cyclic diguanylate; DGCs, diguanylate cyclases;
PDEs, phosphodiesterases; TO, thiazole orange; EPS,
exopolysaccharide; HPLC, high-performance liquid chroma-
tography; CD, circular dichroism; DMSO, dimethyl sulfoxide;
MIC, minimum inhibitory concentration
̀
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H.; Wennemers, H. Functionalized proline-rich peptides bind the
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2018, 57, 7729−7733.
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Ausubel, F. M.; Lory, S. Analysis of Pseudomonas aeruginosa
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characterization of c-di-GMP signaling-related genes in the Probiotic
Lactobacillus acidophilus. Front. Microbiol. 2018, 9, 1935.
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