Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa virulence and biofilm formation

Article abstract of DOI:10.1016/j.bmcl.2018.12.068

Signal molecules are stimulators of multiple quroum-sensing virulence and biofilm formation. Small molecule analogues have been suspected as a potent inhibitor in therapeutic strategy. Herein, we synthesized a series of small molecule compounds from the 2

Full text of DOI:10.1016/j.bmcl.2018.12.068

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa
virulence and biofilm formation
a,1
b,1
a
c
a
a
a
Man-Na Qiu , Fang Wang , Si-Yu Chen , Peng-Cheng Wang , Yu-Hang Fu , Yuan-Yuan Liu ,
a
a
b
a
a
Xuan Wang , Fan-Bei Wang , Chao Wang , Hong-Wen Yang , Yang Wu , Shi-Ying Zhu ,
a
a
a
d,⁎
a,⁎
Hai-Bo Zhou , Wei-Min Chen , Jing Lin , Jun-Xia Zheng , Ping-Hua Sun
a
b
c
Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, College of Pharmacy, Jinan University, Guangzhou 510632, PR China
Pharmacy Department, Qingdao Stomatological Hospital, Qingdao 266000, PR China
Department of Chemistry, Yale University, New Haven, CT 06520, United States
d
School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, PR China
A R T I C L E I N F O
Keywords:
A B S T R A C T
Signal molecules are stimulators of multiple quroum-sensing virulence and biofilm formation. Small molecule
analogues have been suspected as a potent inhibitor in therapeutic strategy. Herein, we synthesized a series of
small molecule compounds from the 2, 8-bit derivatives of quinoline by Suzuki coupling reaction. We found that
these compounds have the biofilm inhibitory effect in normal condition instead of phosphate limitation state.
Furthermore, lacZ reporter strain assay and rhamnolipids as well as pyocyanin experiments showed that these
compounds did not affect las and pqs system but reduced the expression of rhl. All these results suggest that
quinoline derivatives can be treated as potent inhibitors against biofilm and reduce virulence through the rhl
system. This research will be useful in designing new quorum sensing inhibitors to attenuate the infection of
bacteria.
2
, 8-bit derivatives of quinolines
Biofilm formation
rhl system
Quroum-sensing
In order to adapt the change of environment, especially the wide-
spread use of antibiotics, bacteria develop multiple mechanisms to
prevent themselves from kinds of adverse conditions and maintain their
las system under the phosphate limitation conditions though the target
is not yet clear. This hypothesis explains why so many clinically isolated
1
3
strains with the lost of the las system are still pathogenic. In P. aer-
uginosa, the expression of QS depends on the signal molecules which is
called autoinducers (AIs). The signal molecules are N-(3-oxo-dodeca-
noyl)-l-homoserine lactone (OdHSL), N-butanoyl-homoserine lactone
(BHL), 2-heptyl-3,4-dihydroxyquinoline (PQS), and 2-(2-hydro-
xyphenyl)-thiazole-4-carbaldehyde (IQS) which are regulated by LasI,
RhlI, PqsABCD and AmbBCDE respectively through their transcription
1
pathogenesis. One of the crucial mechanisms is Quorum sensing (QS),
which is a cell-to-cell communication that relies on cell density to
2
control collective behavior. With the regulation of QS, bacteria can
form kinds of virulence such as elastase, pyocyanin, alginate, rham-
3
–5
nolipids and biofilm to survive in disadvantageous circumstance.
Gram-negative bacteria Pseudomonas aeruginosa is a ubiquitous or-
6
14
ganism living on the surface of animals, plants and even human being.
factors LasR, RhlR and PqsR. (Fig. 1) Biofilm is another critical reason
It is a notorious opportunistic pathogen for inducing acute and persis-
tent infection in the patients with cystic fibrosis, neutropenia and burns
for the bacterial resistance. Bacteria embed in a self-produced extra-
cellular matrix that consists of polysaccharide matrix, fibrin, lipid
7
15
or wound. Numerous articles revealed that QS plays an essential role in
protein that can reduce the injury from antibiotics. Solano et al.
6
,7
P. aeruginosa virulence and biofilm formation. Some people suspected
that QS in P. aeruginosa cause the prevalence of chronic lung infections
supposed that QS regulates genes involved in biofilm development.
Recently, people found that signal molecule analogues exhibit ex-
cellent anti-biofilm activity and attenuate quorum-sensing viru-
8
in patients with cystic fibrosis. To date, there are four QS system have
1
6,17
been found in that pathogenic bacteria, including las, rhl, iqs and pqs. It
is believed that QS exist a hierarchy network in bacteria. Traditional
view thinks las system as the top of QS which controls the expression of
lence.
However, only few autoinducer type drugs are used in
clinically treatment. In order to develop more effective drugs to use in
the clinic for the inhibitions of the infection of P. aeruginosa, we de-
signed and synthesized 2, 8-bit derivatives of quinolines based on the
pqs and rhl.9
–11
However, Lee et al. proposed that iqs can replace the
12
⁎
Corresponding authors.
E-mail addresses: junxiazheng@163.com (J.-X. Zheng), pinghuasunny@163.com (P.-H. Sun).
1
These authors contributed to the work equally.
https://doi.org/10.1016/j.bmcl.2018.12.068
Received 22 November 2018; Received in revised form 28 December 2018; Accepted 31 December 2018
0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Qiu, M.-N., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2018.12.068

M.-N. Qiu et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 1. A summary of quorum-sensing system networks in P. aeruginosa and the signal molecules.
Table 2
Structure of 2-(3-(trifluoromethyl)phenyl)quinolin-8-ol derivatives.
Compound
R
Compound
R
Fig. 2. Strategy for structural modification of quinoline derivatives.
H-1
H-5
Table 1
H-2
H-3
H-6
H-7
Structure of 2-(4-(trifluoromethyl)phenyl)quinolin-8-ol derivatives.
H-4
signal molecule. Considering the QS may involve in biofilm formation,
we used crystal violet to evaluate the effect and found that some
compounds decrease the biofilm formation of P. aeruginosa in rich
medium instead of phosphate limitation condition. For further ex-
ploration, we tested the expression of las, rhl and pqs by the bioreporter
assay based on the expression of lacZ in P. aeruginosa PAO1 and thus
had detect these effective compounds took place via rhl system. The
rhamnolipids and pyocyanin assay results confirmed our consequent.
Quinoline has a potent inhibitory activity towards bacteria viru-
Compound
R
Compound
R
Y-2
Y-8
Y-3
Y-4
Y-5
Y-9
Y-10
Y-11
1
8
lence like pyocyanin, hydrogen cyanide and biofilm. C-2 position in
quinoline is an active site for antibacterial activity. It has been re-
1
9,20
ported
that C-2 position fluorine-substituted phenyl groups can
Y-6
Y-7
Y-12
increase the antibacterial activities. The ester and amide bonds at the C-
8
position can increase the biofilm inhibitory activity against Staphy-
lococcus aureus and Staphylococcus epidermidis. The quinoline deriva-
tives play crucial effect on resisting both gram-negative and gram-
2

M.-N. Qiu et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 3
acetylation with different acetyl chloride.
Structure of 2-phenylquinolin-8-yl heptanoate derivatives.
Antimicrobial drugs suppress the growth of bacteria. However, on
the opposite side, bacteria develop multiple resistance mechanisms to
survive. In order to avoid this result, quorum-sensing inhibitors take a
new strategie to inhibit the virulence factor and biofilm formation in-
stead of destroying bacteria. To make sure our compounds do not have
influence on the growth of bacteria, we detected our compounds on the
effect on the growth of P. aeruginosa PAO1 and the minimum inhibitory
concentration (MIC) on two Gram-positive bacteria, the Staphylococcus
aureus (ATCC 25923), Escherichia coli (ATCC 8739) and two Gram-ne-
gative bacteria, the Staphylococcus albus (ATCC 8799), Klebsiella
Pneumoniae (ATCC 13883) by measuring the optical density value at the
wavelength of 600 nm (OD600). As expected, P. aeruginosa PAO1 strain
growth curve was not affected and all the MIC of bacteria
were > 256 μM, which mean that our compounds have no influence on
the growth of bacteria. The growth of bacteria assay and MIC assay
Compound
R1
R2
R3
K-1
K-2
H
F
H
CF
3
H
CF
3
positive bacteria, and perform excellent activity in anti-quorum sensing
and biofilm resistance. Therefore, the C-2 and C8 substituted quinoline
derivatives were synthesized in this work. The strategy of the design of
the library was illustrated in Fig. 2. In order to further elaborate on the
structural features, our compounds are shown in Tables 1–3.
(
Fig. 3) confirmed these compounds are potent to be the quorum sen-
sing inhibitors.
In the C-8 position, we tried to find the relationship between the
activity and the length of optimal chain. Thus, we synthesized 2-(4-
The synthesis of 2,8-substituted quinoline derivatives route was
shown in Scheme 1. Selectively C-8 position mono-acetylation of 2,8-
hydroxyquinoline with acetic anhydride followed by bromination at C-
(
trifluoromethyl)phenyl)quinolin-8-ol with different chains (Table 1, Y-
21
1 to Y-7) and investigated the biofilm inhibition by crystal violet. As
shown in Fig. 4, anti-biofilm activities increased as the alkyl chain
lengthened to 4, 6 or 7, indicating that biofilm inhibitor should keep a
long chain like the AI. Comparing Y-1 to Y-12 and H-1 to H-7, we found
that the anti-biofilm activities are better when the chain is alkyl instead
of aromatic ring. These results may relate to the function of alkyl group
contributes to the van der Waals interaction in the QS receptor hy-
drophobic subpocket while ring structure has no such effect. Since we
2
position with POBr gave compound 1. Then, hydrolysis of compound
3
1
with potassium hydroxide generated compound 2. The resulting free
alcohol was then methylation with CH
3
I yielded compound 3. Suzuki
coupling of compound 3 with different Boron substrates generated
corresponding 8-methoxy-2-phenylquinoline derivatives. After removal
methyl group, 2, 8-quinoline derivatives were finally synthesized after
Scheme 1. Synthesis of 2, 8-quinoline derivatives.
3

M.-N. Qiu et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 3. Growth curve of P. aeruginosa (A) without treated (control) or treated with 10 μM Y-1 to Y-12. (B) without treated (control) or treated with 10 μM H-1 to H-7,
K-1, K-2.
found that the fluorine-substituted phenyl groups at the C-2 position
can increase the antibacterial and antibiosis effects of quinoline com-
pounds, a series of C-2 fluorinated compounds were designed and
Since pyocyanin can be synthesized by P. aeruginosa, pqs genes must be
functionalized. Pqs is a gene which produces a transcription factor that
activates phnAB genes. These genes produce the molecule quinolone.
However, through our study, a strange phenomenon that we can ob-
serve is Y-7 and H-7 had no effect on the production of pyocyanin as
shown in Fig. 6. The possible reason pyocyanin is controlled by both pqs
and rhl system. If we just inhibit one of two systems, bacteria can im-
prove the other pathway to maintain its virulence. Combined the results
of rhamnolipids and pyocyanin assay, we realized that our compounds
only repressed the rhl system thus the production of rhamnolipids was
1
9,20
tested.
The graphic of Fig. 4 reveal that the 4′-position were more
favorable for inhibition of biofilm.
In order to determine the quorum sensing activity of our synthesized
compounds, we investigated the effect of our compounds with 10 μM
concentration on the expression of lasB-lacZ, rhlA-lacZ, pqsA-lacZ in the
2
2
P. aeruginosa. If the lacZ is down regulated, the production of β-ga-
lactosidase will decrease and then we can detect it by o-Nitrophenyl-β-
D-Galactopyranoside (ONPG). Based on that result, we detected the
relation between our compounds and the promoter of las, rhl and pqs
system. To our surprise, the rhlA-lacZ had obviously been inhibited
while lasB-lacZ, pqsA-lacZ were not affected. So, we speculated that the
anti-biofilm activities of our synthesized compounds are realized
through the rhl system. Since Y-7 and H-7 showed the best inhibition
activity of QS report strains among the synthesized compounds, they
were used as our model molecules in the mechanism study (See Fig. 5).
To verify our speculation, we tested the effect of our compounds on
2
3,24
rhl system by the rhamnolipids and pyocyanin assay.
Rhamnolipids
is known as a condition for the development of chronic infection which
2
5
were detected in the sputum from cystic fibrosis (CF) patients. By
regulating the enzymes for the biosynthesis of rhamnolipids, rhl system
controls the rhamnolipids virulence factor directly. So, we monitor the
rhamnolipids through orcinol. Consistent with our speculation, the
production of rhamnolipids was significantly reduced by Y-7 and H-7.
Then we can make a conclusion that our compounds indeed inhibit the
expression of rhl system. To further evidence, we tried to detect the
Fig. 5. Effect of Y-7 (10 μM) and H-7 (10 μM) on the different QS monitors,
PAO1-lasB-lacZ, PAO1-rhlA-lacZ, PAO1-pqsA-lacZ. Experiments were done in
triplicate.
2
6
production of pyocyanin strictly adjusted via pqs and rhl pathway.
Fig. 4. Biofilm formation of P. aeruginosa in ABTGC medium (A) without treated (control) or treated with 10 μM Y-1 to Y-12. (B) Without treated (control) or treated
with 10 μM H-1 to H-7, K-1 to K-2.
4

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Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 6. The virulenc assay in P. aeruginosa without treated (control), P aeruginosa lasI rhlI double mutant (Mutant), P. aeruginosa treated with Y-7 (10 μM) and H-7
(
10 μM) (A) rhamnolipids assay (B) pyocyanin assay. Experiments were done in triplicate.
River S&T Nova Program of Guangzhou, China (201610010100), We
sincerely thanks Dr. Liang Yang from the Singapore Centre for
Environmental Life Sciences Engineering, Nanyang Technological
University, Singapore for gifting the P. aeruginosa PAO1.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2018.12.068.
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