Low-toxicity amphiphilic molecules linked by an aromatic nucleus show broad-spectrum antibacterial activity and low drug resistance

Article abstract of DOI:10.1039/c9cc00857h

Amphiphilic molecules linked by an aromatic nucleus were developed that showed high selectivity toward bacteria over mammalian cells, and low drug resistance. A promising compound 4g exhibited strong bactericidal activity against a panel of sensitive and resistant bacteria, low toxicity, the ability to reduce cell viability in biofilms, stability in mammalian fluids, rapid killing of pathogens, and high in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA).

Full text of DOI:10.1039/c9cc00857h

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Reactivity differences of Sc
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DOI: 10.1039/C9CC00857H
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Low-toxicity Amphiphilic Molecules linked by an Aromatic
Nucleus Show Broad-spectrum Antibacterial Activity and Low
Drug Resistance †
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Wenchao Chu,a‡ Yi Yang,a‡ Shangshang Qin, a, c‡ Jianfeng Cai, Mengmeng Bai, Hongtao
b
a
www.rsc.org/
a
a, c
Kong, En Zhang,*
Amphiphilic molecules linked by an aromatic nucleus were membrane binding (electrostatic interactions) and membrane
developed that showed high selectivity toward bacteria over insertion/permeation, eventually leading to cell death1
2,
13
.
mammalian cells, and low drug resistance. Promising Despite their antimicrobial properties, some disadvantages limit
compound 4g exhibited strong bactericidal activity against a the use of AMPs including unknown toxicity, susceptibility to
panel of sensitive and resistant bacteria, low toxicity, the degradation by proteases, and the high cost of manufacture1
4, 15
.
ability to reduce cell viability in biofilms, stability in These inherent limitations of AMPs have driven substantial
mammalian fluids, rapid killing of pathogens, and high in vivo efforts worldwide to develop synthetic mimics of AMPs. Many
efficacy against methicillin-resistant Staphylococcus aureus AMP-mimetics based on the inherent attributes of AMPs provide
(MRSA)
.
broad-spectrum antibacterial activity and have been shown to
overcome bacterial resistance1
6, 17
.
Bacterial resistance and infection present a major challenge in the
H
O
O
1
,
2
N
S
H
field of biomedicine . The rapidly increasing incidence of
antibacterial resistance has caused escalating healthcare costs and
H
H
N
N
O
O
N
S
S
N
S
O
O
O
O
O
N
N
OH
N
O
O
3
high mortality rates ; while the development of new antibiotics has
O
O
OH
OH
OH
O
O
4
O
dramatically decreased . Consequently, there is an immediate need
Penicillin
Nafcillin
Methicillin
Penicillin V
for new agents with novel therapeutic activity against these
resistant pathogens.
O
Cl
O
Br
Br
O
Bacteria have developed several different resistance mechanisms
to respond to a single class of antibiotics such as modification of
the drug target, cell wall permeability, molecular bypass and
H
N
C8H17
C6H13
N
N
N
H
O
N
N
C6H13
H
O
I
II
5
active efflux . In addition, bacteria can synthesise a protective
Fig 1 Antibiotics with a phenoxy group and cationic AMP-mimetics
polysaccharide layer on their surface. This hydrated polymeric
matrix, known as bacterial biofilm, is often the root of resistance
to antimicrobial agents, contributing to chronic bacterial
In our continuing attempt to identify more potent compounds with
great membrane selectivity and high antimicrobial activity against
6
, 7
infections . Targeting the membrane of microorganisms is an
effective and selective antimicrobial approach because bacterial
18-20
19
drug-resistance bacteria
, chalcone derivatives I (Fig. 1) with a 4-
methoxy benzene substituent and moderate length alkane chain (eight
carbon atoms), showed good antibacterial activity (in the range 1–4
μg/mL, Table 1) and moderate haemolytic activity. Inspired by the
innovative discovery of active and low-toxicity cationic small
8
biofilm has subtle differences to the mammalian cell membrane .
The role of antimicrobial peptides (AMPs) in innate immunity is
to kill microorganisms and provide defence against infection9
.
-11
AMPs interact with the target cell membrane via two steps,
21
molecules by Haldar’s group (compound II ) and antibiotics with a
phenoxy group (nafcillin, methicillin, and penicillin V showed better
2
2
activity than penicillin, Fig. 1, ), we sought to find more effective
antibacterial peptide mimics with low toxicity. The introduction of
amide constructs could enhance the stability and the antibacterial
activity of soft antimicrobial agents . In view of the above
considerations, amphiphilic molecules linked by an aromatic nucleus
were designed and synthesized.
Compounds were designed to investigate the effects of an aromatic
core, the length of the alkane chain, and the middle section on their
membrane-selectivity. The general synthetic routes are shown in
a.
School of Pharmaceutical Sciences; Institute of Drug Discovery and Development;
Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of
China; Zhengzhou University, Zhengzhou 450001, PR China. E-mail:
zhangen@zzu.edu.cn
Department of Chemistry, University of South Florida, 4202 E. Fowler Ave,
Tampa, Florida 33620, United States of America.
Collaborative Innovation Center of New Drug Research and Safety Evaluation,
Henan Province, Zhengzhou 450001, PR China.
Electronic Supplementary Information (ESI) available: [details of any
2
3
b.
c.
†
supplementary information available should be included here]. See Schemes S1 and S2 (ESI†). Intermediates 1a–1f reacted with 3a–3h
DOI: 10.1039/x0xx00000x.
to obtain the final molecules 4a–4w, using a previously reported
‡
Co-first authors

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1
9
View Article Online
DOI: 10.1039/C9CC00857H
method . Compounds 5a–5d were synthesized to verify the role of compounds against the NDM and KPC-producing strains were
symmetry. Compounds 5e–5j were obtained to investigate the in the range of 1−8 μg/mL (Table S1, ESI†).
influence of an aromatic core on antibacterial activity. The molecules
Table 1 MIC and HC50 values of compounds I and II, molecules 4a–4w and 5a–
5j, and commercial antibiotics
O
Br
H
m
N
nO
N
N
O
N
m
Br
H
H
MIC(μg/mL)a
Br
n
N
O
O
N
m
5a: m = 5; 5b: m = 6;
5c: m = 7; 5d: m = 8
HC50f
SIg
Com.
O
S. ab
E. fc
E. cd
S. me
4a: m = 6, n = 2; 4b: m = 7, n = 2; 4c: m = 8, n = 2;
4d: m = 3, n = 3; 4e: m = 4, n = 3; 4f: m = 5, n = 3;
4g: m = 6, n = 3; 4h: m = 7, n = 3; 4i: m = 8, n = 3;
4j: m = 3, n = 4; 4k: m = 4, n = 4; 4l: m = 5, n = 4;
4m: m = 6, n = 4; 4n: m = 7, n = 4; 4o: m = 8, n = 4;
4p: m = 3, n = 5; 4q: m = 4, n = 5; 4r: m = 5, n = 5;
4s: m = 6, n = 5; 4t: m = 7, n = 5; 4u: m = 8, n = 5;
H
N
I19
II21
4a
- f
- f
2
- f
- f
1
O
N
m
1
1
4
2
124
805
124
805
O
Br
H
N
N
O
m
O
1
1
＞1280
617
＞1280
1234
156
5e: m = 5; 5f: m = 6; 5g: m = 7; 5h: m = 8
4
b
0.5
0.5
4
0.25
0.5
32
32
2
1
1
O
m
m
N
N
O
HN
Br
4c
4d
4e
1
1
78
m
Br
O
O
N
m
Br
N
O
O
O
N
m
8
16
64
16
1
＞1280
＞1280
＞1280
1219
287
＞320
＞80
＞160
2438
574
O
N
4v: m= 3; 4w: m = 5
Br
16
8
32
8
HN
i: m = 6; 5j: m = 7
m
5
4
f
Fig 2 The structure of molecules 4a–4w and 5a–5j
4
g
0.5
0.5
0.5
4
2
1
4
h
1
0.5
1
1
were characterized by H NMR, 13C NMR, and HR-MS (Fig. 2). All
of the compounds were confirmed to have ≥95% purity using HPLC.
The antibacterial activity and haemolytic activity (HC50) of
amphiphilic molecules were evaluated against both sensitive bacteria
1
4
i
0.5
16
32
16
1
0.5
4
45
90
4
j
8
＞1280
＞1280
＞1280
819
＞320
＞160
＞640
1638
264
4k
4l
8
16
4
32
16
2
(
Staphylococcus aureus; Enterococcus faecalis; Escherichia coli;
Stenotrophomonas maltophilia) and resistant bacterial strains. Some
of these compounds showed excellent selectivity (HC50/MIC S. aureus
2
4
m
0.5
0.5
1
1
)
4
n
2
1
0.5
2
132
and activity against both negative and positive pathogens (Table 1).
Compounds (4d–4f, 4j–4l, 4p–4r) with a short alkyl chain length (m
4
o
0.5
16
32
2
1
20
20
4
p
4
8
16
8
＞1280
＞1280
764
＞320
＞640
1528
1360
68
=
3, 4, or 5) showed poor activity against the test strains (except 4r),
while the HC50 values for these compounds were more than 1280
μg/mL (except 4r: 764 μg/mL). Compounds (4a–4c, 4g–4i, 4m–4o,
4q
4r
2
8
0.5
0.25
0.5
0.5
4
1
4
4
s–4u) with a moderate alkyl chain length (m = 6, 7, or 8) were
effective against the four sensitive strains (MIC in the range of 0.25–
μg/mL). For example, compounds 4s–4u showed high activity
4
s
0.5
0.25
1
0.5
1
1
340
4
t
1
34
2
4
u
1
2
14
28
against S. aureus (MIC in the range 0.25–0.5 μg/mL) and E. coli (MIC
in the range 0.5–1 μg/mL). The MIC values of these compounds (4a–
4
v
4
64
2
32
4
＞1280
132
＞320
264
4
w
0.5
64
16
8
1
4
u) increased with increasing chain length. Whereas the HC50 values
of these compounds (4a–4u) decreased with increasing chain length.
For example, the HC50 values of molecules 4d–4i were >1280, >1280,
5a
128
32
8
128
64
32
16
2
128
128
64
16
2
＞1280
＞1280
616
＞20
＞80
77
5
b
>
1280, 819, 287, and 45 μg/mL, respectively (Table 1). Compound
5
c
4
g (m=6; n=3) showed the best selectivity against S. aureus over
5
d
4
4
438
109.5
＞2560
812
erythrocytes.
5
e
0.5
0.5
0.5
1
4
＞1280
406
The length of the middle section has a great impact on antibacterial or
haemolytic activity. For example, compounds 4a, 4g, 4m, and 4s with
the same alkane chain length (m = 6) showed similar antibacterial
activity, while their haemolytic activities were different (>1280, 1219,
5
f
1
1
1
5g
5h
0.5
0.5
1
1
2
50
100
2
2
20
20
8
19, and 340 μg/mL, respectively), and compounds 4f, 4l, and 4r with
5
i
0.5
1
2
2
103
206
the same alkane chain length (m = 5) showed different antibacterial
activity (8, 2, and 0.5 μg/mL against S. aureus, respectively).
Compounds 4v and 4w with a branched chain were synthesized to
increase hydrophobicity. The antibacterial activity of 4v was slighter
higher than that of 4d. While the antibacterial activity of 4w was
5
j
1
2
4
21
21
V h
M i
1
-f
-j
-j
-j
-j
-j
-j
-j
-j
0.0625
-j
a
Minimum inhibitory concentration; b S. aureus; c E. faecalis; d E. coli; e S.
higher than that of 4f, suggesting that hydrophobicity has a positive maltophilia; f the concentration that causes 50% haemolysis of red blood cells
influence on activity. The antibacterial activity of molecules 5a–5d
was impaired compared with that of amphipathic compounds 4f–4i,
indicating that symmetry plays an important role in the antibacterial
activity. Molecules 5e–5j were synthesized linked by naphthalene-
RBCs); g Selectivity index, HC50/MICS. aureus; h vancomycin; meropenem; not
i
j
(
determined.
Stability in complex mammalian fluids is an important property that
limits the application of AMPs. Compound 4g was pre-incubated in
1
,6-diol or naphthalene-2,3-diol. In particular, compound 5e (MICS.
5
0% plasma for 2, 4, and 6 h at 37 °C. The minimum bactericidal
concentration (MBC) values of 4g were 2 μg/mL in control media and
μg/mL in plasma after different treatment times (Fig. S1a, ESI†).
aureus: 0.5 μg/mL) showed better antibacterial activity than 4f (MICS.
aureus: 8 μg/mL), and higher selectivity (HC50/MICS. aureus>2560) than
8
4
g (HC50/MICS. aureus>2438), indicating that introduction of the
The MBC value of compound 4g against MRSA was 32 μg/mL both
in 50% serum and 50% blood (Fig. S1b, ESI†). Compound 4g showed
better bactericidal activity in 50% plasma (8 μg/mL) than in other
solutions. The MBC and MIC values of 4g at different concentrations
of trypsin were evaluated and showed good activity (MBC: 8 μg/mL;
naphthalene nucleus had promising results. Compounds 5e, 5f, 5g,
and 5i showed excellent antibacterial activity (MICS. aureus: 0.5 μg/mL).
The MICs of compounds 4a, 4b, 4g, 4m, 4r, 4s, 5e, and 5f were
in the range of 0.5−4 μg/mL against 10 methicillin-resistant S.
aureus isolates (Table S1, ESI†). The MIC values of these
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J. Name., 2013, 00, 1-3 | 2
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MIC: 1 μg/mL at a trypsin concentration of 80 μg/mL) (Fig. S1c, Bacteria can form a protective biofilm that acts as a barrier against the
View Article Online
DOI: 10.1039/C9CC00857H1
ESI†). The above results indicated that 4g is stable in complex host immune response and provides resistance against antibiotics .
mammalian fluids and that incubation time has no effect on the The cell viability in biofilms decreased with an increasing
bactericidal activity in plasma.
concentration of compound 4g (Fig. 5a). Cell viability decreased to
3.00, 8.70, and 3.70 log10 CFU/mL at 4, 8, and 16 mg/mL,
1
The analysis of time-kill kinetics of optimized compound 4g against
S. aureus was carried out at different concentrations. For the
exponential phase strains, S. aureus could be removed completely by
treatment with compound 4g for 6 h at 3 or 4 μg/mL (Fig. 3a). For the
stationary phase strains, compound 4g removed S. aureus irreversibly
after 16 h treatment at 12 μg/mL (Fig. 3b). While the number of viable
bacteria treated with vancomycin was 10 CFU/mL after 24 h. These
results suggested the superiority of compound 4g over commonly
used antibiotics in killing bacteria. Photographs of the test strains
treated for 6 or 24 h are shown in Fig. S2 (ESI†).
respectively, and 0 log10 CFU/mL at 64 and 128 mg/mL. Whereas the
cell viability treated with buffer solution was 13.47 log10 CFU/mL.
The destructive activity of compound 4g on biofilm was visually
observed by crystal violet staining (Fig. 5b). The minimum biofilm
inhibitory concentrations (MBICs) and minimum biofilm eradication
concentrations (MBECs) were also evaluated (Table S2, ESI†). The
6
.7
7
0% minimal biofilm inhibit concentrations (MBIC70) of compound
4
g were 10.1 and 22.9 μg/mL against S. aureus and E. coli biofilms,
respectively. The MBECs of compound 4g were 8 μg/mL against S.
aureus and 16 μg/mL against E. coli. The MBIC and MBEC values
were in agreement with the results of the biofilm disruption test (Fig.
5
).
Fig 3 Time-dependent killing of pathogens by compound 4g. S. aureus was grown
to (a) exponential phase, and (b) stationary phase, challenged with compound 4g
or Van (vancomycin). The control was treated with sterile water.
Fig 345
Fig 6 FESEM images of cell membrane damage. (a) Untreated S. aureus; (b) S.
aureus treated with compound 4g at 16 μg/mL. Scale bar 1 μm.
The propensity of bacteria to develop drug resistance was assessed
through resistance selection studies after prolonged passage at sub-
inhibitory concentrations (0.5 × MIC). Only a small change was
detected in the MIC of compound 4g against both S. aureus and E.
coli after 16 passages. Whereas, in the case of norfloxacin and colistin,
the MICs started to increase after 1 and 7 days, increasing by a factor
of 256 and 32, respectively (Fig. 4). Thus, compound 4g had major
advantages over conventional antibiotics norfloxacin and colistin and
induced less bacterial resistance.
Field emission scanning electron microscopy (FESEM) images
showed that untreated bacteria retained well-defined morphology and
the smooth surface characteristic of bacteria (Fig. 6a). However, after
treatment with 4g for 3 h, the morphology of the cell membrane
became irregular (Fig. 6b). Red fragments indicated that cells had
been completely destroyed. Thus, the above results indicated that
compound 4g killed bacteria by disrupting their cell membranes.
The ability of compounds to depolarize the bacterial cell membrane
2
4
3
was investigated using Disc (5) . The fluorescence intensity was
significantly enhanced after treatment with selected compounds (Fig.
S3a, b, ESI†), indicating that these molecules were efficient in
depolarizing the cytoplasmic membranes of S. aureus and E. coli. The
membrane permeabilization of Gram-positive and Gram-negative
bacteria was monitored using the nuclear fluorescence dye PI
(propidium iodide). Compounds 4b, 4r, 4s, and 5f exhibited high
permeabilization of the S. aureus inner membrane (Fig. S3c, ESI†),
and compounds 4a, 4b, 4m, 5e, and 5f exhibited high
permeabilization of E. coli (Fig. S3d, ESI†). Thus, the results
Fig 4 Studies on the emergence of resistance in 4g toward (a) S. aureus (norfloxacin suggested that molecules permeabilize the inner membrane of both
as control) and (b) E. coli (colistin as control).
Gram-positive and Gram-negative bacteria. An outer membrane
permeabilization assay was performed using E. coli (Fig. S3e, ESI†).
All compounds tested enhanced the permeability of E. coli.
Compounds 4b, 4s, and 5f showed the highest activity with respect to
permeabilizing the outer membrane of E. coli.
Fig 5 Effect of 4g on bacterial biofilms. (a) Cell viability of S. aureus in biofilms
treated with compound 4g; (b) Images of S. aureus biofilms treated with 4g after
staining with crystal violet.
Fig 7 In vivo efficacy of compound 4g in a mouse model of MRSA-1115041
skin infection. (a) Bacterial counts of skin samples. P-values (*) were 0.041,
0
.0072, and 0.0106 for samples treated with 3.3 mg/kg/d 4g, 6.6 mg/kg/d 4g,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
and 3.3 mg/kg/d vancomycin (Van), compared with the control.
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The toxicity of the most promising compound, 4g, toward Hela cells 4.
A. Infectious Diseases Society of, America, Clin. InfecVti.ewDiAsr.t,ic2l0e1O0n,l5in0e,
081-1083.
DOI: 10.1039/C9CC00857H
I. Yelin and R. Kishony, Cell, 2018, 172, 1136-1136.
1
was evaluated. The electron scanning microscopy images of HeLa cell
5
6
.
.
morphology (Fig. S4b, c, ESI†) were unaltered after treatment with
molecule 4g compared with the control (Fig. S4a, ESI†). HeLa cells
were evaluated by staining with calcein AM and PI to visualize LIVE
J. W. Costerton, P. S. Stewart and E. P. Greenberg, Science, 1999, 284,
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g fluoresced green (Fig. S5d-I, ESI†), and the same phenomenon was 9.
4
10.
A. Menendez, R. B. Ferreira and B. B. Finlay, Nat. Immunol., 2010, 11,
9-50.
observed with the negative control (Fig. S5a–c, ESI†). The results
confirmed the less-toxic nature of the bacterial membrane-disrupted
compound 4g (concentration 64 × MICS. aureus).
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0.
1.
4
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A mouse model of MRSA skin infection was established to evaluate
2
5
the antibacterial activity in vivo (Fig. S6) . Kunming mice were 12.
purchased from the Animal Center of Zhengzhou University
1
3.
(Zhengzhou, China), and all animal procedures were performed in
accordance with the Guidelines for Care and Use of Laboratory
Animals of Zhengzhou University and approved by the Animal Ethics
1
4.
Committee of Zhengzhou University. Compound 4g showed similar 15.
activity at a dose of 6.6 mg/kg/d with vancomycin (3.3 mg/kg/d) (Fig. 16.
7
). The antibacterial activity of compound 4g was improved with an
increased dose. The skin tissue samples were imaged by haematoxylin
and eosin staining. In normal (healthy, uninfected) skin samples, the
1
7.
layers could be seen clearly (Fig. S7a, ESI†); while, MRSA-infected 18.
skin showed a serious inflammatory reaction. The epidermis separated
1
9.
from the dermis, and multifocal areas of inflammatory cell infiltration
of the dermis as well as the subcutaneous tissue were observed (Fig.
S7b, ESI†). The width of the skin was narrower with 4g-treated
2
0.
P. Bai, S. Qin, W. Chu, Y. Yang, D. Cui, Y. Hua, Q. Yang and E. Zhang,
Eur. J. Med. Chem., 2018, 155, 925-945.
sections (Fig. S7c, ESI†) than with the infected samples. 21.
Vancomycin-treated infected skin also showed minimal inflammation
J. Hoque, M. M. Konai, S. S. Sequeira, S. Samaddar and J. Haldar, J.
Med. Chem., 2016, 59, 10750-10762.
2
2.
Drug
Description:nafcillin,
https://www.rxlist.com/nafcillin-
(Fig. S7e, ESI†). These data indicated that 4g was efficient at reducing
drug.htm#indications).
MRSA infection in vivo in a mouse model.
2
3.
R. A. Allen, M. C. Jennings, M. A. Mitchell, S. E. Al-Khalifa, W. M.
Wuest and K. P. C. Minbiole, Bioorg. Med. Chem. Lett., 2017, 27, 2107-
2112.
M. Wu and R. E. Hancock, J. Biol. Chem., 1999, 274, 29-35.
N. Malachowa, S. D. Kobayashi, K. R. Braughton and F. R. Deleo,
Methods Mol. Biol., 2013, 1031, 109-116.
A series of amphiphilic molecules linked by an aromatic nucleus were
designed and synthesized as AMP-mimetics. Some compounds
showed high membrane selectivity (HC50/MICS. aureus of 4g and 5e 24.
2
5.
were 2438 and >2560, respectively) and excellent antibacterial
activity against a panel of bacteria including various multidrug-
resistant isolates. Mechanistic studies and SEM images indicated that
these compounds depolarized and permeabilized bacterial membranes,
leading to irregular cell morphology and the rapid death of bacteria.
Lead compound 4g possesses optimal attributes, such as stability in
plasma and trypsin, the ability to disrupt established S. aureus
biofilms, lower drug resistance, high in vivo efficacy against MRSA,
and no detectable toxicity. Overall, these results suggest that
amphiphilic molecules linked by an aromatic nucleus are highly
promising compounds for development into pharmaceuticals.
This research was supported the National Natural Science Foundation
of China (No. 21702190), the China Postdoctoral Science Foundation
(2018M640685), and Henan province (17A350004, 172102310227).
Conflicts of interest
There are no conflicts of interest to declare.
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DOI: 10.1039/C9CC00857H
Amphiphilic molecules linked by aromatic nucleus, possessing strong bactericidal
activity, high selectivity, less drug resistance, high in vivo efficacy against MRSA, were
developed.