Synthesis and Bioactivities of New Membrane-Active Agents with Aromatic Linker: High Selectivity and Broad-Spectrum Antibacterial Activity

Article abstract of DOI:10.1021/acsinfecdis.9b00078

The worldwide emergence of microbial resistance to antibiotics constitutes an important and growing public health threat, and novel antibiotics are urgently needed. In this report, a series of symmetrical membrane-active agents linked by an aromatic nucle

Full text of DOI:10.1021/acsinfecdis.9b00078

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Article
Synthesis and Bioactivities of New Membrane-active Agents with Aromatic
Linker: High Selectivity and Broad-Spectrum Antibacterial Activity
Wenchao Chu, yi yang, Jianfeng Cai, Hongtao Kong,
Mengmeng Bai, Xiangjing Fu, Shangshang Qin, and en zhang
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00078 • Publication Date (Web): 22 Jul 2019
Downloaded from pubs.acs.org on July 22, 2019
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Synthesis and Bioactivities of New Membrane-active Agents with Aromatic Linker:
High Selectivity and Broad-Spectrum Antibacterial Activity
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Wenchao Chu†, Yi Yang†, Jianfeng Cai‡, Hongtao Kong†, Mengmeng Bai†, Xiangjing
Fu†,§, Shangshang Qin†,§, En Zhang,* †,§
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† 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.
‡ 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.
Corresponding author: En Zhang (Email: zhangen@zzu.edu.cn)
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The worldwide emergence of microbial resistance to antibiotics constitutes an
important and growing public health threat, and novel antibiotics are urgently needed.
In this report, a series of symmetrical membrane-active agents linked by an aromatic
nucleus were designed and synthesized. Some showed high antibacterial activity
against clinical drug-resistant bacterial isolates including methicillin-resistant
Staphylococcus aureus (MRSA), carbapenemase-producing Enterobacter aerogenes
and delhi metallo-β-lactamase-1-producing Enterobacteriaceae (NDM-1), as well as
drug-sensitive bacteria including Staphylococcus aureus, Enterococcus faecalis,
Escherichia coli, and Stenotrophomonas maltophilia. Lead compound 2n, with good
selectivity for S. aureus (minimum inhibitory concentration [MIC] 0.25 μg/mL) versus
mammalian erythrocytes (hemolytic concentration [HC50] 1211 μg/mL), had notable
properties, including stability in complex mammalian fluids, rapid killing of pathogens,
ability to eradicate established biofilms, and little induction of bacterial drug-resistance.
In a mouse MRSA infection model, compound 2n exhibited a similar level of efficacy
to vancomycin in killing bacteria and suppressing inflammation, demonstrating its
therapeutic potential.
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KEYWORDS: Membrane-active Agents; Broad-Spectrum Antibacterial Activity;
Selectivity; Antibiotics; Structure-activity relationships；Low toxicity.
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High levels of antibiotic resistance leading to serious bacterial infections are a severe
global problem1-2. The World Health Organization has released a report about the crisis
in antibiotic resistance3-4 and encouraged global leaders to act5. The Infectious Disease
Society of America report that six bacteria cause two-thirds of nosocomial infections
in the USA (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and
Enterobacter species)6. Therapeutic options for these pathogens are limited and many
antibiotics are associated with significant resistance7. Unfortunately, the number of new
antibiotics approved by the US Food and Drug Administration continues to decline8.
Thus there is an immediate need for new agents with antimicrobial activity.
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To overcome bacterial resistance, antimicrobials with new mechanisms of action or
structure are urgently needed9. Traditional drugs inhibit bacterial growth by interfering
with metabolic pathways. However, these drugs appear to be susceptible to the
development of bacterial resistance. Bacterial resistance to antimicrobials has many
mechanisms10, including destruction or modification of the drug (hydrolysis, group
transfer, and redox mechanisms)11, target site modification and mutation (e.g.,
mutations in RNA polymerase, resulting in resistance to rifamycin)12, membrane
permeability barriers, and active efflux reducing the concentration of the drug at the
target site13. Bacterial biofilms also contribute to resistance and treatment failure14.
Membrane-targeting compounds are considered promising future antibiotics because of
the reduced capacity of bacteria to develop resistance mechanisms to such drugs15. Thus,
many compounds have been designed to target membrane covered by anionic
exopolymeric substances, including the membrane-targeting antibiotics polymyxin B
(Fig. 1), daptomycin and amphotericin B, which have been successfully applied in the
clinic.
Antimicrobial peptides (AMPs) are produced by many tissues and cell types in a variety
of plant and animal species16-17. AMPs are recognized as a promising source of
molecules and inspiration for the treatment of antibiotic-resistant bacteria18, because of
low bacterial resistance to AMPs and their broad spectrum antibacterial activity.
Although AMPs have diverse sequences, one hallmark is their amphiphilic properties
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that appear significant for antibacterial activity . AMPs associate with the anionic
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bacterial membrane through electrostatic interactions, and then hydrophobic residues
promote pore formation and cell death20, so many AMPs are defined as membrane-
active molecules19. The amphiphilic topologies of AMPs provide important information
for the design of synthetic alternatives. Peptoids21, β-peptides22-23, oligoureas24, and
AApeptides25-26 showed potent and broad-spectrum activity against multidrug-resistant
bacterial pathogens. Additionally, cationic steroid antibiotics (such as CSA-13) (Fig.
1)27, cationic AMP-mimics28-29 and amphiphilic xanthone derivatives30-31 have attracted
considerable attention for their antibacterial activities.
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H2N
O
N
O
H
H2N
O
NH
N
H
O
OH
O
NH
H
H
O
O
O
H
N
H
H2N
O
NH2
N
HN
NH2
N
N
H
H
O
O
NH
HN
O
CSA-13
H
NH2
NH2
N
O
NH2
Br
O
H
N
H
H
H
Br
HO
C8H17
N
N
N C8H17
N
N
4
O
O
O
O
Ploymixin B
O
II
Cl
O
Br
Br
O
H
N
N
C6H13
N
O
N
N
N
H
C6H13
H
O
III
I
Figure 1. Structures of membrane-targeting antibiotics and antimicrobial peptide
mimics.
O
O
H
H
N
O
N
H
N
S
S
O
S
O
O
N
O
N
N
O
O
O
OH
OH
O
OH
O
O
Methicillin
Nafcillin
Penicillin V
Br
nN
m
m
N
m
Br
N
O
N
m
O
n
m
O
N
O
O
Br
Br
Br
Figure 2. Design of novel membrane-active molecules.
During the course of our recent investigations into new structures with inhibitory
activity toward metallo-β-lactamase32-33, we discovered several molecules that have
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excellent antimicrobial activity against drug-resistant strains34-36. Chalcone derivatives
I (Fig. 1), amphiphilic compounds with a 4-methoxy benzene substituent and moderate
length of the lateral chain (eight carbon atoms), showed antibacterial activity ( MIC in
the range 0.5–2 μg/mL Table1) against S. aureus, E. faecalis, E. coli, and S. enterica,
and moderate hemolytic activities. The structure–activity relationship of these
molecules illustrated that the phenoxy and hydrophobic moieties played a significant
role in the specificity toward bacteria rather than mammalian cells. Additionally, we
were inspired by Haldar’s major breakthrough in cationic small molecules active
against MRSA 29, 37 (molecules II and III in Fig. 1), as well as the presence of a phenoxy
group in a variety of drugs such as the antibacterials methicillin, nafcillin, penicillin V
(Fig. 2). In view of the above considerations and to develop our previous work, we
sought to find effective antibacterial peptide mimics with low toxicity.
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In the present report, symmetrical compounds linked by an aromatic nucleus were
synthesized. The net positive charge positioned on the scaffold interacts with anionic
exopolymeric substances on the surface of membrane. Some compounds in this series
showed lower toxicity and higher selectivity (e.g., compound 2n, selectivity index 4844
against S. aureus over erythrocytes) than those previously reported29, 38-40. Further
biological characterization of selected compounds showed their stability in mammalian
fluids and rapid bactericidal ability; no obvious drug-resistance was induced, and they
eradicated bacterial biofilms. Compound 2n, which had the best antibacterial activity,
showed an equal effect to that of vancomycin in a mouse model of MRSA skin infection.
Results and Discussion
Synthesis and Characterization.
Membrane-active symmetrical molecules were synthesized in a very straightforward
protocol, as shown in Scheme 1. The desired products 2a–2r with modifications of the
length of the alkyl chains (m) and the length of the middle (n) were obtained in good
yield (32%-87%) from 1a-1e and variouss N, N-dimethylalkylamines. To determine
the influence of symmetry on the activity, unilateral compounds 3a–3d (Yield 68%-
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83%) were synthesized and compared with the symmetrical compounds. We also
attempted to replace the hydroquinone linker with various aromatic nuclei, compounds
3e–3l (Yield 23%-80%) were obtained from the reactions of 1f–1j and various N, N-
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dimethylalkylamines. All the products were characterized by H NMR, 13C NMR,
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HPLC and mass spectroscopy.
Br
O
2
HO
O
2
1
4
O
nBr
a
b
6
5
2
3
Br
O
HO
OH
2
Br
2
n
O
O
OH
1,4-diether:
1b: n = 3; 1c: n = 4;
1d: n = 5; 1e: n = 6;
1,3-diether: 1f: n = 5;
1,2-diether: 1g: n = 5;
1a
Br
b
1
4
2
3
O
O
HO
OH
6
5
Br
1,6-diether: 1h
2,3-diether: 1i
1,5-diether: 1j
N
O
n
c
m
Br
nN
1a-1e
N
+
m
m
Br
O
2a: m = 5, n = 2; 2b: m = 7, n = 2;
2c: m = 9, n = 2; 2d: m = 11,n = 2;
2e: m = 5, n = 3; 2f: m = 7, n = 3;
2g: m = 9, n = 3; 2h: m = 11, n = 3;
2i: m = 5, n = 4; 2j: m = 7, n = 4;
2k: m = 9, n = 4; 5l: m = 11, n = 4;
2m: m = 5, n = 5; 2n: m = 7, n = 5;
2o: m = 9, n = 5; 2p: m = 11, n = 5;
2q: m = 8, n = 4; 2r: m = 6, n = 6
c
+
Br
N
m
m
O
Br
O
N
3a: m = 5; 3b: m = 7; 3c: m = 9; 3d: m = 11
c
m
1
2
m
1h-1j
N
+
O
N
N
O
3
6
m
5
4
Br
Br
1,6-diether: 3e: m = 5; 3f: m = 7; 3g: m = 9
2,3-diether: 3h: m = 7; 3i: m = 9
1,5-diether: 3j: m = 7
1
7
Br
c
2
3
1f,1g
N
+
O
N
N
O
7
7
Br
1,3-diether: 3k
1,2-diether: 3l
Scheme 1. Synthesis of membrane-active derivatives 2a–2r and 3a–3l.
Reaction conditions: a) CBr4, PPh3, CH3CN, rt, 4 h; b) Br(CH2)nBr, K2CO3, acetone,
reflux, 24 h; c) EtOH, 85 °C, pressure tube, 24 h.
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In Vitro Antibacterial and Hemolytic Activities of Synthesized Compounds
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Table 1. MIC and HC50 of molecules I, II, III, membrane-active derivatives and
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commercial antibiotics.
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MIC (μg/mL) [c]
[h]
Compd. m[a] n[b]
HC50
SI [i]
S. a [d] E. f [e] E. c [f]
S. m [g]
ND
ND
ND
64
1
I35
II37
III29
1
2
ND [l]
ND
ND
128
4
4
4
124
255
124
127.5
805
1
2
805
2a
2b
2c
2d
2e
2f
5
7
2
2
2
2
3
3
3
3
4
4
4
4
5
5
5
5
4
6
3
3
3
32
0.5
0.5
1
64
0.5
1
＞1280
＞40
＞1280 ＞2560
9
0.25
1
0.5
2
108
31
216
31
11
5
1
16
1
128
4
64
1
32
0.5
0.5
4
＞1280
＞80
7
＞1280 ＞1280
2g
2h
2i
9
0.25
1
0.5
1
0.5
4
231
1
924
1
11
5
2
16
4
8
16
2
＞1280
＞640
2j
7
1
2
＞1280 ＞1280
2k
2l
9
0.5
1
0.25
0.25
2
1
1
149
1
298
1
11
5
1
2
2m
2n
2o
2p
2q
2r
3a
3b
3c
2
4
16
1
＞1280
1211
94
＞640
4844
188
0.5
7
0.25
0.5
2
0.5
1
0.5
1
9
1
11
8
0.5
0.5
1
2
8
1
0.5
0.5
64
16
2
1
1
434
868
6
2
1
＞1280 ＞2560
5
128
32
4
128
64
8
＞128
128
16
＞1280
＞1280
295
＞20
＞80
147.5
7
9
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3d
3e
11
5
3
3
3
3
3
3
3
5
5
0.5
8
0.5
64
1
2
4
64
2
70
＞1280
1018
34
140
＞160
2036
68
64
3f
7
0.5
0.5
1
2
3g
9
0.5
2
1
2
10
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3h
7
4
4
592
12
592
12
3i
9
1
1
1
2
3j
7
0.5
0.5
1
1
2
1
2
265
409
473
ND
530
818
473
ND
ND
3k
7
1
1
3l
7
4
4
4
VAN[j]
MEM[k]
1
ND
ND
ND
0.0625
ND
ND
ND
ND
[a] the number of the CH2 in the side chain; [b] the number of the CH2 in the middle
chain; [c] Minimum inhibitory concentration; [d] Staphylococcus aureus; [e]
Enterococcus faecium; [f] Escherichia coli; [g] Stenotrophomonas maltophilia; [h] The
concentration that causes 50% hemolysis of red blood cells; [i] Selectivity index,
HC50/MICS. aureus; [j] Vancomycin; [k] Meropenem; [l] Not determined. All
experimental data was repeated three times.
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Figure 3. MIC values of molecules 2a–2p against S. aureus and HC50 values.
The membrane-active derivatives were tested for their antimicrobial activity against
Gram-positive bacteria S. aureus and E. faecalis, Gram-negative bacteria E. coli and S.
maltophilia, and drug-resistant bacteria including MRSA, carbapenemase-producing
Enterobacter aerogenes (blaKPC), and NDM-1-producing Enterobacteriaceae (blaNDM-
1). The antibacterial efficacy is expressed in terms of the MIC that was required to
inhibit the growth of the bacteria. In general, the compounds (except for 2a, 2e, 3a, 3b
and 3e) displayed antibacterial potency against both Gram-positive and Gram-negative
bacteria. Some compounds showed activities that were comparable to that of the
glycopeptide antibiotic vancomycin against S. aureus.
Data in Table 1 and Figure 3 allow us to perform structure–activity relationship analysis
of the length of the alkyl chains (m), the length of the middle (n), the influence of
naphthalene/benzene modifications, and symmetry of the drug molecule. The length of
the alkyl chains was varied between hexyl chain and dodecyl chain (hexyl chain [m =
5] to dodecyl chain [m = 11]). When the compounds are grouped by the value of m,
those with m = 5, i.e. 2a, 2e, 2i, 2m, had the worst MIC values. Compounds 2b, 2c, 2f,
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2g, 2j, 2k, 2n and 2o (m = 7 or 9) showed good antibacterial activity. However, long
alkyl chains markedly increased the hemolytic activity toward red blood cells, notably
on going from octyl chain (m = 7) to decyl chain (m = 9): the HC50 values of compounds
2b, 2f, 2j, and 2n (all m = 7) were >1280, >1280, >1280, and 1211 μg/mL, respectively,
while those of 2c, 2g, 2k, and 2o (m = 9) were 108, 231, 149, and 94 μg/mL,
respectively. Thus, octyl chain was identified as the optimal side chain, essentially
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consistent with our previous research34-35
.
To understand the effect of the length of the middle (n) on the antibacterial properties
of the compounds, the length was changed from two to five carbon atoms. The change
of this alkyl-linker (n) had little effect on the antibacterial profile. For instance,
compounds 2b, 2f, 2j, and 2n had similar activities to each other, as did 2c, 2g, 2k, and
2o, and 2d, 2h, 2l, and 2p (Figure 3). Compound 2n (m = 7, n = 5) showed higher
antibacterial activity and similar hemolytic activity compared with compounds 2b (m
= 7, n = 2), 2f (m = 7, n = 3), and 2j (m = 7, n = 4), leading to the conclusion that 2n
(HC50/MIC S. aureus: 4844) was the optimal molecule.
Amphipathy contributes significantly to the activity of AMPs as electrostatic
interactions are important for destruction of microbial membranes. To discern the
impact of symmetry on the amphipathy and activity of the test compounds, the
antibacterial activities of compounds 3a, 3b, 3c, and 3d were evaluated (Table 1).
Compounds 3c and 3d showed good antibacterial activities but were highly hemolytic.
Compounds 3a and 3b showed low hemolytic activity, but their antibacterial activities
were poor. These four compounds thus showed a poor balance between antimicrobial
activity and toxicity toward mammalian cells. The symmetry of the compounds in series
2a–2p may contribute to their desirable combination of high antibacterial activity and
low toxicity.
The aromatic linker in 2e–2g was replaced with naphthalene in compounds 3e–3j.
Compound 3e showed poor antibacterial activity, while compounds 3f–3j showed good
activity (Table 1). Indeed, compounds 3e and 3f (HC50/MIC S. aureus: 2036) showed
improved activity compared with 2e and 2f, respectively. Compounds 3f showed
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similar activity but lower hemolytic compared with 3h and 3j, indicating that the
introduction of the 1,6-diether naphthalene nucleus had promising results. Compounds
2g, 2q, 2n and 2r showed good activity with the same total carbon (12), but the values
of HC50 were increased with the increase of the middle (n). Compounds 2n showed
better activity and lower hemolytic compared with 3k and 3l with the different
substituted benzene, indicating para-substitution could be helpful to the activity.
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Compounds with high selectivity and good antibacterial activity (2b, 2f, 2j, 2n, 3f and
3h) were selected for further evaluation against clinical isolates of drug-resistant
bacteria (Table 2). All the compounds retained much of their activity against clinical
MRSA isolates (10 strains, MIC 0.25–4 μg/mL). Some compounds also showed good
activity toward NDM-1-producing Enterobacteriaceae and carbapenemase-producing
E. aerogenes.
Table 2. MICs of selected compounds against clinical drug-resistant bacterial isolates
MIC (μg/mL)
Strains
2b
1
0.5
0.5
2
2
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1
2f
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1
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4
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2n
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3h
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1
1
(G+) [a] MRSA-1114044
(G+) MRSA-1115041
(G+) MRSA-1202610
(G+) MRSA-1202628
(G+) MRSA-20130910162
(G+) MRSA-20130911034 sall
(G+) MRSA-831164
0.5
0.5
0.5
1
1
0.5
4
0.25
0.5
1
4
1
1
1
1
1
4
0.5
0.5
1
0.5
0.5
1
1
1
1
(G+) MRSA-826084
2
(G+) MRSA-905132
2
4
4
1
1
1
(G+) MRSA-904142
0.5
0.5
0.5
0.5
0.5
1
(G-) [b] K. peneumoniae-13-10
2
4
4
2
4
4
(blaNDM-1
)
(G-) E. coli-13-22 (blaNDM-1
(G-) E. coli-13-33 (blaNDM-1
(G-) C. freundii-13-61 (blaNDM-1
(G-) E. cloacae-14-56 (blaNDM-1
(G-) E. aerogenes-15-92 (blaKPC
)
)
2
1
1
1
1
16
2
2
2
2
8
2
4
2
2
2
1
1
1
1
4
2
2
2
2
8
4
8
16
4
)
)
)
[a] (G+): Gram positive bacteria; [b] (G-): Gram negative bacteria. All experimental
data was repeated three times.
Plasma Stability and Bactericidal Activity in Mammalian Fluids
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Figure 4. The stability and bactericidal activity of compound 2n. (a) Compound 2n
was incubated in 50% plasma for different times, then its bactericidal activity against
S. aureus was evaluated; (b) Compound 2n was also evaluated against MRSA strain
1115041 in different culture media (50% plasma, 50% serum and 50% blood). All
experimental data was repeated three times.
Drugs are very likely to interact with proteins because of their physicochemical
properties41. The interaction of drugs with proteins can reduce their free concentration
in plasma and thus decrease their antibacterial efficacy. An indirect approach for
evaluating the antibacterial stability of a molecule is to determine its MBC (minimum
bactericidal concentration) value in the presence of mammalian fluids. S. aureus was
completely killed by 1 μg/mL of compound 2n in Mueller Hinton broth (MHB). The
MBC value of compound 2n increased slightly to 8 µg/mL after interaction with 50%
plasma in MHB for 2 or 4 h (Fig. 4a). The susceptibility of MRSA to compound 2n
was tested in MHB diluted with 50% plasma, 50% serum or 50% blood. The addition
of plasma, serum or blood to MHB increased the MBC value of compound 2n 16, 32,
and 32-fold, respectively, relative to that in MHB alone (Fig. 4b). Mammalian fluids
can reduce, but not eliminate, the efficacy of compound 2n.
Bactericidal Time-Kill Kinetics
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Figure 5. Time-kill kinetics of pathogens by compound 2n. (a) S. aureus early
exponential phase, challenged with compound 2n (at 2, 3, 4 μg/mL) or vancomycin
(Van, 3 μg/mL); (b) S. aureus late exponential phase, challenged with compound 2n (at
6, 8, 12 μg/mL) or Van (8 μg/mL); (c) E. coli early exponential phase, challenged with
compound 2n (at 2, 3, 4 μg/mL) or moxalactam (Mox, 3 μg/mL); (d) E. coli late
exponential phase, challenged with compound 2n (at 6, 8, 12 μg/mL) or Mox (8 μg/mL).
The control was treatment with sterile water. The detection limit of the experiment was
≥ 102 CFU/mL.
Time-kill curves were constructed to probe the bactericidal effect of compound 2n.
Bacteria were grown in the presence of 2n or, as controls, vancomycin (S. aureus) or
moxalactam (E. coli). Samples were taken and plated at 0.5, 1, 2, 3, 4, 5, 6, 16, 18, 20
and 24 h. The time-kill kinetics of 2n showed similar trends to those of the control
antibiotics when the bacteria were in the early exponential phase (Fig. 5a, c).
Compound 2n (2, 3, 4 μg/mL) or vancomycin (3 μg/mL) could clearly kill S. aureus
after treatment for 5 or 6 h. early exponential phase E. coli could be completely killed
by compound 2n (3, 4 μg/mL) or moxalactam (3 μg/mL) after treatment for 4 h.
In the late exponential phase, the initial concentrations of S. aureus and E. coli were
approximately 106 colony-forming units (CFU)/mL and 108 CFU/mL respectively. Cell
viability decreased to about 104 CFU/mL after treatment with 8 μg/mL vancomycin (S.
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aureus) or moxalactam (E. coli) for 24 h. However, the bacteria could be eliminated by
exposure to 2n (6, 8 or 12 μg/mL), thus 2n was superior to vancomycin and moxalactam
in killing efficiency in these conditions. Photographs showing the results of S. aureus
and E. coli treatment with 2n and the control antibiotics are shown in Figure S1.
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Multipassage Resistance Selection
Figure 6. Emergence of resistance studies for 2n tested with S. aureus and E. coli. (a)
Compound 2n against S. aureus, where the antibiotic norfloxacin was used as the
control. (b) Compound 2n against E. coli, where the lipopeptide colistin was used as
the control. All experimental data was repeated three times.
Bacteria cannot easily develop resistance to AMPs because AMPs collapse bacterial
membrane. To evaluate the probability of compound 2n eliciting resistance in bacteria,
S. aureus and E. coli were exposed to subinhibitory concentrations (0.5 × MIC) of 2n,
and as controls norfloxacin (S. aureus) and the lipopeptide colistin (E. coli), during 16
successive culture. As shown in Figure 6, the MICs of 2n against both bacteria
remained very similar up to the end of the experiment. In contrast, the MIC values of
norfloxacin and lipopeptide colistin increased by 256- and 32-fold, respectively. Thus,
compound 2n did not easily induce resistance in bacteria.
Activity of Disrupting Biofilms
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Figure 7. Images of E. coli biofilms stained with crystal violet after treatment with 2n
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at different concentrations.
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Table 3. Minimum biofilm inhibitory concentration (MBIC) and minimum biofilm
eradication concentration (MBEC) of compound 2n against S. aureus and E. coli.
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Biofilm activity (μg/mL)
Strains
MBIC50 MBIC70 MBIC80 MBEC
S. aureus
E. coli
1.7
11.2
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51.8
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Pathogens can grow in matrix-encased communities called biofilms. Biofilms make
microorganisms more resistant to antibacterial treatment, the human immune system,
42
and harsh environments . One of the current strategies to prevent the formation of
biofilms is treatment with AMPs. These molecules bind electrostatically with
negatively-charged lipopolysaccharide (LPS) and disrupt the biofilms. To test the
properties of the AMP-mimic 2n toward biofilms, in-depth analyses were performed.
Biofilm formation was quantified by crystal violet (CV) microtiter plate assay, the color
value showed an obvious trend as the concentration of compound 2n increased (Fig. 7).
The data show that 2n has the ability to kill the bacteria in biofilm (Fig. S2). Minimum
biofilm inhibitory concentrations (MBIC) and minimum biofilm eradication
concentrations (MBEC) were also evaluated (Table 3). S. aureus biofilms proved to be
highly sensitive to the eradication activities of 2n (MBIC50 = 1.7 μg/mL; MBEC = 4
μg/mL); compound 2n also showed good activity against E. coli biofilms (MBIC50 =
10.7 μg/mL; MBEC = 16 μg/mL). Thus, 2n is able both to inhibit the formation of, and
to disintegrate, biofilms.
Scanning Electron Microscopy Study of Cell Integrity
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Figure 8. Field Emission Scanning Electron Microscope images of cell membrane
damage. (a) Untreated S. aureus; (b) S. aureus treated with compound 2n at 16 μg/mL
for 3h; (c) Untreated E. coli; (d) E. coli treated with compound 2n at 16 μg/mL for 3h.
Next, we addressed the effect of 2n on bacterial cell morphology and integrity.
Scanning electron microscopy images were collected after exposure of S. aureus and
E. coli to compound 2n for 3h (Figure 8). The interaction of 2n with the cells resulted
in major destruction of the S. aureus and E. coli integrity (Fig. 8b, d). whereas normal
bacterial morphology was observed for untreated S. aureus and E. coli. These images
are visual proof of the membrane damaging properties of compound 2n.
Mechanism Study
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Figure 9. Mechanism of antibacterial action of membrane-active derivatives. (a), (b)
Cytoplasmic membrane depolarization of S. aureus and E. coli, respectively, at 10
μg/mL test compound. (c) Inner membrane permeabilization of E. coli, respectively, at
10 μg/mL test compound. (d) Outer membrane permeabilization of E. coli, at 10 μg/mL
test compound. The control was treatment with sterile water.
Figure 9 shows that compounds produced in this work acted antimicrobially by
targeting bacterial cell membranes, depolarizing the cytoplasmic membrane and
permeabilizing the inner/outer membrane. Cytoplasmic membrane depolarization was
assessed using the membrane potential-sensitive dye 3,5′-dipropylthiacarbocyanine
iodide (DiSC3-5). DiSC3-5 distributes between the cells and the medium depending on
the membrane potential gradient; the fluorescence intensity increases if there is a loss
in membrane potential. Treatment of S. aureus with compounds 2n, 2f, and 3f led to
dramatically increased fluorescence. Meanwhile, treatment of S. aureus with
compounds 2b, 2j, 3h led to a moderate increase in fluorescence intensity (Fig. 9a). All
the compounds depolarized the membrane of E. coli, with 2n having the greatest effect
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(Fig. 9b). Taken together, these data suggest that the test compounds kill bacteria via
membrane depolarization.
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Amphipathic AMPs generally act by causing pore formation in cell membranes or
disrupting bacterial membranes. Permeabilization of the membrane of S. aureus and E.
coli by compounds developed in this work was tested by the propidium iodide (PI)
uptake assay. The dye PI only enters cells with broken membranes, and it fluoresces
upon binding to nucleic acids. Figure 9c shows the strong ability of compound 2f and
2n to permeabilize the E. coli inner membrane; the effect of the other compounds was
moderate. Compounds 3f and 3h showed a significant capacity to permeabilize the S.
aureus membrane (Fig. S3).
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Outer membrane permeabilization refers to disruption of membrane integrity, which
facilitates the uptake of antibiotics. The possibility of the test compounds affecting
outer membrane permeabilization was investigated by a specific fluorescence assay
using 1-N-phenyl-naphthylamine (NPN). NPN fluoresces strongly only in a
hydrophobic environment like the interior of a membrane, but weakly in aqueous
environments. The Gram-negative bacteria E. coli was exposed to test compounds at
10 μg/mL. All the compounds caused a time-dependent rise in fluorescence intensity
due to increased membrane permeabilization (Fig. 9d). The rise in fluorescence signal
was greatest for compounds 3f, 3h, 2b and 2n. These results indicated that all the
compounds permeated the E. coli outer membrane, but to different extents.
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Figure 10. Confocal microscopy images of S. aureus and E. coli after treatment with
2n (16 μg/mL) for 3 h and staining with DAPI and PI. (a–b) Non-treated S. aureus; (c–d)
S. aureus treated with 2n; (e–f) Non-treated E. coli; (g–h) E. coli treated with 2n. Scale
bar 5 μm.
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To evaluate the influence of 2n on the bacterial membranes, fluorescent dyes 4’,6-
diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were used to visualize the
influence of compound 2n on membranes of S. aureus and E. coli. DAPI could stain
membranes of live bacteria and PI could only stain DNA in the broken membranes.
The red fluorescence shown in Figure 10d and 10h indicated that the membranes of S.
aureus and E. coli were impaired, after incubation with 2n for 3 h.
Electron Scanning and Fluorescence Microscopy Analysis of HeLa Cells
Figure 11. Electron scanning microscopy images of HeLa cells after treatment with
compound 2n for 24 h. (a) Non-treated cells (negative control). Cells treated with 2n at
(b) 64× the MIC and (c) 16× the MIC of S. aureus. (d) Cells treated with 0.1% Triton
X (positive control). Scale bar 200 μm.
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Figure 12. Fluorescence microscopy images of HeLa cells after treatment with 2n for
24 h and staining with calcein AM and propidium iodide (PI). (a–c) Non-treated cells
(negative control). (d–f) Cells treated with 2n (64× the MIC of S. aureus). (g–i) Cells
treated with 2n (16× the MIC of S. aureus). (j–l) cells treated with 0.1% Triton-X
(positive control). Scale bar 200 μm.
The toxicity of compound 2n toward HeLa cells was investigated. HeLa cells were
imaged by optical microscopy to visualize their morphology after incubation with
compound 2n at 64× and 16× the MIC toward S. aureus, respectively. Treated cells
were intact (Fig. 11b, c) and showed similar morphology to untreated cells (Fig. 11a).
In contrast, cells treated with Triton-X were found to have damaged cell morphology
(Fig. 11d). The LIVE/DEAD staining method was used to visualize cells treated with
compound 2n. Untreated HeLa cells (Fig. 12a, b, c) showed green fluorescence,
indicating most of the cells were alive. Cells treated with 2n at different concentrations
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also showed green fluorescence (Fig. 12d, e, f, g, h, i). In contrast, cells treated with 0.1%
Triton-X only showed red fluorescence, illustrating that Triton-X was highly toxic to
the HeLa cells. This in vitro cytotoxicity study showed that 2n has no obvious effect
on HeLa cell viability at 64× the MIC toward S. aureus after treatment for 24 h.
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In Vivo Toxicity and Antibacterial Activity
Figure 13. In vivo efficacy of compound 2n in a mouse model of MRSA-1115041 skin
infection. (a) The process of the experiment. (b) Bacterial counts of treated and
untreated infected skin samples. P-values (*) were 0.0083, 0.0003, 0.0106 for samples
treated with 3.3 mg/kg/d 2n, 6.6 mg/kg/d 2n, and 3.3 mg/kg/d vancomycin compared
with the control.
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Figure 14. Pathological assay based on hematoxylin and eosin staining. (a) Untreated
skin; (b) Infected skin without any antibacterial treatment; (c), (d) Infected skin treated
with 6.6 mg/kg/d and 3.3 mg/kg/d 2n, respectively; (e): Infected skin treated with 3.3
mg/kg/d vancomycin. Scale bar 50 μm.
The therapeutic capacity of compound 2n was evaluated in a mouse model of MRSA
skin infection (Fig. 13a)43. It was found that 2n had excellent antibacterial activity. Skin
and soft tissue infections are common and easily lead to deep-seated infection44. MRSA
is associated with significantly high morbidity and mortality rates45.
First, acute dermal toxicity of 2n was assessed. The treated mouse showed no sign of
irritation or unnatural behavior when the applied concentration of 2n was <1500 μg/mL.
Cyclophosphamide was used to reduce the neutrophile granulocyte of mouse before
infection with 60 μL of ~109 CFU/mL MRSA. After inoculation of MRSA for 24 h, the
antibacterial agents were injected subcutaneously once with 12 h intervals. The number
of viable bacteria was quantified after last treatment. Compound 2n performed well
cure effect at both doses (Fig. 13b, 6.6 mg/kg/d and 3.3 mg/kg/d). Indeed, the curative
effect of vancomycin was less than that of 2n at dose 3.3 mg/kg/d. The antibacterial
activity of 2n showed a dose-dependent relationship. A significant difference in MRSA
count was observed between the 2n-6.6 mg/kg/d-treated group and the vancomycin-
treated group (p = 0.0053).
The infected skin regions were subjected to histological (hematoxylin and eosin)
staining. In normal (healthy, uninfected) skin samples, the layers can be seen clearly
(Fig. 14a). Compared with this control, MRSA-infected skin showed a serious
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inflammatory reaction. The epidermis separated from the dermis, and multifocal areas
of inflammatory cell infiltration of the dermis as well as the subcutaneous tissue were
observed (Fig. 14b). In contrast, 2n-treated sections showed minimal inflammation and
a narrower width of the skin than in the infected sample, especially for the high-dose
2n group (Fig. 14c). Vancomycin-treated infected skin is shown in Fig. 14e. The data
indicate that 2n was more efficient than vancomycin in reducing MRSA infection in
this in vivo mouse model.
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Conclusion
A series of membrane-active compounds with several aromatic linkers was designed
and synthesized. Compound 2n was identified as a potent agent with antibacterial
activity (MIC 0.25–1 μg/mL against S. aureus, E. coli, E. faecalis and S. maltophilia)
and low toxicity (HC50 toward red blood cells = 1211 μg/mL; selectivity index = 4844).
The aromatic linker and the length of the alkyl chains were key factors in the
antibacterial potency of the membrane-active compounds in this work. Compounds 2b,
2f, 2j, 2n, 3f, 3h showed good activity toward MRSA (MIC 0.25–4 μg/mL), NDM-1-
producing Enterobacteriaceae, and carbapenemase-producing Enterobacter aerogenes.
Mechanistic studies suggest that the antibacterial mechanism of action of these
compounds involves depolarization of the cytoplasmic membrane and permeabilization
of the inner/outer membrane, leading to bacterial death. Leading compound 2n
possesses optimal attributes, such as stability in plasma; the ability to disintegrate
established E. coli biofilms; little induction of drug resistance in treated bacteria; high
in vivo efficacy against MRSA; and no obvious observed toxicity. These symmetrical
compounds with aromatic linkers could be beneficial for treatment of infections
induced by drug-resistant bacteria such as MRSA.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website
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at DOI:
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General procedure for the synthesis compounds 1a-1j, 2a-2r, 3a-3l; Detail
characterization (Yield, NMR, HR-MS) of compounds 1a-1j, 2a-2r, 3a-3l; Detail
description of the all biological experiment (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: zhangen@zzu.edu.cn.
ORCID
En Zhang: 0000-0002-4818-756X
Jianfeng Cai: 0000-0003-3106-3306
Notes
There are no conflicts of interest to declare.
ACKNOWLEDGEMWNT
Project supported by the National Natural Science Foundation of China (No.
21702190), the China Postdoctoral Science Foundation (2018M640685, 001803014,
2019T120642) and Henan province (17A350004).
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Synthesis and Bioactivities of New Membrane-active Agents with Aromatic Linker:
High Selectivity and Broad-Spectrum Antibacterial Activity
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