Meta -Substituted benzenesulfonamide: A potent scaffold for the development of metallo-β-lactamase ImiS inhibitors

Article abstract of DOI:10.1039/c9md00455f

Metallo-β-lactamase (MβL) ImiS contributes to the emergence of carbapenem resistance. A potent scaffold, meta-substituted benzenesulfonamide, was constructed and assayed against MβLs. The twenty-one obtained molecules specifically inhibited ImiS (IC₅₀ = 0.11-9.3 μM); 2g was found to be the best inhibitor (IC₅₀ = 0.11 μM), and 1g and 2g exhibited partially mixed inhibition with K_i of 8.0 and 0.55 μM. The analysis of the structure-activity relationship revealed that the meta-substitutes improved the inhibitory activity of the inhibitors. Isothermal titration calorimetry (ITC) assays showed that 2g reversibly inhibited ImiS. The benzenesulfonamides exhibited synergistic antibacterial effects against E. coli BL21 (DE3) cells with ImiS, resulting in a 2-4-fold reduction in the MIC of imipenem and meropenem. Also, mouse experiments showed that 2g had synergistic efficacy with meropenem and significantly reduced the bacterial load in the spleen and liver after a single intraperitoneal dose. Tracing the ImiS in living E. coli cells by RS at a super-resolution level (3D-SIM) showed that the target was initially associated on the surface of the cells, then there was a high density of uniform localization distributed in the cytosol of cells, and it finally accumulated in the formation of inclusion bodies at the cell poles. Docking studies suggested that the sulfonamide group acted as a zinc-binding group to coordinate with Zn(ii) and the residual amino acid within the CphA active center, tightly anchoring the inhibitor at the active site. This study provides a highly promising scaffold for the development of inhibitors of ImiS, even the B2 subclasses of MβLs.

Full text of DOI:10.1039/c9md00455f

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RESEARCH ARTICLE
meta-Substituted benzenesulfonamide: a potent
scaffold for the development of metallo-β-
lactamase ImiS inhibitors†
Cite this: DOI: 10.1039/c9md00455f
*
Ya Liu, Cheng Chen, Le-Yun Sun, Han Gao, Jian-Bin Zhen and Ke-Wu Yang
Metallo-β-lactamase (MβL) ImiS contributes to the emergence of carbapenem resistance. A potent
scaffold, meta-substituted benzenesulfonamide, was constructed and assayed against MβLs. The twenty-
one obtained molecules specifically inhibited ImiS (IC50 = 0.11–9.3 μM); 2g was found to be the best
inhibitor (IC50 = 0.11 μM), and 1g and 2g exhibited partially mixed inhibition with Ki of 8.0 and 0.55 μM. The
analysis of the structure–activity relationship revealed that the meta-substitutes improved the inhibitory
activity of the inhibitors. Isothermal titration calorimetry (ITC) assays showed that 2g reversibly inhibited
ImiS. The benzenesulfonamides exhibited synergistic antibacterial effects against E. coli BL21 (DE3) cells
with ImiS, resulting in a 2–4-fold reduction in the MIC of imipenem and meropenem. Also, mouse
experiments showed that 2g had synergistic efficacy with meropenem and significantly reduced the
bacterial load in the spleen and liver after a single intraperitoneal dose. Tracing the ImiS in living E. coli cells
by RS at a super-resolution level (3D-SIM) showed that the target was initially associated on the surface of
the cells, then there was a high density of uniform localization distributed in the cytosol of cells, and it
finally accumulated in the formation of inclusion bodies at the cell poles. Docking studies suggested that
the sulfonamide group acted as a zinc-binding group to coordinate with ZnIJII) and the residual amino acid
within the CphA active center, tightly anchoring the inhibitor at the active site. This study provides a highly
promising scaffold for the development of inhibitors of ImiS, even the B2 subclasses of MβLs.
Received 21st September 2019,
Accepted 26th November 2019
DOI: 10.1039/c9md00455f
rsc.li/medchem
metallo-β-lactamases (MβLs), which use one or two ZnIJII) ions
at active sites to mediate the hydrolysis of the β-lactam ring.7
Introduction
β-Lactam antibiotics remain the most important and
frequently used antimicrobial agents, constituting more than
50% of the antibiotics prescribed worldwide.1 However, the
effectiveness of β-lactam antibiotics, including penicillins,
cephalosporins and carbapenems, has been threatened by the
emergence of drug-resistant bacteria that produce
β-lactamases.2,3 β-Lactamases are enzymes that inactivate
β-lactam antibiotics by breaking the C–N bond of the
β-lactam ring and render the drugs ineffective.4 According to
the primary sequence homologies, β-lactamases have been
categorized into four classes, A–D.5 Class A, C, and D enzymes
are called serine β-lactamases (SβLs), which use a common
catalytic mechanism where an active site serine
nucleophilically attacks the β-lactam carbonyl, leading to a
cleaved β-lactam ring.6 Class B enzymes are known as
MβLs are further divided into subclasses B1–B3, based on the
amino acid sequence homology and ZnIJII) content.8
The B1 and B3 subclasses MβLs hydrolyze almost all known
β-lactam antibiotics, leading to multiple-drug resistance in
bacteria. In contrast, the B2 subclass enzymes have a narrow
substrate profile including carbapenems, which have been
called one of the “last resort” antibiotics.9 To combat bacterial
drug-resistance, the development of β-lactamase inhibitors to
restore the efficacy of the existing β-lactam antibiotics is an
essential strategy. The co-administration of β-lactam antibiotics
with β-lactamase inhibitors, such as clavulanic acid,
tazobactam, and sulbactam, has been successfully used for the
treatment of the bacterial infections mediated by SβLs.10
However, there are no MβL inhibitors available for clinical
purposes to date.11 Therefore, the development of MβL
inhibitors is urgently needed.
Given the biomedical importance of MβLs, significant
efforts have been made to develop inhibitors of these
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of
Ministry of Education, College of Chemistry and Materials Science, Northwest
University, Xi'an 710127, P. R. China. E-mail: kwyang@nwu.edu.cn
† Electronic supplementary information (ESI) available: 1H, 13C NMR and MS
characterization, Over-expression and purification of MβLs, Docking studies,
cytotoxicity assay. See DOI: 10.1039/c9md00455f
enzymes,12
such
as
azolylthioacetamides,13
triazolylthioacetamides,14 bisthiazolidines15 and maleic acid
derivatives,16 which exhibit inhibitory activities by binding to
the ZnIJII) ions of the target enzymes. Chelating inhibitors,
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such as aspergillomarasmine A17 and [S,S]-ethylenediamine-N,
N′-disuccinic acid (EDDS),18 inactivate clinically-relevant
MβLs like VIM-2 and NDM-1 by sequestering the metals of
each enzyme. Covalent inhibitors, such as ebselen, inactivate
the B1 and B2 subclass MβLs by the formation of a Se–S
bond with the cysteine residue at the active site of the
enzymes.19 Martin Everett et al. first reported that ANT431, a
sulfonamide compound, exhibited inhibition efficacy on
MβLs VIM-2, NDM-1 and IMP-1.20 Recently, our studies
revealed that azolylthioacetamide was a highly promising
scaffold for the development of MβL inhibitors with IC50
values in the submicromolar grade.21
target enzyme, which confers the best inhibitory effect.
Second, phenylamide was grafted with different substituents
on the molecule to ensure different electronic and lipophilic
environments, which could manipulate the activity of the
molecules. With these two strategies, twenty-one
benzenesulfonamides 1a–j, 2a–h, 3e–f and RS (Fig. 1) were
designed and synthesized with previously reported
methods.28,29 Briefly, the appropriate benzoic acid was
refluxed in SOCl2 for 3 h for conversion into the substituted
benzoyl
aminobenzensulfonamide in the presence of pyridine to give
the desired benzenesulfonamides. All compounds
chloride,
which
reacted
with
ImiS is a representative of the B2 subclass MβLs; therefore,
significant effort has been made in the structural,
spectroscopic, mechanistic and inhibition studies on this
enzyme.22–24 Recently, our studies showed that the thiazole-
substituted azolylthioacetamides specifically inhibited ImiS,
with Ki values in the range of 1.2–3.6 μM using imipenem as
the substrate,23 and the N-heterocyclic dicarboxylic acid was a
competitive inhibitor of ImiS with a Ki value of 3.5 μM.24 The
synthesized were characterized by 1H and 13C NMR and
confirmed by HRMS (see ESI†). These compounds were
tested as inhibitors with the purified MβLs NDM-1, ImiS and
L1; their inhibitory modes were investigated by generating
Lineweaver–Burk plots and isothermal titration calorimetry
(ITC). Also, the antimicrobial activities of these inhibitors in
combination with the existing antibiotics against antibiotic-
resistant strains were evaluated, and molecular docking was
performed to investigate the interactions of inhibitor
molecules with the target enzyme.
acetazolamide
benzenesulfonamide
(ACZ),25
(PBS),26
pyrrolidinone-substituted
and hydrazido
benzenesulfonamide (HBS)27 (Fig. 1) were reported to bind
the ZnIJII) ions at the active sites of carbonic anhydrase (CA)
with their sulfonamide group, therefore exhibiting strong
enzymatic inhibition.27 CA, a monozinc enzyme like ImiS,
indicated that the sulfonamides may be used for the
development of MβLs, particularly ImiS inhibitors. Our goal is
to develop specific inhibitors of MβLs and to use these
inhibitors in combination with β-lactam antibiotics to combat
bacterial infections mediated by MβLs. Towards this goal,
benzenesulfonamides with various aromatic substituents
(Fig. 1) were designed, synthesized and characterized.
The benzenesulfonamides were designed based on two
strategies: first, the ortho, meta, and para positions of the
aromatic substituents on the benzene ring, relative to the
sulfonamide group, were adjusted to define the optimal
position for the compound to bind to the active site of the
Results and discussion
Activity evaluation of benzenesulfonamides
To test whether these sulfonamides were MβL inhibitors, the
inhibition experiments under steady-state conditions were
conducted on an Agilent UV8453 spectrometer using
imipenem (40 μM) as the substrate for ImiS, and cefazolin
(40 μM) for NDM-1 and L1. The concentrations of inhibitors
were varied between 0 and 20 μM. The hydrolysis of
imipenem and cefazolin was monitored at 300 and 262 nm,
respectively. The initial reaction rates were determined in the
absence and presence of inhibitors in triplicate, and the
average values were recorded.
The percent inhibition, defined as enzyme activity without
inhibitor (100%) minus residual activity with inhibitor, of
benzenesulfonamide derivatives on ImiS, NDM-1 and L1 is
shown in Fig. 2. It can be clearly observed that the
sulfonamides exhibited more than 70% inhibitory activities
against ImiS at a concentration of 20 μM; 2g in particular
Fig. 2 Percent inhibition of benzenesulfonamide derivatives (20 μM)
against MβLs. Imipenem was used as substrate for ImiS, and cefazolin
was used as substrate for NDM-1 and L1.
Fig. 1 Structures of the synthesized benzenesulfonamides and the
reported sulfonamides.
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showed 95% inhibition. However, the compounds had less
than 20% inhibition against NDM-1 and L1 at
a
concentration of up to 20 μM. Therefore, for the remainder
of this work, we focused on inhibition studies of ImiS.
The inhibitor concentrations causing 50% decrease in the
enzyme activity (IC50) of all sulfonamides against ImiS were
determined in 30 mM Tris (pH 7.0) using imipenem as the
substrate. The collected IC50 data (Table 1) indicated that all
of these compounds had inhibitory efficacy against ImiS,
exhibiting an IC50 value range of 0.11–9.3 μM, and 2g was
found to be the most potent inhibitor (IC50 = 0.11 μM). It
should be noted that 2a–h had an inhibitory potency on ImiS
(IC50 < 1 μM) that was an order of magnitude higher as
compared to 1a–1j (IC50 = 2.7–9.3 μM) and 3e–3f (IC50 = 4.6–
5.4 μM). These IC50 values of sulfonamides are consistent
with their percent inhibitions, as shown in Fig. 2. The
analysis of these IC50 data revealed a structure–activity
relationship (SAR), where the meta-substitutes of the phenyl
ring more significantly improved the inhibitory activity of
benzenesulfonamides against ImiS as compared to the same
substitutes at the ortho- and para-positions.
Fig.
3
Time- and concentration-dependent inhibition of ImiS by
benzenesulfonamide 2g (A), and the inhibition curve of ImiS by 2g at a
concentration between 0.01 and 10 μM (B).
protein binding by titrating one solution into another, has
been applied in enzyme kinetic studies.30 A MicroCal-ITC200
was employed to investigate the inhibition of imipenem
hydrolysis with ImiS by sulfonamide in single injection mode
at 25 °C, as previously reported.31 The concentrations of the
enzyme and imipenem were 100 nM and 0.5 mM,
respectively, and the concentration of the inhibitor was
varied between 0 and 10 μM. ITC was used to monitor the
inhibition of imipenem hydrolysis with ImiS by 2g, as shown
in Fig. 4. The thermopower (dq/dt) gradually decreased with
the increase in the inhibitor concentration from 0 to 10 μM,
confirming that 2g inhibited ImiS in a dose-dependent form
thermodynamically. It should be noted that the addition of
2g did not result in changes in the total heat (Q) released
(Fig. 4, inset), revealing that the sulfonamide reversibly
inhibited ImiS, similar to the inhibition mode of D-captopril
and azolylthioacetamide (ATAA) on NDM-1, as previously
reported.31 This is likely due to the formation of the enzyme–
inhibitor complex (EI) that slowed down substrate hydrolysis.
Given the best potency of sulfonamide 2g, its time- and
concentration-dependent inhibitions on ImiS were assayed
(Fig. 3). It can be observed that in the presence of
sulfonamide at
a concentration of 0.5, 1, and 5 μM,
respectively, the residual activity of ImiS decreased with the
increase in the inhibitor concentration (Fig. 3A). Also, the
residual activity of the enzyme decreased with the increase in
the inhibitor incubation time with the enzyme, and the
molecule showed maximum inhibition at a concentration of
around 5.0 μM and after incubation with ImiS for about 3 h.
The concentration-dependent inhibition of 2g on ImiS was
assayed, and the resulting inhibition curve (Fig. 3B) showed
that more than 92% activity (<8% residual activity) of the
enzyme was inhibited by the molecule at a concentration of
around 10 μM.
Inhibition mode of benzenesulfonamides
To further identify the inhibition mode of the sulfonamides
against ImiS, 1g and 2g were chosen to determine the Ki
values. The concentrations of the inhibitors were varied
between
0
and 10 μM, and substrate (imipenem)
Inhibition assay of ImiS by ITC
concentrations were varied between 20 and 100 μM. All
experimental hydrolytic rates were determined in triplicate.
The inhibition mode was assayed by generating Lineweaver–
Isothermal titration calorimetry (ITC), an essential approach
to measure the heat (Q) released or absorbed during ligand–
Table 1 The inhibitory activities (IC50, μM) of benzenesulfonamide derivatives against MβL ImiSa
P-Substituted compd.
IC50
M-Substituted compd.
IC50
O-Substituted compd.
IC50
1a
1b
1c
1d
1e
1f
1g
1h
1i
7.5 0.3
2.7 0.4
4.8 0.1
9.3 0.3
5.1 0.5
3.3 0.1
7.3 0.2
3.5 0.2
5.6 0.7
4.1 0.6
2a
2b
2c
2d
2e
2f
0.15 0.04
0.14 0.07
0.86 0.04
0.31 0.05
0.44 0.01
0.23 0.08
0.11 0.02
0.16 0.08
3e
3f
RS
5.4 0.2
4.6 0.2
6.8 0.5
2g
2h
1j
a
The substrate used was imipenem and inhibitor concentrations were varied between 0.01 and 20 μM. Enzyme and inhibitors were
preincubated for 3 h before testing.
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Antibacterial activity assays in vitro
The capability of benzenesulfonamides to restore the
antibacterial activity of imipenem and meropenem against E.
coli producing ImiS was investigated by measuring the
minimum inhibitory concentration (MIC) changes in the
antibiotics in the absence and presence of sulfonamides
using a previously reported method.33 The E. coli BL21 (DE3)
with and without ImiS were used for the evaluation of the
inhibitors. The concentration of inhibitors used was 16 μg
mL−1. The collected MIC data (Table 2) indicated that all
sulfonamides tested increased the antimicrobial effect of
both imipenem and meropenem on E. coli expressing ImiS,
resulting in a 2–4-fold reduction in MICs, which is consistent
with their inhibitory effect on ImiS. However, the inhibitors
alone did not inhibit the bacterial growth at a concentration
of up to 64 μg mL−1. Also, no synergistic antibacterial effect
of the sulfonamides on E. coli without ImiS was observed
using the same dose of inhibitor, suggesting that the
capability of sulfonamides to restore antibiotic activity is due
to their inhibition toward ImiS in living bacterial cells.
Fig. 4 Overlayed heat flow curves of imipenem (0.5 mM) hydrolysis by
ImiS in the absence and presence of benzenesulfonamide 2g in the
concentration range of 0–10 μM; the concentration of ImiS was 100 nM.
Burk plots, and the Ki values were obtained by fitting initial
velocity versus substrate concentration at each inhibitor
concentration using SigmaPlot 12.0. The Lineweaver–Burk
plots of imipenem hydrolysis catalyzed by ImiS in the
absence and presence of two benzenesulfonamides are
shown in Fig. 5. The Lineweaver–Burk plots along with
slope-intercept replots (Fig. S1 and S2†) suggest that both
inhibitors are partial mixed-type inhibitors, implying that
the ESI† can yield the product without the release of I.32
The Ki values of 1g and 2g were determined to be 8.0 0.3
Real-time tracking of ImiS in living bacterial cells
To track the ImiS inside living bacterial cells, we constructed
a fluorescent sulfonamide RS (Fig. 1), and used it to perform
the super-resolution fluorescence imaging of the E. coli BL21
and 0.55
0.04 μM (average
standard deviation of
cells harboring ImiS on
a 3D-structured illumination
triplicates), respectively.
microscopy (3D-SIM).34 Fig. 6 shows the 3D-SIM images of
ImiS E. coli cells incubated with RS in MHB at a dose of
around 50 μM (final concentration) for different time
periods. The interactions of RS with bacteria were imaged
using a microscopy technique, where a clear visualization of
the interactions of RS and intracellular enzyme (ImiS) was
clearly demonstrated. RS was found to associate at certain
sites on and in the bacteria, depending on the incubation
time. On incubation for 2 h, the inhibitors initially associated
with the surface of the bacteria due to electrostatic
interactions (Fig. 6a–c). On incubation for 8 h, a high density
of bacterial cells with either membrane-associated or uniform
localization of the inhibitors distributed in the cytosol of the
bacterial cells was observed (Fig. 6d–f). On incubation for 16
h, the target ImiS accumulated at the cell poles was observed
(Fig. 6g–i), suggesting the formation of the inclusion bodies
of the enzyme.35
Antibacterial activity assays in vivo
To test whether the sulfonamides were capable of restoring
the activity of meropenem in vivo, 2g was chosen to perform
synergistic antibacterial assays with meropenem in a mice
model with a previously reported method.36 Briefly, the
Kunming mice model of systemic infection was established
by the intraperitoneal injection of E. coli BL21 (DE3) cells
harboring ImiS. Two hours after infection with the bacteria,
the infected mice were intraperitoneally injected with a single
dose of the drug. The effects of meropenem or sulfonamide
2g monotherapy or antibiotic-inhibitor combination therapy
Fig.
5 Lineweaver–Burk plots of the ImiS-catalyzed hydrolysis of
imipenem in the absence and presence of benzenesulfonamides 1g (A)
and 2g (B). Inhibitor concentrations were 0 (●), 1 (○), 5 (▼), and 10 μM (▽).
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Table 2 MICs of carbapenems, imipenem (A) and meropenem (B), against E. coli BL21 (DE3) cells expressing ImiS in the absence and presence of
a
benzenesulfonamides at a concentration of 16 μg mL−1
A
Compd.
MIC
1a
8
1b
8
1c
8
1d
8
1e
8
1f
8
1g
8
1h
8
1i
8
1j
8
Compd.
MIC
2a
4
2b
4
2c
4
2d
4
2e
4
2f
4
2g
4
2h
4
3e
8
3f
8
RS
8
B
Compd.
MIC
Compd.
MIC
1a
32
2a
16
1b
32
2b
16
1c
32
2c
16
1d
32
2d
16
1e
32
2e
16
1f
32
2f
1g
32
2g
16
1h
32
2h
16
1i
1j
32
3e
32
32
3f
32
RS
32
16
a
MICs of imipenem and meropenem alone on E. coli BL21 (DE3) with ImiS were 16 and 64 μg mL−1, respectively. The MICs of imipenem and
meropenem on E. coli without ImiS in the absence and presence of benzenesulfonamides were 0.25 and 0.0375 μg mL−1, respectively.
on bacterial load in tissues were evaluated. The assay results
are shown in Fig. 7. The bacterial load in the tissues treated
with sulfonamide alone was almost unaffected. However,
compared with meropenem monotherapy, the synergistic
therapy of sulfonamide with meropenem significantly
reduced the bacterial load in the spleen (Fig. 7A) and liver
(Fig. 7B) after a single intraperitoneal dose, indicating that
sulfonamide had synergistic antibacterial efficacy in vivo.
and one of the oxygen atoms was coordinated to the ZnIJII)
ion at the catalytic site (1.9 Å for CphA/2a), another oxygen
atom formed two hydrogen bonds with two amino acid
residues, namely, His196 (2.2 Å) and ASN233 (2.4 Å), and a
hydrogen atom on the amino group formed a hydrogen
bond with ASP120 (2.2 Å). The amide group of the
sulfonamide also interacted with the amino acid residue
ASN233 (2.4 Å). As a result, the sulfonamide functional
group coordinated with the ZnIJII) ion and residual amino
acid within the CphA active center, tightly anchoring the
inhibitor in the active site. This metal-binding mode of
sulfonamide revealed by docking studies is similar to that
of the azolylthioacetamide for CphA.37
Docking study
To further explore the binding of sulfonamides to the target
enzyme, 2a was selected for molecular docking. Since there
is no available crystal structure of ImiS, CphA (PDB code
2QDS) that shares 96% sequence identity with ImiS was
used instead. The lowest energy-docking conformations of
these clusters are shown in Fig. 8, and the binding energy
of the CphA/2a complex is −6.87 kcal mol−1. The
sulfonamide group acted as a ZnIJII)-binding group (ZBG)
Fig. 7 Kunming mice were injected intraperitoneally with E. coli BL21
cells harboring ImiS. Groups of mice were treated with a single dose of
meropenem (10 mg kg−1), inhibitor 2g (10 mg kg−1), a combination of
meropenem (10 mg kg−1) and 2g (10 mg kg−1), and PBS buffer (as
control) by intraperitoneal injection. The bacterial loads in the spleen
(A) and liver (B) were determined by selective plating.
Fig. 6 Confocal microscopic real-time imaging of E. coli BL21 cells
harboring ImiS after treatment with RS (50 μM) for 2 (a–c), 8 (d–f) and
16 h (g–i). Scale bars, 0.5 μm.
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that the sulfonamide reversibly inhibited ImiS. The
identification of Ki showed that 1g and 2g were partially
mixed enzymatic inhibitors. MIC tests demonstrated that the
sulfonamides restored a 2–4-fold antimicrobial activity of
imipenem and meropenem on E. coli expressing ImiS. Mouse
experiments showed that 2g had synergistic efficacy with
meropenem, and significantly reduced the bacterial load in
the spleen and liver after a single intraperitoneal dose.
Tracing the ImiS in living E. coli cells through labeling it with
RS at a super-resolution level using 3D-SIM showed that the
target was initially associated on the surface of cells, then
there was a high density of uniform localization distributed
in the cytosol of cells, and it finally accumulated in the
formation of inclusion bodies at the cell poles. Docking
studies suggested that the sulfonamide group acted as the
zinc-binding group (ZBG) to coordinate with the ZnIJII) and
the residual amino acid within the CphA active center, tightly
anchoring the inhibitor at the active site. Cytotoxicity assays
indicated that the sulfonamides had low cytotoxicity.
Fig.
8 Lowest-energy docking conformation of sulfonamide 2a
docked into the active site of CphA (PDB code 2QDS). The enzyme
backbone is shown as a cartoon in green, and selected residues are
shown as sticks colored by elements (H, white; C, yellow; N, blue; O,
red; S, orange). The ZnIJII) ion is shown as a magenta sphere; 2a is
shown as sticks with the same color code as amino acid residues.
Characteristic short distances between the inhibitor and the protein
are indicated by dashed lines. These figures were generated with
PyMOL.
Cytotoxicity assays
Experimental
The potential toxicity of enzyme inhibitors is a principal
problem for clinical medical applications. Four sulfonamides
1e, 2e, 2g and 3f with different working concentrations (6.25,
12.5, 25, 50, 100, 200 μM) were subjected to a cytotoxicity
Chemicals
Sulfanilamide, thionyl chloride and substituted benzoic acid
were purchased from Sigma-Aldrich (Shanghai) Trading Co.
Ltd. All other starting materials were purchased from
commercial sources and were purified using standard
methods. Analytical thin layer chromatography (TLC) was
performed on silica gel GF254 plates with visualization by
assay with mouse fibroblast cells (L929), based on
a
previously reported method.38 As shown in Fig. 9, more than
80% of the cells tested maintained viability in the presence
of the inhibitors at a concentration of up to 200 μM,
suggesting that these sulfonamides have low cytotoxicity.
ultraviolet radiation. H and 13C NMR spectra were recorded
1
on
a Bruker Avance III 400 MHz NMR spectrometer.
Chemical shifts are given in parts per million (ppm) on the
delta scale. The peak patterns are reported as singlet (s),
doublet (d), triplet (t), quartet (q), doublet (dd), and multiplet
(m). The spectra were recorded with TMS as the internal
standard. Coupling constants ( J) were reported in Hertz (Hz).
Mass spectra were recorded on a micro TOF-Q (BRUKER)
mass spectrometer. The activity evaluation of inhibitors was
performed on an Agilent 8453 UV-vis spectrometer.
Conclusions
Twenty-one benzenesulfonamides (1a–j, 2a–h, 3e–f and RS)
were synthesized and characterized via NMR and MS.
Biological assays revealed that all these compounds
specifically inhibited ImiS, exhibiting IC50 values in the range
of 0.11–9.3 μM, and 2g was found to be the best inhibitor
(IC50 = 0.11 μM). The structure–activity relationship analysis
revealed that the meta-substitutes of the phenyl ring improved
the activity of sulfonamides on ImiS. ITC assays indicated
General procedure for the preparation of
benzenesulfonamides (1a–j, 2a–h, 3e–f and RS)
The acyl chlorides were synthesized as previously reported.28
To a solution of the corresponding benzoic acid (4 mmol) in
SOCl2 (20 mL) DMF was added dropwise for the catalytic
reaction. The reaction mixture was stirred and refluxed at 85
°C for 3 h. After the reaction was completed, the solvent was
removed by rotary evaporation under a reduced pressure, and
the resulting residue was dissolved in acetone (10 mL) for the
next step reaction without further purification. The
benzenesulfonamides were synthesized using a previously
reported method.29 The as-prepared acyl chloride solution
was added dropwise over 30 min to a stirred solution of
sulfanilamide (2.9 mmol, 500 mg) and pyridine (5.8 mmol,
467 mL) in dry acetone (10 mL) at 0 °C. The reaction mixture
was stirred at room temperature for about 5 h, and the
Fig. 9 Percent cell viability (relative to the absence of sulfonamide) of
L-929 mouse fibroblastic cells in the presence of 1e, 2e, 2g and 3f at
concentrations of 6.25, 12.5, 25, 50, 100, and 200 μM, respectively.
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resulting products were filtered or evaporated under reduced
pressure. The crude product was purified via flash column
chromatography on silica gel (ethyl acetate/petroleum ether,
2 : 1) to give the sulfonamides 1a–j, 2a–h and 3e–f.
substrate were 100 nM and 0.5 mM, respectively, and the
concentrations of the inhibitor were varied between 0 and 10
μM. Before starting measurements, the enzyme sample was
pre-incubated with different concentrations of 2g for 3 h and
then delivered into the sample cell (210 μL). The reaction in
the absence of inhibitor was also monitored as the control.
The stirring rate of the syringe was set to 750 rpm. The system
was equilibrated at the desired 25 °C. Each reaction was
automatically initiated by adding imipenem (0.5 mM) in the
form of a single 38 μL injection into the enzyme-containing
sample cell. Heat flow was recorded as a function of time.
Data were collected every 1 s until the signal reached the
baseline and continued to be recorded for the appropriate
time to generate the final baseline. The progress curves of
imipenem hydrolysis catalyzed by ImiS in the absence and
presence of 2g at different concentrations are shown in Fig. 5.
The fluorescent sulfonamide RS was synthesized via a
previously reported method.19 To
a stirred solution of
4-hydroxybenzenesulfonamide (2.0 eq.) in DCM was added
DCC (1.3 eq.), DMAP (0.13 eq.) and rhodamine B (1.0 eq.) at
0 °C. The reaction mixture was stirred at 0 °C for 10 min,
and then at room temperature for 4 h; the solvent was
removed under vacuum and extracted with DCM (3 × 10 mL).
The combined organic phase was dried over anhydrous
Na2SO4, distilled under reduced pressure, and the crude
product was purified via flash column chromatography on
silica gel (CHCl3/CH3OH, 10 : 1) to afford RS.
Determination of IC50
Determination of MIC
MβLs from subclasses B1 (NDM-1), B2 (ImiS) and B3 (L1)
were over-expressed and purified using a previously described
method,39–41 and the details are provided in the ESI.† The
inhibitor concentration causing 50% decrease in the enzyme
activity (IC50) was determined at 25 °C using imipenem as
the substrate for ImiS. The sulfonamides 1a–j, 2a–h, 3e–f and
RS were dissolved in a small volume of DMSO and diluted
with 30 mM Tris (pH 7.0), and ImiS sample and imipenem
were mixed in 30 mM Tris (pH 7.0). The final concentrations
of DMSO in inhibition experiments were below 0.5%, and
control experiments verified that 0.5% DMSO had no
inhibition against ImiS. All inhibitors were assayed at six
different concentrations between 0 and 20 μM, and substrate
concentration was 40 μM. The hydrolysis of imipenem was
monitored at 300 nm on an Agilent UV8453 spectrometer,
and the hydrolytic rates were determined in triplicate. The
IC50 values were calculated by plotting the average percentage
inhibition against inhibitor concentration and the linear
fitting of the data.
A single colony of E. coli BL21 (DE3) containing plasmids
pET26b-ImiS on LB agar plates was transferred to 5 mL of a
Mueller-Hinton (MH) liquid medium. Strains grown in the
MH medium to OD600 of 0.5 were used as inoculum after an
84-fold dilution to 1 × 105 CFU mL−1 in the MH medium. MIC
values were determined using the Clinical and Laboratory
Standards Institute (CLSI) macrodilution (tube) broth
method.31 The antibiotic was dissolved in the MH medium to
prepare 128, 64, 32, 16, 8, 4, 2, and 1 μg mL−1 stock solutions,
respectively. The sulfonamides 1a–j, 2a–h, 3e–f and RS were
dissolved in the DMSO and MH media to prepare 64 μg mL−1
(initial concentration) stock solutions. The as-prepared
solutions with different antibiotic concentrations (50 μL) and
inhibitor solution (50 μL) were added to a 96-hole plate, and
then 100 μL inoculum was added sequentially to the as-
prepared solutions. MIC was interpreted as the lowest
concentration of the drug that completely inhibited the visible
growth of bacteria after incubating plates at 37 °C for at least
16 h. Each measurement was performed in triplicate in at
least two independent experiments and the highest MIC value
was reported.
Determination of Ki
The inhibition mode of sulfonamides was determined by
generating Lineweaver–Burk plots, and the Ki values were
determined by fitting initial velocity versus substrate
concentration at each inhibitor concentration using
SigmaPlot 12.0. The concentrations of imipenem used were
20, 40, 60, 80 and 100 μM, respectively. The initial hydrolysis
rate of the substrate was determined by the method for
measuring IC50, as described above. All hydrolytic rates were
determined in triplicate.
Sample preparation for cell imaging
The cell imaging was performed on
a Nikon N-SIM
instrument at 25 °C. E. coli BL21 cells harboring ImiS (OD600
= 0.5) were incubated with the fluorescent sulfonamide RS at
a dose of around 50 μM (final concentration) in tubes at 37
°C for different times. After incubation, the cells were washed
five times with PBS buffer (10 000 rpm, 5 min) to remove any
adsorbed RS probe on the cell surface. The cells were re-
suspended in PBS to give the final samples for confocal
microscopic imaging.
ITC assays
Isothermal titration calorimetry (ITC) experiments were
performed on a Malvern MicroCal iTC 200 instrument at 25
°C using a single injection mode. During all experiments, the
reference cell was filled with degassed water. The MβLs,
substrates, and inhibitor 2g were prepared in 30 mM Tris with
0.5% addition of DMSO. The concentrations of ImiS and
Mouse experiments
Healthy male and female KM mice, 20 2 g, were supplied
by the Experimental Animal Center, Medical College of Xi'an
Jiaotong University, China. Mouse experiments were
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performed in the Xi'an Jiaotong University Health Science
11 G. Cornaglia, H. Giamarellou and G. M. Rossolini, Lancet
Infect. Dis., 2011, 11, 381–393.
Center. The animals were provided with
a standard
commercial mouse pellet diet and ultrapure water ad libitum.
Mice were randomly divided into four groups with six mice
in each group, with half being male and the other half
female. All mice were treated with 200 μL bacteria suspension
(OD600 = 1.0) via intraperitoneal injection. After infection for
2 h, the infected mice in groups 1, 2, 3 and 4 were subjected
to intraperitoneal treatment with PBS, 10 mg kg−1
meropenem, and 10 mg kg−1 sulfonamide 2g, 10 mg kg−1
meropenem plus 10 mg kg−1 2g, respectively. All experimental
mice were euthanized 24 h post-infection, and 0.1 g each of
the spleen and liver were collected and homogenized into 1
mL of sterile PBS. Organ homogenates were then serially
diluted in PBS, and plated on LB-agar for c.f.u. enumeration.
12 J. D. Docquier and S. Mangani, Drug Resist. Updates,
2018, 36, 13–29.
13 Y. Xiang, Y. N. Chang, Y. Ge, J. S. Kang, Y. L. Zhang and X. L.
Liu, Bioorg. Med. Chem. Lett., 2017, 27, 5225–5229.
14 L. Zhai, Y. L. Zhang, J. S. Kang, P. Oelschlaeger, L. Xiao and
S. S. Nie, ACS Med. Chem. Lett., 2016, 7, 413–417.
15 M. M. González, M. Kosmopoulou, M. F. Mojica, V. Castillo,
P. Hinchliffe and I. Pettinati, ACS Infect. Dis., 2015, 1,
544–554.
16 Y. Ishii, M. Eto, Y. Mano, K. Tateda and K. Yamaguchi,
Zhongguo Ganran Yu Hualiao Zazhi, 2011, 54, 3625–3629.
17 A. M. King, S. A. Reid-Yu, W. Wang, D. T. King, G. De Pascale
and N. C. Strynadka, Nature, 2014, 510, 503–506.
18 A. Proschak, J. Kramer, E. Proschak and T. A. Wichelhaus,
J. Antimicrob. Chemother., 2017, 73, 425–430.
19 C. Chen, Y. Xiang, K. W. Yang, Y. Zhang, W. M. Wang and
J. P. Su, Chem. Commun., 2018, 54, 4802–4805.
20 M. Everett, N. Sprynski and A. Coelho, et al., Antimicrob.
Agents Chemother., 2018, 62, 00074.
21 Y. L. Zhang, K. W. Yang and Y. J. Zhou, ChemMedChem,
2015, 9, 2445–2448.
Ethical statement
All animal procedures were performed in accordance with
the Guidelines for Care and Use of Laboratory Animals of
Xi'an Jiaotong University and experiments were approved by
the Animal Ethics Committee of Xi'an Jiaotong University,
(approval ID: NO. XJTULAC2019-1234).
22 A. L. Costello, N. P. Sharma, K. W. Yang, M. W. Crowder and
D. L. Tierney, Biochemistry, 2006, 45, 13650–13658.
23 S. K. Yang, J. S. Kang, P. Oelschlaeger and K. W. Yang, ACS
Med. Chem. Lett., 2015, 6, 455–460.
Conflicts of interest
There are no conflicts to declare.
24 F. Lei, K. W. Yang, L. S. Zhou, J. M. Xiao, X. Yang and L.
Zhai, Bioorg. Med. Chem. Lett., 2012, 22, 5185–5189.
25 T. D. Groot, A. P. Sinke, M. L. A. Kortenoeven, M. Alsady, R.
Baumgarten and O. Devuyst, J. Am. Soc. Nephrol., 2015, 27,
2082–2091.
26 I. Vaškevičienė, V. Paketurytė, N. Pajanok, B. Sapijanskaitė,
K. Kantminienė and V. Mickevičius, Bioorg. Med. Chem.,
2018, 27, 322–337.
27 M. F. Abo-Ashour, W. M. Eldehna, N. Alessio, H. S. Ibrahim, B.
Silvia and S. M. Abou-Seri, Eur. J. Med. Chem., 2018, 157, 28–36.
28 M. Kumar, K. Ramasamy, V. Mani, R. K. Mishra, A. B. A.
Majeed and E. D. Clercq, Arabian J. Chem., 2014, 7, 396–408.
29 A. Ammazzalorso, A. Carrieri, F. Verginelli, I. Bruno, G.
Carbonara and A. D'Angelo, Eur. J. Med. Chem., 2016, 114,
191–200.
30 P. Draczkowski, A. Tomaszuk, P. Halczuk, M. Strzemski, D.
Matosiuk and K. Jozwiak, Biochim. Biophys. Acta, 2016, 1860,
967–974.
Acknowledgements
This work was supported by Grant (21572179) from the
National Natural Science Foundation of China and Grant
(2019KW-068) from the Shaanxi Province International
Cooperation Project.
References
1 S. M. Drawz and R. A. Bonomo, Clin. Microbiol. Rev.,
2010, 23, 160–201.
2 A. L. Demain and R. P. Elander, Antonie Van Leeuwenhoek,
1999, 75, 5–19.
3 A. A. Medeiros, Clin. Infect. Dis., 1997, 24(Suppl 1), S19.
4 D. T. King, S. Sobhanifar and N. C. J. Strynadka, Protein Sci.,
2016, 25, 787–803.
5 R. P. Ambler, Philos. Trans. R. Soc., B, 1980, 289, 321–331.
6 M. Galleni, J. Lamotte-Brasseur, X. Raquet, A. Dubus, D.
Monnaie and J. R. Knox, Biochem. Pharmacol., 1995, 49,
1171–1178.
31 Y. J. Zhang, W. M. Wang, P. Oelschlaeger, C. Chen, J. E. Lei
and M. Lv, ACS Infect. Dis., 2018, 4, 1671–1678.
7 M. F. Mojica, R. A. Bonomo and W. Fast, Curr. Drug Targets,
2016, 17, 1029–1050.
32 I. H. Segal, Enzyme Kinetics, Wiley-Interscience, New York,
1993, ISBN: 0-471-30309-7.
8 G. Garau, I. Garcia-Saez, C. Bebrone, C. Anne, P. Mercuri
and M. Galleni, Antimicrob. Agents Chemother., 2004, 48,
2347–2349.
9 K. M. Papp-Wallace, A. Endimiani, M. A. Taracila and R. A.
Bonomo, Antimicrob. Agents Chemother., 2011, 55, 4943–4960.
10 D. E. Ehmann, H. Jahic, P. L. Ross, R. F. Gu, J. Hu and T. F.
Durand-Reville, J. Biol. Chem., 2013, 288, 27960–27971.
33 F. R. Cockerill, Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically: Approved
standard, Clinical and Laboratory Standards Institute, 2000.
34 J. L. Shu, N. M. O'Briensimpson, N. Pantarat, A. Sulistio,
E. H. H. Wong and Y. Y. Chen, Nat. Microbiol., 2016, 1, 16162.
35 A. Rokney, M. Shagan, M. Kessel, Y. Smith, I. Rosenshine
and A. B. Oppenheim, J. Mol. Biol., 2009, 392, 589–601.
RSC Med. Chem.
This journal is © The Royal Society of Chemistry 2020

View Article Online
RSC Medicinal Chemistry
Research Article
36 A. M. King, S. A. Reid-Yu, W. Wenliang, D. T. King, D. P.
Gianfranco and N. C. Strynadka, Nature, 2014, 510,
503–506.
37 D. Gao, A. Li, L. Guan, X. Zhang and L. Y. Wang, Dyes Pigm.,
2016, 129, 163–173.
39 H. Yang, M. Aitha, A. M. Hetrick, T. K. Richmond, D. L.
Tierney and M. W. Crowder, Biochemistry, 2012, 51, 3839–3847.
40 P. A. Crawford, N. Sharma, S. Chandrasekar, T. Sigdel, T. R.
Walsh and J. Spencer, Protein Expression Purif., 2004, 36,
272–279.
38 S. K. Yang, J. S. Kang, P. Oelschlaeger and K. W. Yang, ACS
Med. Chem. Lett., 2015, 6, 455–460.
41 M. W. Crowder, T. R. Walsh, L. Banovic, M. Pettit and J.
Spencer, Antimicrob. Agents Chemother., 1998, 42, 921–926.
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