Thiosemicarbazones exhibit inhibitory efficacy against New Delhi metallo-β-lactamase-1 (NDM-1)

Article abstract of DOI:10.1038/s41429-021-00440-3

The superbug infection caused by metallo-β-lactamases (MβLs) carrying drug-resistant bacteria, specifically, New Delhi metallo-β-lactamase (NDM-1) has become an emerging threat. In an effort to develop novel inhibitors of NDM-1, thirteen thiosemicarbazones (1a-1m) were synthesized and assayed. The obtained molecules specifically inhibited NDM-1, with an IC₅₀ in the range of 0.88–20.2 μM, and 1a and 1f were found to be the potent inhibitors (IC₅₀ = 1.79 and 0.88 μM) using cefazolin as substrate. ITC and kinetic assays indicated that 1a irreversibly and non-competitively inhibited NDM-1 in vitro. Importantly, MIC assays revealed that these molecules by themselves can sterilize NDM-producing clinical isolates EC01 and EC08, exhibited 78-312-fold stronger activities than the cefazolin. MIC assays suggest that 1a (16 μg ml^?1) has synergistic antimicrobial effect with ampicillin, cefazolin and meropenem on E. coli producing NDM-1, resulting in MICs of 4-32-, 4-32-, and 4-8-fold decrease, respectively. These studies indicate that the thiosemicarbazide is a valuable scaffold for the development of inhibitors of NDM-1 and NDM-1 carrying drug-resistant bacteria.

Full text of DOI:10.1038/s41429-021-00440-3

The Journal of Antibiotics (2021) 74:574–579
https://doi.org/10.1038/s41429-021-00440-3
ARTICLE
Thiosemicarbazones exhibit inhibitory efficacy against New Delhi
metallo-β-lactamase-1 (NDM-1)
Ying Ge1,2 Peng-Wei Kang1 Jia-Qi Li1 Han Gao1 Le Zhai3 Le-Yun Sun1 Cheng Chen1 Ke-Wu Yang1
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Received: 8 September 2020 / Revised: 7 June 2021 / Accepted: 17 June 2021 / Published online: 7 July 2021
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2021
Abstract
The superbug infection caused by metallo-β-lactamases (MβLs) carrying drug-resistant bacteria, specifically, New Delhi
metallo-β-lactamase (NDM-1) has become an emerging threat. In an effort to develop novel inhibitors of NDM-1, thirteen
thiosemicarbazones (1a-1m) were synthesized and assayed. The obtained molecules specifically inhibited NDM-1, with an
IC50 in the range of 0.88–20.2 µM, and 1a and 1f were found to be the potent inhibitors (IC50 = 1.79 and 0.88 μM) using
cefazolin as substrate. ITC and kinetic assays indicated that 1a irreversibly and non-competitively inhibited NDM-1 in vitro.
Importantly, MIC assays revealed that these molecules by themselves can sterilize NDM-producing clinical isolates EC01
and EC08, exhibited 78-312-fold stronger activities than the cefazolin. MIC assays suggest that 1a (16 μg ml−1) has
synergistic antimicrobial effect with ampicillin, cefazolin and meropenem on E. coli producing NDM-1, resulting in MICs of
4-32-, 4-32-, and 4-8-fold decrease, respectively. These studies indicate that the thiosemicarbazide is a valuable scaffold for
the development of inhibitors of NDM-1 and NDM-1 carrying drug-resistant bacteria.
Introduction
including penicillins, cephalosporins and carbapenems.
Commonly, bacteria become resistant to β-lactam anti-
biotics by producing β-lactamases [2]. β-Lactamases are
enzymes that inactivate β-lactam antibiotics by breaking
the C-N bond of β-lactam ring and render the drugs
ineffective [3].
Since the discovery of penicillin in the last century,
β-lactams, as the most effective and successful antibiotics,
have been widely used for the treatment of bacterial
infections [1]. However, the overuse of β-lactam anti-
biotics has resulted in a large number of bacteria
that produce β-lactamases, and the resulting bacteria
are resistant to the most commonly-used antibiotics,
So far, there have been more than 2800 distinct β-
lactamases identified [4], and these enzymes have been
categorized into classes A, B, C and D, based on their
amino acid sequence homologies and action mechanism.
Classes A, C and D enzymes are referred to as serine-β-
lactamases (SβLs), which utilize an active site serine to
nucleophilically attack the β-lactam carbonyl, ultimately
leading to a cleaved β-lactam ring. Class B enzymes, also
called metallo-β-lactamases (MβLs), use either 1 or 2
equivalents Zn(II) to catalyze β-lactams hydrolysis. MβLs
are further divided into subclasses B1, B2, and B3, based on
their amino acid sequence homologies and Zn(II) content
[5]. MβLs belonging to the B1 and B3 subclasses can
hydrolyze almost all known β-lactam antibiotics. In con-
trast, the B2 subclass enzymes have a narrow substrate
profile including carbapenems, which have been called one
of the “last resort” antibiotics [6]. There are no known
clinical inhibitors of the MβLs to date [7].
These authors contributed equally: Ying Ge, Peng-Wei Kang
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1038/s41429-
021-00440-3.
* Ke-Wu Yang
kwyang@nwu.edu.cn
1
Key Laboratory of Synthetic and Natural Functional Molecule
Chemistry of Ministry of Education, Chemical Biology Innovation
Laboratory, College of Chemistry and Materials Science,
Northwest University, Xi’an, P. R. China
2
Medical College, Xijing University, Xian, China
3
Shaanxi Key Laboratory of Phytochemistry, College of Chemistry
Given the enormous biomedical importance of MβLs, a
large amount of effort have been made to develop inhibitors
and Chemical Engineering, Baoji University of Arts and Sciences,
Baoji, Shaanxi Province, P. R. China

Thiosemicarbazones exhibit inhibitory efficacy against New Delhi...
575
H
Fig. 1 Structures of the synthetic
thiosemicarbazones
NH2
N
R
N
S
OH
HO
R=
N
N
HO
HO
N
1f
1g
1c
1b
1d
1e
1a
S
O2N
Cl
Cl
Cl
Cl
1j
1l
1k
1m
1h
1i
of the enzymes [8], such as azolylthioacetamides [9], bis-
thiazolidines [10], and maleic acids [11], which exhibit inhi-
bition effect by binding to Zn(II) of the enzymes; chelating
inhibitors, represented as aspergillomarasmine A (AMA) [12],
and [S,S]-ethylenediamine-N,N’-disuccinic acid (EDDS) [13],
which inactivate MβLs like VIM-2 and NDM-1 by seques-
tering the metals of each enzyme; and covalent inhibitors,
such as ebselen, which inactivate the B1 and B2 subclass
MβLs by formation of a Se-S bond with a cysteine residue at
active site of enzymes [14]. Recently, our studies revealed
that the azolylthioacetamide is a highly promising scaffold for
the development of MβLs inhibitors with IC50 values in
submicromolar range [15].
combination with β-lactam antibiotics to combat bacterial
infections. Towards this goal, the thiosemicarbazones with
various aromatic substituents (Fig. 1) were synthesized
and characterized by NMR and MS. These compounds
were tested as inhibitors with the purified enzyme NDM-1,
their inhibitory mode was investigated by generating
Lineweaver–Burk plots and isothermal titration calorimetry
(ITC). Also, the antimicrobial activities of these inhibitors
were evaluated in combination with the existing antibiotics
against antibiotic-resistant strains.
Materials and methods
New Delhi metallo-β-lactamase-1 (NDM-1), a B1 subclass
MβL, hydrolyzes almost all β-lactam antibiotics including
carbapenems [16]. The plasmid-encoded NDM-1 gene has
been shown to horizontally transfer to other pathogenic bac-
teria, and therefore led to multiple drug resistance and caused
spread of the resistant bacteria around the world [17]. Given
the enormous importance of NDM-1, a large number of
NDM-1 inhibitors have been reported, such as metal com-
plexing reagent [18], binary carboxylic acid [19], D- and
L-captopril [20], and ebuprofen derivative [21]. Recently,
ebselen has been reported to be the covalent inhibitor of
NDM-1 by forming a S–Se bond with the Cys221 residue at
active site of the enzyme [3, 22].
General Information
General chemicals were purchased from TCI (Tokyo
Chemical Industry, Tokyo, Japan), Sigma-Aldrich (US),
Energy-Chemical (China) and Aladdin (China). The
instruments used in experiments include TOF-Q mass
spectrometer, agilent-8453 UV-Vis spectrophotometer,
AKTA purifier 10, FD-1C freeze dryer and IKA magnetic
stirrer. The plasmid of NDM-1 and Escherichia coli
BL21 cells were donated by Professor Michael Crowder at
Miami University, USA. The plasmid of NDM-1 was
transformed into BL21 E. coli to offer the engineering
bacteria E. coli-NDM-1 that only produce NDM-1 enzyme
for protein expression and MIC evaluation. Analytical data
for all synthesized compounds can be found in the sup-
plementary file. The clinical strains E. coli harboring NDMs
(EC01, EC08) were obtained from the First Affiliated
Hospital of Xi’an Jiaotong University.
Thiosemicarbazone, an inhibitor of urease and tyrosinase,
has been widely used for antitumor and antibacterial studies
[23, 24].
The urease and tyrosinase, both dizinc-containing enzymes
like B1 and B3 subclasses MβLs, reminded us of that the
thiosemicarbazone may be a potential scaffold for the devel-
opment of MβLs, specifically NDM-1 inhibitors. In addition,
Spyrakis, et al, reported that the compounds bearing a thiol, a
thiosemicarbazide or thiosemicarbazone moiety had a good
inhibitory effect on NDM-1 [25]. Our goal is to develop
specific inhibitors of MβLs and to use these inhibitors in
Inhibition studies
To test whether these thiosemicarbazones were NDM-1
inhibitors, the inhibition experiments under steady-state

576
Y. Ge et al.
conditions were conducted on an Agilent UV8453 spectro-
meter using cefazolin as substrate. The thiosemicarbazones,
NDM-1 and substrates samples were prepared with 30 mM
Tris, pH 7.0. The final enzyme concentration was 13 nM and
the concentration of cefazolin was 50 µM. The concentrations
of inhibitors were varied between 0 and 20 µM. Enzyme and
inhibitor were pre-incubated for 20 min before adding sub-
strate. Hydrolysis of cefazolin was monitored at 265 nm.
The initial reaction rates were determined in the absence
and presence of inhibitors in triplicate, and the average
values were recorded. The IC50 values were linear fitted by
plotting the average percentage inhibition against inhibitor
concentration.
To further identify the inhibition mode of the thiosemi-
carbazones on NDM-1, 1a was chosen to determine Ki
values. Concentrations of the inhibitor were varied between
0 and 10 μM, and substrate (cefazolin) concentrations were
varied between 20 and 100 µM. All experimental hydrolytic
rates were determined in triplicate. The inhibition mode was
assayed by generating Lineweaver–Burk plots, and Ki
values were obtained by fitting the initial velocity versus
substrate concentration at each inhibitor concentration using
SigmaPlot 12.0.
bacteria was MIC value. Each measurement was repeated
three times.
Results and discussion
Synthesis of thiosemicarbazones
Thirteen thiosemicarbazones 1a-1m (Fig. 1) were synthe-
sized by previously reported methods [28]. Briefly, the
aromatic aldehyde and thiosemicarbazide were refluxed in
ethanol with 0.1 equivalent pyrrolidine as catalyst for 5 h,
and the reaction mixture was gradually cooled to room
temperature, filtrated, washed with cold ethanol, and dried
to give the product thiosemicarbazones, with a yield of
more than 90%.
Inhibitory activity assay
Percent inhibition, defined as enzyme activity without
inhibitor (100%) minus residual activity with inhibitor, of
thiosemicarbazones on NDM-1 is shown in Fig. S1. The
results show that all compounds exhibited more than 34%
inhibition against NDM-1 at a concentration of 10 µM,
especially 1a and 1f showed more than 86% inhibition.
The inhibitor concentrations causing 50% decrease of
enzyme activity (IC50) of all thiosemicarbazones against
NDM-1 were determined in 30 mM Tris (pH 7.0) using
cefazolin as substrate. These kinetic experiments were done in
triplicate, and the average values standard deviations are
reported in Table 1. The collected IC50 data indicated that all
of these compounds had inhibitory efficacy on NDM-1,
exhibiting an IC50 value in the range of 0.88–20.2 μM, and 1a
and 1f were found to be the most potent inhibitors (IC50 =
1.79 and 0.88 μM). The structure-activity relationship analysis
reveals that the thiosemicarbazones with nitrogen heterocyclic
ring or hydroxyl aromatic ring, particularly p-hydroxyl aro-
matic ring has the better inhibition activity than the molecules
with other substitutes. Given the good potency of thiosemi-
carbazone 1a, the time- and concentration-dependent inhibi-
tions of it on NDM-1 were assayed (Fig. S2). NDM-1 and 1a
Isothermal titration calorimetry (ITC) assays
Isothermal titration calorimetry (ITC), an essential approach
to measure the heat (Q) released or absorbed during reaction
by SigmaPlot been applied in enzyme kinetic studies [26].
A MicroCal-ITC200 was employed to investigate the
inhibition of cefazolin hydrolysis with NDM-1 by
thiosemicarbazone in single injection mode at 25 °C as
previously reported [27]. The concentrations of the enzyme
and cefazolin were 100 nM and 0.1 mM, respectively, and
the concentrations of inhibitor were varied between 0
and 120 µM.
Determination of MIC
The MIC values of the antibiotics alone and in the presence
of the enzyme inhibitor against antibiotic-resistant bacteria
were determined using the broth micro-dilution method.
Single colonies of cells were transferred into 5 ml of
Mueller–Hinton (MH) liquid medium. Strains, grown in
MH medium to OD600 = 0.45, were used as inocula after
84-fold dilution to 1×105 CFU ml−1 in MH medium.
Antibiotics and inhibitors were dissolved in MH medium to
prepare different concentration stock solutions. These
solutions, with different antibiotic concentrations (50 μl),
were diluted to 100 μl with 50 μl inhibitor solution, then
100 μl inoculum was added, sequentially, into the prepared
solutions. The mixtures were incubated at 37 °C for 16 h
and the minimum concentration of completely inhibited
Table 1 IC50 values of thiosemicarbazones against NDM-1
Compound
IC50 (μM)
Compound
IC50 (μM)
1a
1b
1c
1d
1e
1f
1.79 0.01
6.81 0.02
8.19 0.02
6.98 0.03
9.11 0.02
0.88 0.01
7.49 0.03
1h
1i
16.7 0.04
19.8 0.03
15.7 0.02
20.2 0.02
9.87 0.02
17.4 0.03
1j
1k
1l
1m
1g

Thiosemicarbazones exhibit inhibitory efficacy against New Delhi...
577
Fig. 2 Lineweaver–Burk plots of
cefazolin hydrolysis catalyzed
by NDM-1 in the absence and
presence of thiosemicarbazone
1a. Inhibitor concentration was
in the range of 0–10 µM
were incubated for different time before adding cefazolin. It
can be observed that, in the presence of 1a at a concentration
of 10 µM, the residual activity of NDM-1 decreased with the
extension of preincubation time, and the molecule showed the
maximum inhibition after incubation with the enzyme for
about 80 min (Fig. S2A), indicating that the compound is a
time-dependent inhibitor. Also, it is observed (Fig. S2B) that
the percent inhibition of 1a on NDM-1 increased with the
increase of inhibitor concentration. The Lineweaver–Burk
plots of cefazolin hydrolysis catalyzed by NDM-1 in the
absence and presence of thiosemicarbazone (Fig. 2) suggest
that 1a is a non-competitive inhibitor, and the Ki value
determined is 1.2 µM, which is lower than the data (10.7 µM)
of thiosemicarbazone moiety reported by Spyrakis, et al [25].
Fig. 3 Overlayed heat flow curves of NDM-1 enzyme catalyzed
hydrolysis of cefazolin in the absence and presence of thiosemi-
carbazone 1a at a concentration in the range of 0–120 µM, using a
single injection mode ITC assay at 25 °C
Inhibition assay by ITC
Monitoring of the inhibition of imipenem hydrolysis with
NDM-1 by 1a is shown in Fig. 3. It is clearly observed that
the thermopower (dq/dt) gradually decreased with the
increase in the inhibitor concentrations from 0 to 120 µM,
confirming that 1a is a dose-dependent inhibitor of NDM-1
thermodynamically. However, the addition of the com-
pound resulted in the decrease of total heat (Q) released,
revealing that the thiosemicarbazone irreversibly inhibited
NDM-1, similar to the inhibition mode of ebselen on NDM-
1, as previously reported [27]. The Q is an important indi-
cator to discriminate the mode of action of inhibitors. When
Q remains constant, the inhibitor is a reversible inhibitor,
but the inhibitor molecule that reduces Q is an irreversible
inhibitor [27]. This irreversible inhibition is probably due to
the binding of 1a to two zinc ions at active site of NDM-1.
MIC determination
The sterilizing ability and synergistic antimicrobial activity
with antibiotic of thiosemicarbazones were investigated by
determining the minimum inhibitory concentrations (MICs)
in the absence and presence of 1a-1m as previously reported
[29]. The drug-resistant strains BL21 E. coli producing
NDM-1 (E. coli-NDM-1), and clinical isolates E. coli har-
boring NDMs (EC01, EC08) were used to assay the inhibi-
tors, and the antibiotics used were cefazolin, ampicillin, and
meropenem. The collected MIC data (Table 2a) indicated that
all of the tested thiosemicarbazides alone exhibited anti-
microbial effect against EC01 and EC08, with a MIC value
that is 4-312-fold lower than that of cefazolin, but 1a, 1d, 1f,

578
Y. Ge et al.
Table 2 Antibacterial activities (MICs, μg ml−1) of thiosemicarbazones
1a-1m against three drug-resistant strains expressing β-lactamases (a),
and MICs of 1a and 1f (16 μg ml−1) synergize three antibiotics (b)
synergistic antimicrobial effect with ampicillin, cefazolin and
meropenem on E. coli producing NDM-1 and EC08, resulting
in MICs of 4-32-, 4-64-, and 4-16-fold decrease, respectively.
a
Acknowledgements This research was funded by the Grants
(22077100 and 2019KW-068 to K.W.Y) from the National Natural
Science Foundation of China and Shaanxi Province International
Cooperation Project and grant (17JS007 to L.Z.) from the Shaanxi
Education Commission, China.
compd. E.coli- EC01 EC08 compd. E.coli- EC01 ECO8
NDM-1
NDM-1
1a
1b
1c
1d
1e
1f
64
64
64
1h
1i
1024
256
64
1024 1024
256
256
128
256
32
256
128
128
256
64
256
256
128
256
32
256
64
256
64
1j
Compliance with ethical standards
1k
1l
32
64
32
256
32
256
128
256
64
Conflict of interest The authors declare no competing interests.
1m
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
1g
256
256
256
cefazolin 256
20000 5000
b
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