N-Aryl mercaptoacetamides as potential multi-target inhibitors of metallo-β-lactamases (MBLs) and the virulence factor LasB fromPseudomonas aeruginosa

Article abstract of DOI:10.1039/d1md00187f

Increasing antimicrobial resistance is evolving to be one of the major threats to public health. To reduce the selection pressure and thus to avoid a fast development of resistance, novel approaches aim to target bacterial virulence instead of growth. Another strategy is to restore the activity of antibiotics already in clinical use. This can be achieved by the inhibition of resistance factors such as metallo-β-lactamases (MBLs). Since MBLs can cleave almost all β-lactam antibiotics, including the “last resort” carbapenems, their inhibition is of utmost importance. Here, we report on the synthesis andin vitroevaluation ofN-aryl mercaptoacetamides as inhibitors of both clinically relevant MBLs and the virulence factor LasB fromPseudomonas aeruginosa. All testedN-aryl mercaptoacetamides showed low micromolar to submicromolar activities on the tested enzymes IMP-7, NDM-1 and VIM-1. The two most promising compounds were further examined in NDM-1 expressingKlebsiella pneumoniaeisolates, where they restored the full activity of imipenem. Together with their LasB-inhibitory activity in the micromolar range, this class of compounds can now serve as a starting point for a multi-target inhibitor approach against both bacterial resistance and virulence, which is unprecedented in antibacterial drug discovery.

Full text of DOI:10.1039/d1md00187f

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N-Aryl mercaptoacetamides as potential multi-
target inhibitors of metallo-β-lactamases (MBLs)
and the virulence factor LasB from Pseudomonas
aeruginosa†
Cite this: DOI: 10.1039/d1md00187f
Samir Yahiaoui, ‡a Katrin Voos, ‡b Jörg Haupenthal, a Thomas A. Wichelhaus,c
d
Denia Frank, c Lilia Weizel,d Marco Rotter,d Steffen Brunst,d Jan S. Kramer,
ae
Ewgenij Proschak,d Christian Ducho
b and Anna K. H. Hirsch
*
*
Increasing antimicrobial resistance is evolving to be one of the major threats to public health. To reduce
the selection pressure and thus to avoid a fast development of resistance, novel approaches aim to target
bacterial virulence instead of growth. Another strategy is to restore the activity of antibiotics already in
clinical use. This can be achieved by the inhibition of resistance factors such as metallo-β-lactamases
(MBLs). Since MBLs can cleave almost all β-lactam antibiotics, including the “last resort” carbapenems, their
inhibition is of utmost importance. Here, we report on the synthesis and in vitro evaluation of N-aryl
mercaptoacetamides as inhibitors of both clinically relevant MBLs and the virulence factor LasB from
Pseudomonas aeruginosa. All tested N-aryl mercaptoacetamides showed low micromolar to
submicromolar activities on the tested enzymes IMP-7, NDM-1 and VIM-1. The two most promising
compounds were further examined in NDM-1 expressing Klebsiella pneumoniae isolates, where they
restored the full activity of imipenem. Together with their LasB-inhibitory activity in the micromolar range,
this class of compounds can now serve as a starting point for a multi-target inhibitor approach against both
bacterial resistance and virulence, which is unprecedented in antibacterial drug discovery.
Received 4th June 2021,
Accepted 23rd July 2021
DOI: 10.1039/d1md00187f
rsc.li/medchem
priority by the World Health Organization (WHO).1 This
opportunistic pathogen is responsible for fatal lung infections
Introduction
Due to the emergence of drug-resistant bacteria and the lack
of new antibiotics in the pipeline, novel effective and
innovative treatment options are urgently needed, in
particular to cure infections with Gram-negative pathogens.
One of the highly problematic Gram-negative bacteria is
Pseudomonas aeruginosa, which has been assigned critical
in cystic fibrosis patients and many hospital-acquired
infections.2,3 P. aeruginosa is a versatile human pathogen that
easily acquires resistance against multiple classes of
antibacterial agents, including aminoglycosides, quinolones,
polymyxins, and β-lactams.4,5
The β-lactams are still the most commonly used group of
antibiotics.6 One of the main mechanisms of resistance in
Gram-negative pathogens including P. aeruginosa is the
production of β-lactamases.7–9 These enzymes can inactivate
β-lactam antibiotics by the hydrolysis of their four-membered
β-lactam ring.10,11 Based on their mechanism of action, two
main groups of β-lactamases (BLs) were identified: the serine-
β-lactamases (SBLs) and the metallo-β-lactamases (MBLs).
SBLs act via a nucleophilic serine residue that covalently
binds to the carbonyl unit of the β-lactam ring to achieve its
hydrolysis. They have a variable substrate spectrum ranging
from small-, broad- to extended-spectrum, and even
carbapenems can be degraded by some SBLs. Additionally,
several effective SBL inhibitors (e.g., clavulanic acid) are
already in clinical use in combination with β-lactam
antibiotics to overcome resistance.12
a Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) – Helmholtz
Centre for Infection Research (HZI), Campus E8 1, 66123 Saarbrücken, Germany.
E-mail: Anna.Hirsch@Helmholtz-hips.de
b Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland
University, Campus C2 3, 66123 Saarbrücken, Germany.
E-mail: Christian.Ducho@Uni-Saarland.de
c Institute of Medical Microbiology and Infection Control, University Hospital
Frankfurt, Paul-Ehrlich-Straße 40, 60596 Frankfurt, Germany
d Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-
Laue-Straße 9, 60438, Frankfurt, Germany
e Department of Pharmacy, Saarland University, Campus Building E8 1, 66123
Saarbrücken, Germany
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00187f
‡ Equal contribution.
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tissues such as elastin and collagen.32,33 The protease was
also shown to promote biofilm formation through the
regulation of rhamnolipids.34 Moreover, it was found to alter
the human immune response by degrading host defense
components such as cytokines,35 antimicrobial peptides
(AMP),36 and surfactant proteins. These multiple roles of
LasB in the infection process of P. aeruginosa make this
protease an attractive antivirulence target.37,38
Recently, we have demonstrated the potential of
thiocarbamates as prodrugs of N-aryl mercaptoacetamides
(Scheme 1) as promising inhibitors of several bacterial
proteases. Interestingly, these compounds are potent
inhibitors of P. aeruginosa LasB and collagenases from
Clostridium and Bacillus species.39,40 Regarding LasB, in
addition to the elucidation of the binding mode of our hit
compound (1, Scheme 1), we demonstrated the in vivo
efficacy of the same derivative in a Galleria mellonella larvae
infection model. Moreover, compound 1 shows no cytotoxic
effects on HepG2, HEK293 and A549 cells (see ESI,† Table S1)
and its thiocarbamate prodrug 1a (see Scheme 1) remarkably
displays high selectivity over several human matrix
metalloproteases (MMPs).39
It has been demonstrated that several thiol-containing drugs
such as captopril (an angiotensin converting enzyme (ACE)
inhibitor which is used for the treatment of hypertension),
thiorphan (the active metabolite of racecadotril used for the
treatment of diarrhoea), and tiopronin (which is used in
patients with cystinuria, Fig. 2), display promising in vitro
inhibitory activities toward different MBLs.41 The shown three
inhibitors all consist of the same core structure, that is, a
2-substituted (2-mercaptoacetyl)- or (2-mercatopropanoyl)
glycine. Substantial synthetic work has already been done on
this scaffold to obtain novel MBL inhibitors, and co-
crystallization experiments have been reported.42–46
Fig.
1 Chemical structures and reported Ki/IC50 values against
different MBLs of QPX772820,21 and VNRX-5133 (taniborbactam),22–24
two cyclic boronates that are in clinical trials.
MBLs can be divided into classes B1 to B3 which differ in
their amino acid sequence homology and, as a result, their
zinc coordination.13 Class B2 (e.g., CphA) has only one
catalytically active zinc ion and a very narrow substrate
profile,14 while classes B1 (e.g., NDM) and B3 (e.g., AIM)
coordinate two zinc ions and show a very broad substrate
spectrum.14–16 In particular the B1 class is of high clinical
relevance.17–19 So far, two MBL inhibitors having a cyclic
boronate as core structure (see Fig. 1) are in clinical trials.
QPX7728 is in phase I (NCT04380207, alone and with
QPX2014) and VNRX-5133 (taniborbactam) is in phase III in
combination with cefepime (NCT03840148).20–24 However, no
clinically approved MBL inhibitors have been reported to
date.25,26
It is known that bacterial pathogenesis and infectivity are
mediated by microbial components called virulence factors,
which play pivotal roles in adhesion, biofilm formation,
invasion of host tissues, and immune evasion.27,28 Targeting
these factors is a newly emerging approach in the field of
anti-infective drug discovery. This so-called antivirulence
strategy is intended to disarm the bacteria rather than killing
them. Several bacterial virulence factors have been identified
including toxins, extracellular enzymes needed for cell
invasion and biofilm formation, cell-surface proteins that
mediate bacterial attachment, secretion systems, and
quorum-sensing systems that regulate bacterial cell-to-cell
communication.27,29–31
Inspired by this finding, and with respect to our slightly
similar mercaptoacetamide core structure, we explored the
effect of our thiol-based LasB inhibitor (compound 1) toward
a class B1 MBL called imipenemase 7 (IMP-7). Interestingly,
our LasB inhibitor showed a strong inhibitory effect with a
sub-micromolar IC50 value (IC50 = 0.86 0.06 μM).39
One of the major virulence factors of P. aeruginosa is the
elastase LasB. This extracellular zinc-containing protease is
responsible for the invasion of host tissues, biofilm
formation, and immune evasion. It has been demonstrated
that LasB can cleave important components of the connective
Based on the encouraging inhibitory activity of compound
1 against IMP-7, this study aimed to further investigate the
inhibitory profile of the N-aryl mercaptoacetamide class
against various MBLs. Aside from that, another goal was to
explore the potential of using this class as multi-target
Scheme 1 Hydrolysis of thiocarbamate prodrug 1a to its active thiol form 1 and their corresponding IC50 values reported for the inhibition of
IMP-7.39
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Fig. 2 Chemical structures and reported IC50 values of N-aryl mercaptoacetamides, compound 1,39 thiorphan,41 tiopronin41 and captopril47 for
the inhibition of different MBLs.
inhibitors tackling both virulence and bacterial resistance. In
this work, we report on the design, synthesis, and in vitro
evaluation of 13 N-aryl mercaptoacetamide derivatives for
inhibition of three clinically important and transferrable
MBLs of the class B1: IMP-7, Verona integrin encoded
metallo-β-lactamase 1 (VIM-1) and New Delhi metallo-β-
lactamase 1 (NDM-1). Furthermore, we tested the two most
active compounds in combination with the carbapenem
antibiotic imipenem against a clinical NDM-1-expressing
Klebsiella pneumoniae isolate and confirmed their activity on
the bacterial level. The selectivity over nine potential human
off-targets was additionally confirmed.
in their thiocarbamate prodrug form.39 However, the co-crystal
structure revealed the free thiol as the active form. This finding
was confirmed by stability tests and in vitro assays.39,40 In this
work, we therefore synthesised the free-thiol forms of four
compounds, which had previously been tested against LasB and
ColH as thiocarbamates, as well as four novel
mercaptoacetamide derivatives. The synthetic access to the
mercaptoacetamide derivatives is outlined in Scheme 2.
A three-step synthetic route afforded target compounds
18–25, starting from the corresponding aniline derivatives. In a
first step, an amide formation was carried out by dropwise
addition of chloroacetyl chloride to a cooled solution of the
corresponding aniline and DIPEA in CH2Cl2 or THF. Second, an
SN2 reaction of the alkyl chlorides 2–9 with potassium
thioacetate in acetone led to the thioacetates 10–17. Compounds
10–17 were hydrolysed with aqueous KOH in methanol to afford
the thiol target molecules 18–25. These as well as the previously
synthesised compounds 1 and 26–29 (Fig. 3) were then
evaluated with respect to their biological activities.
Results and discussion
Synthesis of N-aryl mercaptoacetamides
Here, we report on the biological evaluation of 13 N-aryl
mercaptoacetamides bearing various polar and nonpolar
substituents on the aryl moiety. We have previously
demonstrated that by variation of the substitution pattern on
the aryl ring (and thus the electron density and H-bonding
interactions), we either obtain LasB or ColH inhibitors.
Additionally, the phenyl ring seems to be crucial for the
selectivity of these inhibitors over human MMPs as potential
off-targets.39,40 Therefore, the impact of the same structural
modifications on the inhibition of MBLs should also be
investigated. Eight out of the 13 compounds were newly
synthesised, while five had already been published as LasB or
ColH inhibitors.39,40 The majority of all our previously reported
N-aryl mercaptoacetamides were tested against LasB and ColH
N-Aryl mercaptoacetamides as broad-spectrum MBL
inhibitors
To explore the potential of our thiol-based class to inhibit
MBLs, we first tested the synthesised compounds 18–25 as
well as the previously reported compounds 1 and 26–29
(Fig. 3) in vitro for their inhibitory potency against NDM-1,
VIM-1 and IMP-7.39,40 Based on their in vitro activity, we then
selected several derivatives for further evaluation in bacteria
in combination with imipenem.
Scheme 2 Synthesis of the thiols 18–25 as potential multi-target inhibitors of LasB and metallo-β-lactamases.
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Fig. 3 Previously reported thiols 26–29 as potential multi-target inhibitors of bacterial virulence and resistance.39,40
MBL activity assays
and as observed for IMP-7, the nonpolar groups were slightly
preferred. Concerning VIM-1, all compounds showed
comparable activities with IC50 values ranging between 2.3
μM (26) and 5.9 μM (24). Additionally, for mono-substituted
We used an activity assay based on kinetic fluorescence using
fluorocillin as a substrate41 to evaluate the inhibitory activity
of the mercaptoacetamide derivatives against NDM-1, VIM-1
and IMP-7. The results can be found in Table 1.
Remarkably, all derivatives were active against all three
MBL enzymes and consequently displayed broad-spectrum
effects in vitro (Table 1). Compound 1 showed a sub-
compounds featuring
a chlorine or methyl group, the
position of the substituent did not have a significant effect
on the biological response.
Growth-inhibition assays
micromolar activity against both IMP-7 (IC50 = 0.86
0.06
μM)39 and NDM-1 (IC50 = 0.65 0.04 μM) and an IC50 of 2.2
0.3 μM against VIM-1. All the other derivatives showed low
micromolar activities against the three tested MBLs.
Regarding many previously reported MBL inhibitors (a
selection can be found in Fig. 2 and 4),41–43,48–50 our
compounds can be considered equipotent. Only the class of
cyclic boronates (e.g. 34 (Fig. 4), QPX7728, and VNRX-5133
(Fig. 1)) was significantly more active against MBLs (with IC50
values in the low nanomolar range) and has already two
candidates in clinical trials.20,23,50,51
In comparison to compound 1, all structural
modifications of the aryl moiety resulted in a moderate loss
of activity against all MBLs. Regarding IMP-7, the IC50 values
range from 1.9 μM (compound 26) to 11 μM (compound 24)
with better tolerance for the nonpolar substituents. The most
potent derivative against NDM-1 was compound 28 (1.5 μM),
Encouraged by the promising in vitro activities of our N-aryl
mercaptoacetamides, we investigated the effects of
compounds 1 and 26 to restore the antibacterial effect of
imipenem (ESI,† Fig. S1) in a growth inhibition assay. As we
wanted to investigate MBL activity without the influence of
LasB inhibition, and as our compounds were the most active
on NDM-1, we selected a clinical imipenem-resistant K.
pneumoniae isolate (strain T2301) that is able to express
NDM-1 but not LasB. K. pneumoniae is one of the known
ESKAPE pathogens and is rated critical by the WHO on their
global priority list of antibiotic-resistant bacteria.52,53
Nosocomial infections with K. pneumoniae have caused
several outbreaks in multiple locations with very high
mortality rates, i.e., up to 72%.54–56 NDM-1 (and other
antibiotic resistance genes) spread between several pathogens
via horizontal transfer,57–59 and NDM-1-expressing K.
Table 1 Chemical structures, MBL and LasB inhibition of N-aryl mercaptoacetamides and their thiocarbamate prodrug forms
a
a
a
a
Thiol IC50
Thiol IC50
Thiol IC50
Thiol IC50
Thiocarbamate
Cp.
R
[μM] IMP-7
[μM] NDM-1
[μM] VIM-1
[μM] LasB
IC50a [μM] LasB
1
3,4-DiCl
H
2-Cl
3-Cl
4-Cl
2-Me
3-Me
4-Me
4-OMe
4-C2H4OH
4-SO2NH2
4-Ac
0.86 0.06
4.7 0.7
1.9 0.5
4.1 1.6
2.4 0.3
6.4 2.0
4.4 1.5
5.4 1.5
8.6 1.8
0.65 0.04
3.6 0.9
1.8 0.1
2.7 0.4
2.1 0.1
6.0 0.5
3.4 0.2
3.4 0.3
5.0 2.1
6.7 3.0
6.3 1.5
1.5 0.2
8.2 2.6
2.2 0.3
5.1 1.3
2.3 0.2
3.6 1.2
4.7 1.8
4.9 1.3
4.1 0.8
5.0 1.6
5.9 1.3
5.9 2.0
4.1 0.9
3.5 0.8
4.0 0.7
6.6 0.3 (ref. 39)
6.2 0.3
—
18
26
19
27
20
21
22
23
24
25
28
29
39
11
14
3
1
2
14
19
16
—
48
36
48
—
—
73
—
1
1
1
21 1 (ref. 39)
14
38
19
24
52
54
72
1
3
1
1
3
3
5
2
1
1
11
4
7.7 1.2
5.4 2.5
2.7 0.7
3
3-Cl-2,6-diMe
12 1 (ref. 39)
a
Means of at least two independent measurements standard deviation.
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Fig. 4 A selection of the structural variations of reported MBL inhibitors and their corresponding inhibitory activities in vitro (30,48 31,43 32,49 33,42
and 3450).
pneumoniae have caused several outbreaks in hospitals in the
last decade.60–62 Growth curves of the bacterial isolate with
imipenem were determined in the presence and absence of
the named compounds (two separate experiments, see
Fig. 5A and B). Compounds 1 and 26 were selected based on
their promising activities against MBLs.
Inhibitory activity towards ColH was not determined, as the best
two compounds on MBLs (1 and 26) had already been tested in
their thiocarbamate form against ColH and did just show
micromolar activities, which is more than three orders of
magnitude less potent than the ColH-inhibiting hit compound
28.40
As shown in the growth curves in Fig. 5, the presence of
imipenem (at 8 μg mL−1, i.e., 0.5× MIC) delayed the initiation
of bacterial growth by a few hours, but after 24 hours, no
relevant differences between the growth of imipenem-treated
bacteria and the imipenem-free control were observed.
Remarkably, when MBL inhibitors 1 (Fig. 5A, at 50 μg mL−1 ( =
21 μM)) or 26 (Fig. 5B, at 50 μg mL−1 ( = 25 μM)) were
combined with imipenem (at 8 μg mL−1), the inhibitory
potential of imipenem was fully restored, and no growth of
bacteria occurred over 24 hours. MBL inhibitors alone did not
exhibit any intrinsic antibacterial activity at 50 μg mL−1. As an
additional control experiment, we employed EDTA as an MBL
inhibitor, with MBL inhibition being caused by the metal-
chelating properties of EDTA (ESI,† Fig. S2). The resultant
growth curves were rather similar to those obtained with MBL
inhibitors 1 and 26, respectively. Overall, this highlights the
potency of the reported MBL inhibitors not just on the
enzymatic, but also on the cellular level, making them
attractive candidates for further development.
The evaluation of compounds 1, 19, 21, 26, and 27 in a
functional LasB inhibition assay expectedly39,40 showed similar
activities compared to their thiocarbamate prodrug derivatives
(Table 1). However, compounds 22 and 23 were two-fold more
active than their thiocarbamate congeners. Regarding the novel
mercaptoacetamides, the unsubstituted derivative 18 as well as
the 2-methyl compound 20 showed decreased IC50 values
relative to compound 1. Moreover, as expected based on our
previous studies,39 compounds 24 and 25 were the least active
derivatives in the series due to the presence of polar
substituents that are unfavourable for LasB inhibition.
Selectivity over other proteases
As most of our compounds all show activity on LasB and the
three tested MBLs, and as it is known that 28 additionally is a
nanomolar collagenase H (ColH) inhibitor,40 it is crucial to
exclude that our compounds are just non-selective zinc-
chelators. Thiocarbamates 1a and 28a had already been
evaluated for their selectivity over a range of six MMPs which
had been chosen with respect to their structural variations in
their S1′ binding pockets.39,40 We decided to test our hit
N-Aryl mercaptoacetamides as multi-target inhibitors of LasB
and MBLs
compound
1 accordingly and, additionally, expand the
The
promising
antibacterial
effects
of
N-aryl
selectivity panel with two histone deacetylases (HDACs) and the
tumour necrosis factor α converting enzyme (TACE). Those
three enzymes are all zinc-dependent proteases.63–65 HDACs
play a crucial role in epigenetic regulation by modification of
gene expression and thus in cell proliferation.66,67 TACE, also
known as “a disintegrin and metalloprotease 17” (ADAM17), is
mercaptoacetamides in combination with imipenem (Fig. 5)
prompted us to further investigate the potential of this class as
multi-target inhibitors targeting both virulence and resistance.
Therefore, all synthesised compounds were evaluated for their
potential inhibition of LasB as a bacterial virulence factor.
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Fig. 5 Growth-inhibition assay. Growth curves of NDM-1-expressing K. pneumoniae (T2301) over time (h) in the absence and presence of
imipenem at 8 μg mL−1 (i.e., 0.5× MIC) MBL inhibitors at 50 μg mL−1 ( = 21 μM (1) or 25 μM (26)); compound 1 (A) and compound 26 (B).
important for a functional immune system, for the nervous
system and tissue generation.68–71 Therefore, these enzymes
represent important antitargets. As controls, batimastat (ESI,†
Fig. S3) as unselective MMP inhibitor, trichostatin A (ESI,† Fig.
S4), a known inhibitor of HDACs, and ilomastat as TACE
inhibitor (ESI,† Fig. S5) were chosen. Compound 1 was tested at
a concentration of 100 μM for its inhibitory activity on those
nine enzymes. The results are reported in Table 2.
Table 2 Inhibition values of six MMPs, TACE and two HDACs in the presence of 100 μM 1, 0.5 μM trichostatin A and IC50 values of batimastat.40,72
Ilomastat resulted in 91 4% inhibition of TACE activity at 1 μM
1 inhibition at 100 μMa [%]
Batimastat IC50a [nM]
Trichostatin A inhibition at 0.5 μMa [%]
MMP-1
MMP-2
MMP-3
MMP-7
MMP-8
MMP-14
TACE
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
40
9
11
2.2 0.1
1.8 0.1
5.6 0.9
7.0 0.2
0.7 0.2
2.8 0.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4
5
HDAC-3
HDAC-8
7
n.d.
n.d.
94
95
3
2
a
Means and SD of at least two independent measurements are displayed; n.d. = not determined; n.i. = no inhibition (if ≤10%).
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Compound 1 showed no inhibition of the six tested MMPs
and the two HDACs. A slight inhibition of TACE (40 4%) at
a rather high concentration of 100 μM was found, but this
still corresponds to a selectivity factor of more than five,
which should be considered sufficient in particular in this
early phase of development.
the reported compounds on virulence factors can make
bacteria less pathogenic and more vulnerable towards
immune response and/or low doses of antibiotics. Even
though no improvement of the in vitro inhibitory activities of
compound 1 has been achieved so far, the reported results
indicate that some structural variations in the aryl moiety
appear to be feasible. This opens the way to a future
optimisation of this compound class, also with respect to
ADME-T properties.
Cytotoxicity assays
It had already been demonstrated in our previous studies
that compound 1 did not show any cytotoxicity against the
three tested cell lines HepG2, HEK293 and A549 at a
concentration of 100 μM. The results can be found in the
ESI,† Table S1.39,73
Additionally, compound 1 already had been subjected to a
more complex in vivo toxicity model in zebrafish embryos
(ESI,† Table S2).73 Here the maximum tolerated concentration
(MTC) was 10 μM. At higher concentrations (e.g. 30 μM), the
compound precipitated in this assay and the zebrafish
embryos died.73 Further studies to determine whether the
toxicity derived from the compound's in vivo effect itself or
from the formation of crystals will be performed in the
course of future hit optimisation.
Experimental section
Chemistry
Compounds 1,39 26,40 27,39,40 2840 and 2939 were previously
reported. All other compounds were synthesised according to
Scheme 1 as described in detail in the ESI.†
IMP-7, NDM-1 and VIM-1 in vitro inhibition assays
The assays were performed according to
a previously
described procedure.41 They were carried out at room
temperature in black polystyrol 96-well plates (Corning) using
fluorocillin (prepared as described by Rukavishnikov et al.74),
as substrate. The fluorescence emitted by the fluorescent
product difluorofluorescein was kinetically monitored using
a Tecan fluorescent plate reader Infinite F200. IC50 values
were calculated using data obtained from measurements with
at least six different inhibitor concentrations, applying a
sigmoidal dose–response (variable slope with four
parameters) equation using GraphPad Prism 5 (GraphPad
software, La Jolla, CA, USA) software.
Conclusions
In summary, based on the encouraging inhibitory activity of
a previously reported LasB inhibitor (compound 1) against
IMP-7, we designed, synthesised and evaluated a series of
N-aryl mercaptoacetamide derivatives against the three
metallo-β-lactamases IMP-7, VIM-1 and NDM-1. Interestingly,
all tested compounds showed promising inhibition of all
three MBLs. Compound 1 displayed the strongest inhibitory
effects with submicromolar IC50 values toward both IMP-7
and NDM-1, and low micromolar activity against VIM-1.
To confirm the potential of our mercaptoacetamide
derivatives as MBL inhibitors, compounds 1 and 26 were
tested in combination with imipenem against a clinical NDM-
1-expressing K. pneumoniae isolate. Remarkably, at 50 μg
mL−1 (∼20 μM), both compounds were able to restore the
activity of imipenem. Additionally, compound 1 showed high
selectivity over a total of nine potential off-targets (six MMPs,
TACE and two HDACs) at a concentration of 100 μM, at which
it already previously had demonstrated to also have no
cytotoxic effects on human cell lines.39,73 These promising
effects of our N-aryl mercaptoacetamides against MBLs, their
previously confirmed anti-LasB properties and their
demonstrated high selectivity over human off-targets inspires
the use of this class as multi-target inhibitors tackling both
virulence and resistance of pathogens. This is a new approach
that is unprecedented in antibacterial drug discovery.
Growth inhibition assay
A
computerised incubator (Tecan Infinite M200 pro,
Crailsheim, Germany) was used to obtain the growth curves
over a time course. Optical density (OD600) was measured for
evaluating bacterial (i.e., K. pneumoniae NDM-1) density in
suspension. Prior to each experiment, bacteria were cultured
to the exponential phase (OD600 = 0.6–0.8) in cation-adjusted
Mueller Hinton broth (Becton Dickinson, Heidelberg,
Germany). 5 μL of diluted bacterial suspension and 100 μL of
cation-adjusted Mueller Hinton broth either with or without
imipenem (Arcos Organics, Schwerte, Germany) and/or the
respective MBL inhibitor were added into wells of a 96 well
plate to give a final bacterial inoculum of approx. 5 × 105
CFU mL−1. The temperature was adjusted to 37 °C. A
permanent shaking speed of 450 rpm was used and only
interrupted before OD600 measurement. The optical densities
of samples were recorded on-line every 10 minutes for 24
hours. Experiments were determined in triplicate.
Representative growth curves are shown. For quality control,
bacterial strain E. coli ATCC 25922 was used.
Although further investigations will be needed to improve
the inhibitory potencies of our inhibitors, our results
demonstrate an attractive new multi-targeting approach,
which can enhance the efficacy of currently used antibiotics
against resistant MBL-expressing pathogens. In addition to
restoring the sensitivity to antimicrobial agents, the effect of
LasB in vitro inhibition assay
The purification of LasB as well as the performance,
measurement and evaluation of the FRET-based in vitro
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inhibition assay were carried out as previously described by
Treatment, Drug Resist. Updates, 2000, 3(4), 247–255, DOI:
10.1054/drup.2000.0152.
Kany et al.39
3 D. M. Ramsey and D. J. Wozniak, Understanding the Control
of Pseudomonas Aeruginosa Alginate Synthesis and the
Prospects for Management of Chronic Infections in Cystic
Fibrosis: P. Aeruginosa Pathogenesis, Mol. Microbiol.,
2005, 56(2), 309–322, DOI: 10.1111/j.1365-2958.2005.04552.x.
4 Z. Pang, R. Raudonis, B. R. Glick, T.-J. Lin and Z. Cheng,
Antibiotic Resistance in Pseudomonas Aeruginosa: Mechanisms
and Alternative Therapeutic Strategies, Biotechnol. Adv.,
2019, 37(1), 177–192, DOI: 10.1016/j.biotechadv.2018.11.013.
5 M. Bassetti, A. Vena, A. Croxatto, E. Righi and B. Guery, How
to Manage Pseudomonas Aeruginosa Infections, Drugs
Context, 2018, 7, 1–18, DOI: 10.7573/dic.212527.
MMP in vitro inhibition assay
The assays were performed as previously described.39,40
TACE in vitro inhibition assay
ADAM-17 (TACE) inhibitor screening assay kit was purchased
from Sigma-Aldrich. The assay was performed according to
the guidelines of the manufacturer. Fluorescence signals were
measured in a CLARIOstar plate reader (BMG Labtech).
HDAC in vitro inhibition assay
6 K. Bush and P. A. Bradford, β-Lactams and β-Lactamase
Inhibitors: An Overview, Cold Spring Harb. Perspect. Med.,
2016, 6(8), a025247, DOI: 10.1101/cshperspect.a025247.
7 D. J. Wolter and P. D. Lister, Mechanisms of β-Lactam
Resistance Among Pseudomonas Aeruginosa, Curr. Pharm.
Des., 2013, 19, 209–222, DOI: 10.2174/138161213804070311.
8 D. Rawat and D. Nair, Extended-Spectrum β-Lactamases in
Gram Negative Bacteria, J. Global Infect. Dis., 2010, 2(3),
263–274, DOI: 10.4103/0974-777X.68531.
9 A. A. Medeiros, Evolution and Dissemination of
β-Lactamases Accelerated by Generations of β-Lactam
Antibiotics, Clin. Infect. Dis., 1997, 24(Supplement 1),
S19–S45, DOI: 10.1093/clinids/24.supplement_1.s19.
10 A. A. Medeiros, β-Lactamases: Quality and Resistance, Clin.
Microbiol. Infect., 1997, 3, 4S2–4S9, DOI: 10.1016/S1198-
743X(14)65030-8.
11 M. I. Page and A. P. Laws, The Mechanism of Catalysis and
the Inhibition of β-Lactamases, Chem. Commun.,
1998, 1609–1617, DOI: 10.1039/A803578D.
HDAC3 and HDAC8 inhibitor screening kits were purchased
from Sigma-Aldrich. The assay was performed according to
the guidelines of the manufacturer.
Author contributions
Samir Yahiaoui, investigation, writing – original draft; Katrin
Voos, investigation, validation, visualization, writing – original
draft;
Jörg
Haupenthal,
conceptualization,
project
administration, validation, writing – review & editing; Thomas
A. Wichelhaus, project administration, resources, supervision,
validation, visualization, writing – review & editing; Denia Frank,
investigation; Lilia Weizel, investigation; Marco Rotter,
investigation; Steffen Brunst, investigation; Jan S. Kramer,
investigation; Ewgenij Proschak, project administration,
supervision, validation, writing – review & editing; Christian
Ducho, conceptualization, project administration, supervision,
validation, writing – review & editing; Anna K. H. Hirsch,
conceptualization, funding acquisition, project administration,
supervision, validation, writing – review & editing.
12 K. A. Toussaint and J. C. Gallagher, β-Lactam/β-Lactamase
Inhibitor Combinations: From Then to Now, Ann. Pharmacother.,
2015, 49(1), 86–98, DOI: 10.1177/1060028014556652.
13 T. Palzkill, Metallo-β-Lactamase Structure and Function, Ann.
N. Y. Acad. Sci., 2013, 1277(1), 91–104, DOI: 10.1111/j.1749-
6632.2012.06796.x.
Conflicts of interest
There are no conflicts to declare.
14 C. Bebrone, Metallo-β-Lactamases (Classification, Activity,
Genetic Organization, Structure, Zinc Coordination) and
Their Superfamily, Biochem. Pharmacol., 2007, 74(12),
1686–1701, DOI: 10.1016/j.bcp.2007.05.021.
Acknowledgements
The authors thank Jeannine Jung and Jelena Konstantinović
for excellent technical support. A. K. H. Hirsch gratefully
acknowledges funding from the Helmholtz-Association's
Initiative and Networking Fund. E. Proschak thanks German
Research Foundation (DFG, Heisenberg-Professur PR1405/7-1)
for financial support.
15 M. I. Abboud, C. Damblon, J. Brem, N. Smargiasso, P.
Mercuri, B. Gilbert, A. M. Rydzik, T. D. W. Claridge, C. J.
Schofield and J.-M. Frère, Interaction of Avibactam with
Class B Metallo-β-Lactamases, Antimicrob. Agents Chemother.,
2016, 60(10), 5655–5662, DOI: 10.1128/AAC.00897-16.
16 M.-N. Lisa, A. R. Palacios, M. Aitha, M. M. González, D. M.
Moreno, M. W. Crowder, R. A. Bonomo, J. Spencer, D. L.
Tierney, L. I. Llarrull and A. J. A. Vila, General Reaction
Mechanism for Carbapenem Hydrolysis by Mononuclear and
Binuclear Metallo-β-Lactamases, Nat. Commun., 2017, 8(1),
538, DOI: 10.1038/s41467-017-00601-9.
Notes and references
1 World Health Organization, Antibacterial Agents in Clinical
Development, in An Analysis of the Antibacterial Clinical
Development Pipeline, Including Tuberculosis, The World
Health Organization, Geneva, 2017.
2 R. E. W. Hancock and D. P. Speert, Antibiotic Resistance in
Pseudomonas Aeruginosa: Mechanisms and Impact on
17 P. Nordmann, L. Poirel, T. R. Walsh and D. M.
Livermore, The Emerging NDM Carbapenemases, Trends
RSC Med. Chem.
This journal is © The Royal Society of Chemistry 2021

View Article Online
RSC Medicinal Chemistry
Research Article
Microbiol., 2011, 19(12), 588–595, DOI: 10.1016/j.
tim.2011.09.005.
Surface Proteins of Staphylococcus Aureus, Nat. Rev.
Microbiol., 2014, 12(1), 49–62, DOI: 10.1038/nrmicro3161.
28 J. W. Newman, R. V. Floyd and J. L. Fothergill, The
Contribution of Pseudomonas Aeruginosa Virulence Factors
and Host Factors in the Establishment of Urinary Tract
Infections, FEMS Microbiol. Lett., 2017, 364(15), 1–11, DOI:
10.1093/femsle/fnx124.
18 M. F. Mojica, R. A. Bonomo and W. Fast, B1-Metallo-β-
Lactamases: Where Do We Stand?, Curr. Drug Targets, 2016, 17(9),
1029–1050, DOI: 10.2174/1389450116666151001105622.
19 A. U. Khan, L. Maryam and R. Zarrilli, Structure, Genetics
and Worldwide Spread of New Delhi Metallo-β-Lactamase
(NDM):
A
Threat to Public Health, BMC Microbiol.,
29 K. Tam and V. J. Torres, Staphylococcus aureus Secreted
Toxins and Extracellular Enzymes, in Gram-Positive
Pathogens, ed. V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A.
Portnoy, M. Braunstein and J. I. Rood, ASM Press,
Washington, DC, USA, 2019, pp. 640–668, DOI: 10.1128/
9781683670131.ch40.
30 S. T. Rutherford and B. L. Bassler, Bacterial Quorum
Sensing: Its Role in Virulence and Possibilities for Its
Control, Cold Spring Harb. Perspect. Med., 2012, 2(11),
a012427–a012427, DOI: 10.1101/cshperspect.a012427.
31 M. B. Calvert, V. R. Jumde and A. Titz, Pathoblockers or
Antivirulence Drugs as a New Option for the Treatment of
Bacterial Infections, Beilstein J. Org. Chem., 2018, 14,
2607–2617, DOI: 10.3762/bjoc.14.239.
32 B. Wretlind and O. R. Pavlovskis, Pseudomonas Aeruginosa
Elastase and Its Role in Pseudomonas Infections, Clin.
Infect. Dis., 1983, 5(Supplement_5), S998–S1004, DOI:
10.1093/clinids/5.Supplement_5.S998.
33 L. W. Heck, K. Morihara, W. B. McRae and E. J. Miller,
Specific Cleavage of Human Type III and IV Collagens by
2017, 17(1), 101, DOI: 10.1186/s12866-017-1012-8.
20 S. J. Hecker, K. R. Reddy, O. Lomovskaya, D. C. Griffith, D.
Rubio-Aparicio, K. Nelson, R. Tsivkovski, D. Sun, M. Sabet, Z.
Tarazi, J. Parkinson, M. Totrov, S. H. Boyer, T. W. Glinka, O. A.
Pemberton, Y. Chen and M. N. Dudley, Discovery of Cyclic
Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of
Serine and Metallo-β-Lactamases, J. Med. Chem., 2020, 63(14),
7491–7507, DOI: 10.1021/acs.jmedchem.9b01976.
21 O. Lomovskaya, D. Rubio-Aparicio, K. Nelson, D. Sun, R.
Tsivkovski, M. Castanheira, J. Lindley, J. Loutit and M. Dudley,
In Vitro Activity of the Ultrabroad-Spectrum Beta-Lactamase
Inhibitor QPX7728 in Combination with Multiple Beta-Lactam
Antibiotics against Pseudomonas Aeruginosa, Antimicrob. Agents
Chemother., 2021, 65(6), e00210-21, DOI: 10.1128/AAC.00210-21.
22 J. C. Hamrick, J.-D. Docquier, T. Uehara, C. L. Myers, D. A. Six,
C. L. Chatwin, K. J. John, S. F. Vernacchio, S. M. Cusick,
R. E. L. Trout, C. Pozzi, F. De Luca, M. Benvenuti, S. Mangani,
B. Liu, R. W. Jackson, G. Moeck, L. Xerri, C. J. Burns, D. C.
Pevear and D. M. Daigle, VNRX-5133 (Taniborbactam), a
Broad-Spectrum Inhibitor of Serine- and Metallo-β-
Lactamases, Restores Activity of Cefepime in Enterobacterales
and Pseudomonas Aeruginosa, Antimicrob. Agents Chemother.,
2020, 64(3), e01963-19, DOI: 10.1128/AAC.01963-19.
Pseudomonas
Aeruginosa
Elastase,
Infect.
Immun.,
1986, 51(1), 115–118, DOI: 10.1128/IAI.51.1.115-118.1986.
34 H. Yu, X. He, W. Xie, J. Xiong, H. Sheng, S. Guo, C. Huang,
D. Zhang and K. Zhang, Elastase LasB of Pseudomonas
Aeruginosa Promotes Biofilm Formation Partly through
Rhamnolipid-Mediated Regulation, Can. J. Microbiol.,
2014, 60(4), 227–235, DOI: 10.1139/cjm-2013-0667.
35 M. Parmely, A. Gale, M. Clabaugh, R. Horvat and W. W.
Zhou, Proteolytic Inactivation of Cytokines by Pseudomonas
Aeruginosa, Infect. Immun., 1990, 58(9), 3009–3014, DOI:
10.1128/IAI.58.9.3009-3014.1990.
36 A. Schmidtchen, I.-M. Frick, E. Andersson, H. Tapper and L.
Björck, Proteinases of common pathogenic bacteria degrade
and inactivate the antibacterial peptide LL-37, Mol.
Microbiol., 2002, 46(1), 157–168, DOI: 10.1046/j.1365-
2958.2002.03146.x.
37 Z. Kuang, Y. Hao, B. E. Walling, J. L. Jeffries, D. E. Ohman
and G. W. Lau, Pseudomonas Aeruginosa Elastase Provides
an Escape from Phagocytosis by Degrading the Pulmonary
Surfactant Protein-A, PLoS One, 2011, 6(11), e27091, DOI:
10.1371/journal.pone.0027091.
23 B. Liu, R. E. L. Trout, G.-H. Chu, D. McGarry, R. W. Jackson,
J. C. Hamrick, D. M. Daigle, S. M. Cusick, C. Pozzi, F. De
Luca, M. Benvenuti, S. Mangani, J.-D. Docquier, W. J. Weiss,
D. C. Pevear, L. Xerri and C. J. Burns, Discovery of
Taniborbactam (VNRX-5133): A Broad-Spectrum Serine- and
Metallo-β-Lactamase Inhibitor for Carbapenem-Resistant
Bacterial Infections, J. Med. Chem., 2020, 63(6), 2789–2801,
DOI: 10.1021/acs.jmedchem.9b01518.
24 S. Mushtaq, A. Vickers, M. Doumith, M. J. Ellington, N.
Woodford and D. M. Livermore, Activity of β-Lactam/
Taniborbactam
(VNRX-5133)
Combinations
against
Carbapenem-Resistant Gram-Negative Bacteria, J. Antimicrob.
Chemother., 2021, 76(1), 160–170, DOI: 10.1093/jac/dkaa391.
25 Faridoon and U. I. Nazar, An Update on the Status of Potent
Inhibitors of Metallo-β-Lactamases, Sci. Pharm., 2013, 81(2),
309–328, DOI: 10.3797/scipharm.1302-08.
26 Z. Cheng, C. A. Thomas, A. R. Joyner, R. L. Kimble, A. M.
Sturgill, N.-Y. Tran, M. R. Vulcan, S. A. Klinsky, D. J. Orea,
C. R. Platt, F. Cao, B. Li, Q. Yang, C. J. Yurkiewicz, W. Fast
38 J. F. Alcorn and J. R. Wright, Degradation of Pulmonary
Surfactant Protein D by Pseudomonas Aeruginosa Elastase
Abrogates Innate Immune Function, J. Biol. Chem.,
2004, 279(29), 30871–30879, DOI: 10.1074/jbc.M400796200.
39 A. M. Kany, A. Sikandar, J. Haupenthal, S. Yahiaoui, C. K.
Maurer, E. Proschak, J. Köhnke and R. W. Hartmann,
Binding Mode Characterization and Early in Vivo Evaluation
of Fragment-Like Thiols as Inhibitors of the Virulence Factor
and M. W. Crowder, MBLinhibitors.Com,
a
Website
Resource Offering Information and Expertise for the
Continued Development of Metallo-β-Lactamase Inhibitors,
Biomolecules, 2020, 10(3), 459, DOI: 10.3390/biom10030459.
27 T. J. Foster, J. A. Geoghegan, V. K. Ganesh and M. Höök,
Adhesion, Invasion and Evasion: The Many Functions of the
This journal is © The Royal Society of Chemistry 2021
RSC Med. Chem.

View Article Online
Research Article
RSC Medicinal Chemistry
LasB from Pseudomonas Aeruginosa, ACS Infect. Dis.,
2018, 4(6), 988–997, DOI: 10.1021/acsinfecdis.8b00010.
40 E. Schönauer, A. M. Kany, J. Haupenthal, K. Hüsecken, I. J.
Hoppe, K. Voos, S. Yahiaoui, B. Elsässer, C. Ducho, H.
Brandstetter and R. W. Hartmann, Discovery of a Potent
Inhibitor Class with High Selectivity toward Clostridial
Collagenases, J. Am. Chem. Soc., 2017, 139(36), 12696–12703,
DOI: 10.1021/jacs.7b06935.
41 F.-M. Klingler, T. A. Wichelhaus, D. Frank, J. Cuesta-Bernal,
J. El-Delik, H. F. Müller, H. Sjuts, S. Göttig, A. Koenigs, K. M.
Pos, D. Pogoryelov and E. Proschak, Approved Drugs
Containing Thiols as Inhibitors of Metallo-β-Lactamases:
Strategy To Combat Multidrug-Resistant Bacteria, J. Med.
Chem., 2015, 58(8), 3626–3630, DOI: 10.1021/jm501844d.
42 J. Ma, Q. Cao, S. M. McLeod, K. Ferguson, N. Gao, A. L.
Breeze and J. Hu, Target-Based Whole-Cell Screening by 1 H
NMR Spectroscopy, Angew. Chem., Int. Ed., 2015, 54(16),
4764–4767, DOI: 10.1002/anie.201410701.
50 J. Brem, R. Cain, S. Cahill, M. A. McDonough, I. J. Clifton,
J.-C. Jiménez-Castellanos, M. B. Avison, J. Spencer, C. W. G.
Fishwick and C. J. Schofield, Structural Basis of Metallo-β-
Lactamase, Serine-β-Lactamase and Penicillin-Binding
Protein Inhibition by Cyclic Boronates, Nat. Commun.,
2016, 7(1), 12406, DOI: 10.1038/ncomms12406.
51 S. T. Cahill, R. Cain, D. Y. Wang, C. T. Lohans, D. W.
Wareham, H. P. Oswin, J. Mohammed, J. Spencer, C. W. G.
Fishwick, M. A. McDonough, C. J. Schofield and J. Brem,
Cyclic Boronates Inhibit All Classes of β-Lactamases,
Antimicrob. Agents Chemother., 2017, 61(4), e02260-16, DOI:
10.1128/AAC.02260-16.
52 E. Tacconelli, N. Magrini, Y. Carmeli, S. Harbarth, G.
Kahlmeter, J. Kluytmans, M. Mendelson, C. Pulcini, N. Singh
and U. Theuretzbacher, Global Priority List of Antibiotic-
Resistant Bacteria to Guide Research, Discovery, and
Development of New Antibiotics, World Health Organization,
2017, vol. 27, pp. 318–327.
43 Y.-L. Wang, S. Liu, Z.-J. Yu, Y. Lei, M.-Y. Huang, Y.-H. Yan, Q.
Ma, Y. Zheng, H. Deng, Y. Sun, C. Wu, Y. Yu, Q. Chen, Z.
Wang, Y. Wu and G.-B. Li, Structure-Based Development of
(1-(3′-Mercaptopropanamido)Methyl)Boronic Acid Derived
Broad-Spectrum, Dual-Action Inhibitors of Metallo- and
Serine-β-Lactamases, J. Med. Chem., 2019, 62(15), 7160–7184,
DOI: 10.1021/acs.jmedchem.9b00735.
44 Y. Yan, G. Li and G. Li, Principles and Current Strategies
Targeting Metallo-β-lactamase Mediated Antibacterial Resistance,
Med. Res. Rev., 2020, 40(5), 1558–1592, DOI: 10.1002/med.21665.
45 S. Liu, L. Jing, Z.-J. Yu, C. Wu, Y. Zheng, E. Zhang, Q. Chen,
Y. Yu, L. Guo, Y. Wu and G.-B. Li, (( S )-3-Mercapto-2-
Methylpropanamido)Acetic Acid Derivatives as Metallo-β-
53 L. B. Rice, Federal Funding for the Study of Antimicrobial
Resistance in Nosocomial Pathogens: No ESKAPE, J. Infect.
Dis., 2008, 197(8), 1079–1081, DOI: 10.1086/533452.
54 A. Borer, L. Saidel-Odes, K. Riesenberg, S. Eskira, N. Peled,
R. Nativ, F. Schlaeffer and M. Sherf, Attributable Mortality
Rate for Carbapenem-Resistant Klebsiella Pneumoniae
Bacteremia, Infect. Control Hosp. Epidemiol., 2009, 30(10),
972–976.
55 E. B. Hirsch and V. H. Tam, Detection and Treatment Options
for Klebsiella Pneumoniae Carbapenemases (KPCs): An
Emerging Cause of Multidrug-Resistant Infection, J. Antimicrob.
Chemother., 2010, 65(6), 1119–1125, DOI: 10.1093/jac/dkq108.
56 P. Nordmann, G. Cuzon and T. Naas, The Real Threat of
Klebsiella Pneumoniae Carbapenemase-Producing Bacteria,
Lancet Infect. Dis., 2009, 9(4), 228–236, DOI: 10.1016/S1473-
3099(09)70054-4.
Lactamase
Inhibitors:
Synthesis,
Kinetic
and
Crystallographic Studies, Eur. J. Med. Chem., 2018, 145,
649–660, DOI: 10.1016/j.ejmech.2018.01.032.
46 G.-B. Li, J. Brem, R. Lesniak, M. I. Abboud, C. T. Lohans, I. J.
Clifton, S.-Y. Yang, J.-C. Jiménez-Castellanos, M. B. Avison, J.
Spencer, M. A. McDonough and C. J. Schofield,
Crystallographic Analyses of Isoquinoline Complexes Reveal a
New Mode of Metallo-β-Lactamase Inhibition, Chem. Commun.,
2017, 53(43), 5806–5809, DOI: 10.1039/C7CC02394D.
47 J. Brem, S. S. van Berkel, D. Zollman, S. Y. Lee, O. Gileadi,
P. J. McHugh, T. R. Walsh, M. A. McDonough and C. J.
Schofield, Structural Basis of Metallo-β-Lactamase Inhibition
by Captopril Stereoisomers, Antimicrob. Agents Chemother.,
2016, 60(1), 142–150, DOI: 10.1128/AAC.01335-15.
48 P. Chen, L. B. Horton, R. L. Mikulski, L. Deng, S. Sundriyal,
T. Palzkill and Y. Song, 2-Substituted 4,5-Dihydrothiazole-4-
Carboxylic Acids Are Novel Inhibitors of Metallo-β-
Lactamases, Bioorg. Med. Chem. Lett., 2012, 22(19),
6229–6232, DOI: 10.1016/j.bmcl.2012.08.012.
57 S. Chatterjee, A. Mondal, S. Mitra and S. Basu, Acinetobacter
Baumannii Transfers the BlaNDM-1 Gene via Outer
Membrane Vesicles, J. Antimicrob. Chemother., 2017, 72(8),
2201–2207, DOI: 10.1093/jac/dkx131.
58 T. Krahn, D. Wibberg, I. Maus, A. Winkler, S. Bontron, A.
Sczyrba, P. Nordmann, A. Pühler, L. Poirel and A. Schlüter,
Intraspecies Transfer of the Chromosomal Acinetobacter
Baumannii Bla NDM-1 Carbapenemase Gene, Antimicrob.
Agents Chemother., 2016, 60(5), 3032–3040, DOI: 10.1128/
AAC.00124-16.
59 H. Lee, J. Shin, Y.-J. Chung, M. Park, K. J. Kang, J. Y. Baek,
D. Shin, D. R. Chung, K. R. Peck, J.-H. Song and K. S. Ko,
Co-Introduction of Plasmids Harbouring the Carbapenemase
Genes, BlaNDM-1 and BlaOXA-232, Increases Fitness and
Virulence of Bacterial Host, J. Biomed. Sci., 2020, 27(1), 8,
DOI: 10.1186/s12929-019-0603-0.
49 J. Brem, S. S. van Berkel, W. Aik, A. M. Rydzik, M. B. Avison,
I. Pettinati, K.-D. Umland, A. Kawamura, J. Spencer, T. D. W.
Claridge, M. A. McDonough and C. J. Schofield, Rhodanine
Hydrolysis Leads to Potent Thioenolate Mediated Metallo-β-
Lactamase Inhibition, Nat. Chem., 2014, 6(12), 1084–1090,
DOI: 10.1038/nchem.2110.
60 L. Poirel, Z. Al Maskari, F. Al Rashdi, S. Bernabeu and P.
Nordmann, NDM-1-Producing Klebsiella Pneumoniae
Isolated in the Sultanate of Oman, J. Antimicrob. Chemother.,
2011, 66(2), 304–306, DOI: 10.1093/jac/dkq428.
61 E. Voulgari, C. Gartzonika, G. Vrioni, L. Politi, E. Priavali, S.
Levidiotou-Stefanou and A. Tsakris, The Balkan Region:
RSC Med. Chem.
This journal is © The Royal Society of Chemistry 2021

View Article Online
RSC Medicinal Chemistry
Research Article
NDM-1-Producing Klebsiella Pneumoniae ST11 Clonal Strain
Causing Outbreaks in Greece, J. Antimicrob. Chemother.,
2014, 69(8), 2091–2097, DOI: 10.1093/jac/dku105.
Lymphocytes, Proc. Natl. Acad. Sci. U. S. A., 1966, 55(4),
805–812, DOI: 10.1073/pnas.55.4.805.
68 F. Zunke and S. Rose-John, The Shedding Protease ADAM17:
Physiology and Pathophysiology, Biochim. Biophys. Acta, Mol.
Cell Res., 2017, 1864(11), 2059–2070, DOI: 10.1016/j.
bbamcr.2017.07.001.
69 M. Gooz, ADAM-17: The Enzyme That Does It All, Crit. Rev.
Biochem. Mol. Biol., 2010, 45(2), 146–169, DOI: 10.3109/
10409231003628015.
70 J. Scheller, A. Chalaris, C. Garbers and S. Rose-John,
ADAM17: A Molecular Switch to Control Inflammation and
Tissue Regeneration, Trends Immunol., 2011, 32(8), 380–387,
DOI: 10.1016/j.it.2011.05.005.
62 J. A. Escobar Pérez, N. M. Olarte Escobar, B. Castro-Cardozo,
I. A. Valderrama Márquez, M. I. Garzón Aguilar, L. Martinez
de la Barrera, E. R. Barrero Barreto, R. A. Marquez-Ortiz,
M. V. Moncada Guayazán and N. Vanegas Gómez, Outbreak
of NDM-1-Producing Klebsiella Pneumoniae in a Neonatal
Unit in Colombia, Antimicrob. Agents Chemother., 2013, 57(4),
1957–1960, DOI: 10.1128/AAC.01447-12.
63 A. Vannini, C. Volpari, G. Filocamo, E. C. Casavola, M.
Brunetti, D. Renzoni, P. Chakravarty, C. Paolini, R. De
Francesco, P. Gallinari, C. Steinkuhler and S. Di Marco,
Crystal Structure of a Eukaryotic Zinc-Dependent Histone
Deacetylase, Human HDAC8, Complexed with a Hydroxamic
Acid Inhibitor, Proc. Natl. Acad. Sci. U. S. A., 2004, 101(42),
15064–15069, DOI: 10.1073/pnas.0404603101.
71 R. Menghini, L. Fiorentino, V. Casagrande, R. Lauro and M.
Federici, The Role of ADAM17 in Metabolic Inflammation,
Atherosclerosis, 2013, 228(1), 12–17, DOI: 10.1016/j.
atherosclerosis.2013.01.024.
64 K. Maskos, C. Fernandez-Catalan, R. Huber, G. P.
Bourenkov, H. Bartunik, G. A. Ellestad, P. Reddy, M. F.
Wolfson, C. T. Rauch, B. J. Castner, R. Davis, H. R. G. Clarke,
M. Petersen, J. N. Fitzner, D. P. Cerretti, C. J. March, R. J.
Paxton, R. A. Black and W. Bode, Crystal Structure of the
Catalytic Domain of Human Tumor Necrosis Factor-α-
Converting Enzyme, Proc. Natl. Acad. Sci. U. S. A., 1998, 95(7),
3408–3412, DOI: 10.1073/pnas.95.7.3408.
65 P. J. Watson, L. Fairall, G. M. Santos and J. W. R. Schwabe,
Structure of HDAC3 Bound to Co-Repressor and Inositol
Tetraphosphate, Nature, 2012, 481(7381), 335–340, DOI:
10.1038/nature10728.
72 A. M. Kany, A. Sikandar, S. Yahiaoui, J. Haupenthal, I.
Walter, M. Empting, J. Köhnke and R. W. Hartmann,
Tackling Pseudomonas Aeruginosa Virulence by
a
Hydroxamic Acid-Based LasB Inhibitor, ACS Chem. Biol.,
2018, 13(9), 2449–2455, DOI: 10.1021/acschembio.8b00257.
73 J. Konstantinović, S. Yahiaoui, A. Alhayek, J. Haupenthal, E.
Schönauer, A. Andreas, A. M. Kany, R. Mueller, J. Köhnke, F.
Berger, M. Bischoff, R. W. Hartmann, H. Brandstetter and
A. K. H. Hirsch, N-Aryl-3-Mercaptosuccinimides as
Antivirulence Agents Targeting Pseudomonas Aeruginosa
Elastase and Clostridium Collagenases, J. Med. Chem.,
2020, 63(15), 8359–8368, DOI: 10.1021/acs.jmedchem.0c00584.
74 A. Rukavishnikov, K. R. Gee, I. Johnson and S. Corry,
Fluorogenic Cephalosporin Substrates for β-Lactamase TEM-
1, Anal. Biochem., 2011, 419(1), 9–16, DOI: 10.1016/j.
ab.2011.07.020.
66 K. Struhl, Histone Acetylation and Transcriptional
Regulatory Mechanisms, Genes Dev., 1998, 12, 599–606.
67 B. G. Pogo, V. G. Allfrey and A. E. Mirsky, RNA Synthesis and
Histone Acetylation during the Course of Gene Activation in
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