Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of Imipenemase-Type Metallo-β-Lactamases

Article abstract of DOI:10.1021/acs.jmedchem.1c00362

In an attempt to exploit the hydrolytic mechanism by which β-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the β-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.

Full text of DOI:10.1021/acs.jmedchem.1c00362

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Article
Novel Cephalosporin Conjugates Display Potent and Selective
Inhibition of Imipenemase-Type Metallo-β-Lactamases
̈
Kamaleddin H. M. E. Tehrani, Nicola Wade, Vida Mashayekhi, Nora C. Bruchle, Willem Jespers,
Koen Voskuil, Diego Pesce, Matthijs J. van Haren, Gerard J. P. van Westen, and Nathaniel I. Martin*
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ABSTRACT: In an attempt to exploit the hydrolytic mechanism by
which β-lactamases degrade cephalosporins, we designed and synthe-
sized a series of novel cephalosporin prodrugs aimed at delivering thiol-
based inhibitors of metallo-β-lactamases (MBLs) in a spatiotemporally
controlled fashion. While enzymatic hydrolysis of the β-lactam ring was
observed, it was not accompanied by inhibitor release. Nonetheless, the
cephalosporin prodrugs, especially thiomandelic acid conjugate (8),
demonstrated potent inhibition of IMP-type MBLs. In addition,
conjugate 8 was also found to greatly reduce the minimum inhibitory
concentration of meropenem against IMP-producing bacteria. The
results of kinetic experiments indicate that these prodrugs inhibit IMP-
type MBLs by acting as slowly turned-over substrates. Structure−activity
relationship studies revealed that both phenyl and carboxyl moieties of 8
are crucial for its potency. Furthermore, modeling studies indicate that
productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and
selectivity.
to rapid oxidation to their corresponding disulfides, leading to
the loss of zinc-binding affinity, MBL inhibition, and
synergistic activity.11 Recently, we reported a cephalosporin
prodrug approach to selectively enable the release of strong
zinc-chelating small molecules upon hydrolysis by MBLs.12 In
doing so, we identified inhibitors with potent activity against
NDM- and VIM-type MBLs. In the present study, we aimed to
apply a similar design strategy employing thiol-based MBL
inhibitors 1−3. As illustrated in Figure 1B, the prodrugs
consist of a cephalosporin core with the thiol-based MBL
inhibitors linked at the 3-position. The hydrolytic action of
MBLs upon such conjugates was envisioned to result in a
cascade reaction, ultimately leading to release of the inhibitor
with both spatial and temporal control. We hypothesized that
in the case of thiol-based inhibitors 1−3, this prodrug strategy
could be effective by addressing not only the selectivity but
also their poor stability stemming from their rapid oxidation.
Here, we describe the preparation of cephalosporin−thiol
conjugates 6 and 8−10 and evaluation of their performance as
INTRODUCTION
■
Despite the growing threat of β-lactam resistance caused by
metallo-β-lactamases (MBLs), there are no approved drugs on
the market that target this class of enzymes. Unlike serine-β-
lactamases, MBLs are metalloenzymes containing one or two
zinc ions in their active site. An activated water molecule,
coordinated by these zinc ions, in turn acts as the nucleophile
in the hydrolysis of all classes of β-lactams (except
monobactams).1−3 MBLs of particular clinical significance
are the New Delhi metallo-β-lactamase (NDM), Verona
integron-encoded metallo-β-lactamase (VIM), and imipene-
mase (IMP) families, all of which possess broad spectrum β-
lactamase activity.4 The previously reported inhibitors of
MBLs have been the subject of several comprehensive review
articles.5−8 Indeed, a wide range of compounds have been
reported as MBL inhibitors with the majority acting by either
sequestering zinc and/or by forming a ternary complex with
metalloenzyme.9,10
Previously, we described the in vitro ability of a selected
group of thiols (1−3, Figure 1A) to inhibit MBLs and in doing
so resensitize a panel of MBL-producing clinical isolates to
meropenem, a potent carbapenem antibiotic.11 Our earlier
studies used isothermal titration calorimetry (ITC) to
demonstrate that thiols 1 and 2 bind zinc with Kd values of
10 and 20 μM, respectively. However, as we also
demonstrated, these thiol-containing compounds are prone
Received: February 28, 2021
Published: June 29, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00362
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Figure 1. (A) Previously reported thiols as MBL inhibitors. (B) Cephalosporin prodrugs of the thiols 1−3 and their related structural analogues.
a
Scheme 1. Chemical Route to the Cephalosporin Conjugates
a
Reagents and Conditions: (a) NaI, DMF, r.t., 30 min; (b) NaHCO3, 1, r.t., 20 h; (c) TFA, Anisole, 0 °C, 1 h; (d) BF3·OEt2, Thiols, acetonitrile,
45 °C, 2 h; (e) phenylacetyl chloride, saturated NaHCO3 solution, acetone, r.t., 20 h.
MBL-inhibitor prodrugs capable of resensitizing MBL-
expressing strains to β-lactam antibiotics.
acid (7-ACA, 7) with the corresponding thiols, followed by
acylation of the 7-amino group (see the Experimental Section
for detailed procedures). Notably, conjugate 8 was prepared as
a diastereomeric mixture, given the stereochemical instability
of the corresponding thiomandelic acid building block 2.13
Compounds 11−13 and 15 were designed and synthesized for
the purpose of structure−activity relationship evaluation of the
thiol conjugates 6 and 8−10.
To assess the zinc-binding properties of the cephalosporin−
thiol conjugates, ITC binding studies were performed, which
revealed no appreciable binding interaction with zinc (data not
shown). This is in contrast with the thiols 1−3 themselves,
RESULTS AND DISCUSSION
■
The cephalosporin−thiol conjugates were synthesized using
two different routes (Scheme 1). Thioalkylation of mercaptoa-
cetophenone with the chloromethyl cephalosporin “GCLE”
(4), a common intermediate used in the industrial synthesis of
cephalosporin antibiotics, yielded intermediate 5, followed by
deprotection with trifluoroacetic acid (TFA) to yield
compound 6. Alternatively, compounds 8−13 were prepared
via the BF3-promoted substitution of 7-aminocephalosporanic
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Table 1. MIC of Meropenem in Combination with the Cephalosporin Conjugates Tested at Multiple Concentrations against
Four Clinical Isolates
meropenem MIC (μg/mL)
compound
concentration (μg/mL)
E. cloacae (IMP-1)
K. pneumoniae (IMP-28)
E. coli (VIM-2)
E. coli (NDM-1)
6
0
32
64
128
0
16
8 (2)
8 (2)
4 (4)
32
4
8
32
a
1 (4)
8 (1)
4 (2)
4 (2)
8
16 (2)
8 (4)
8 (4)
32
0.5 (8)
0.25 (16)
4
8
32
64
128
0
1 (32)
0.5 (64)
0.5 (64)
32
≤0.063 (≥64)
≤0.063 (≥64)
≤0.063 (≥64)
4
4 (2)
2 (4)
1 (8)
8
16 (2)
16 (2)
8 (4)
32
9
32
64
128
0
32
64
128
0
32
64
128
0
32
64
128
0
1 (32)
1 (32)
0.5 (64)
16
8 (2)
8 (2)
4 (4)
32
8 (4)
8 (4)
4 (8)
16
8 (2)
8 (2)
8 (2)
16
≤0.063 (≥64)
≤0.063 (≥64)
≤0.063 (≥64)
4
4 (2)
4 (2)
2 (4)
8
8 (1)
8 (1)
4 (2)
4
2 (2)
2 (2)
1 (4)
8
8 (1)
4 (2)
4 (2)
4
16 (2)
8 (4)
8 (4)
64
32 (2)
32 (2)
16 (4)
32
16 (2)
16 (2)
16 (2)
64
32 (2)
16 (4)
16 (4)
32
10
11
12
13
15
1 (4)
0.5 (8)
0.25 (16)
2
0.5 (4)
0.25 (8)
0.25 (8)
4
0.5 (8)
0.25 (16)
0.25 (16)
2
0.25 (8)
0.25 (8)
0.125 (16)
2
32
64
128
0
8 (2)
8 (2)
4 (4)
16
2 (2)
2 (2)
1 (4)
4
16 (2)
16 (2)
8 (4)
32
32
64
128
0
16 (1)
16 (1)
16 (1)
32
1 (2)
2 (2)
2 (2)
1 (4)
4
16 (2)
16 (2)
16 (2)
32
0.5 (4)
0.25 (8)
2
b
DPA
32
64
128
16 (2)
1 (32)
0.25 (128)
0.125 (16)
≤0.031 (≥64)
≤0.031 (≥64)
≤0.063 (≥64)
≤0.063 (≥64)
≤0.063 (≥64)
0.5 (64)
0.5 (64)
≤0.5 (≥64)
a
b
Fold reduction of MIC shown in brackets. Dipicolinic acid.
which were previously shown to be strong zinc-binders with
low μM Kd values.11 In addition, stability analyses were
performed to test whether inhibitor release occurred
spontaneously. Following overnight incubation in Mueller−
Hinton broth (MHB), HPLC analysis of the conjugates 6 and
8−10 showed that all demonstrated good stability (>95%
intact after 15 h, Table S1).
The compounds were tested for their performance against a
panel of MBL-producing clinical isolates. After it was found
that the cephalosporin conjugates do not inhibit bacterial
growth at concentrations up to 128 μg/mL, their ability to
restore the antibacterial activity of meropenem was tested. The
results showed that compound 8 and 9 were potent synergists,
lowering the minimum inhibitory concentration (MIC) of
meropenem against IMP-producing isolates most effectively
(Table 1).
employed the chromogenic cephalosporin nitrocefin as the
substrate.8 The IC50 data thus obtained revealed conjugates 8
and 9 to be particularly potent and selective IMP inhibitors
(Table 2). These findings are consistent with the trends
observed in the bacterial growth inhibition synergy assays,
where strains possessing IMP-type enzymes were also most
sensitive to meropenem when coadministered with conjugates
8 and 9. Sequence alignment of IMP-1 and IMP-28 does not
reveal any differences in the amino acids comprising the active
sites (Table S2). Overall, the two enzymes share a high
sequence identity (92.28%). Among the few amino acid
differences outside the active site, the His306Gln mutation has
been proposed to be responsible for the overall decreased
hydrolytic efficiency of IMP-28 compared to IMP-1.14
However, as far as the inhibitory profile of the cephalosporins
evaluated in this study is concerned, the two enzymes showed
similar performance.
Encouraged by the promising results against the MBL-
producing clinical isolates, we tested the ability of the
conjugates to inhibit purified IMP-1, IMP-28, VIM-2, and
NDM-1 enzymes. The biochemical assay used for these studies
To investigate the mechanism of inhibition, and more
specifically to assess release of the thiol inhibitors, the most
potent cephalosporin conjugates 8 and 9 were incubated with
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when 8 and 9 were subjected to the same experiment, these
vinylic signals were not detected (Figure 2B), suggesting that
the thiols at the 3-position were not released. The results of
Table 2. IC50 (μM) of Cephalosporin Conjugates Reported
as Mean SD
a
compound
IMP-1
IMP-28
NDM-1
VIM-2
1
these H NMR studies were further corroborated by LC−MS
6
8
9
10
11
12
13
15
DPA
3.3 0.2
0.47 0.08
4.7 0.4
48 0.7
14
1
77 12
>100
94 0.2
76 11
10 0.5
analyses of the enzymatic hydrolysis products of 8 and 9,
which revealed the hydrolyzed β-lactam compounds 8H and
9H as the only detectable products (Figure 2C, see Supporting
Information Figures S2−S4 and S7 for complete NMR and
LC−MS data). Given that previous studies published by our
group and others have detected the release of aromatic thiols
as leaving groups after incubation with MBLs,12,18 we ascribe
the observed lack of inhibitor release in our study to the poor
leaving group quality of the aliphatic thiols we used.
0.46 0.04
1.1 0.2
30
45
4.3 0.8
>100
>100
16
19
1
1
9
5
51
1
43
3
>100
>100
72
73 0.1
49
53 12
57
10 0.8
2.2 0.4
>100
>100
3
2
>100
10 0.1
9
29 0.5
29
5
a
Assay used nitrocefin used as the chromogenic substrate. A detailed
The finding that compounds 8 and 9 demonstrate potent
inhibition of IMP-28 despite not releasing the corresponding
zinc-binding thiol inhibitors upon MBL-mediated β-lactam
hydrolysis was surprising. To better understand the mechanism
of inhibition of these cephalosporin conjugates, we next
determined the kinetic parameters of the hydrolysis of the
cephalosporin conjugates using purified MBL enzymes. In
addition, we evaluated the MBL-inhibitory activity of the
partial hydrolysis products 8H and 9H. The kinetic analysis of
the hydrolysis of the cephalosporins by IMP-28, NDM-1, and
VIM-2 provided valuable insights into the IMP-28 selectivity
and inhibitory potency observed for 8 and 9. These analyses
description of the assay can be found in the Experimental Section.
IMP-28 and analyzed using 1H NMR and LC−MS techniques.
It has been shown previously by our group and others that the
molecular mechanism of cephalosporin hydrolysis can be
probed in situ using NMR techniques.12,15−17 In the present
study, we used the commercially available 7-phenylacetylamide
derivative of 7-ACA (compound 16, Figure 2A) as a positive
control. After incubating compound 16 with IMP-28, the rapid
appearance of vinylic protons corresponding to the elimination
product was detected at ca. 5.50 ppm (Figure 2A). However,
Figure 2. (A) Enzymatic degradation of 16 showing the growth of the signals corresponding to the vinylic protons of 16H resonating as two
singlets ca. 5.5 ppm. (B) Enzymatic degradation of 9 instead leads to 9H. For the purpose of clarity, the segment corresponding to water signal has
been omitted from the NMR spectra. (C) LC−MS analysis of IMP-28-mediated degradation of 9, confirms the exclusive formation of 9H.
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showed that IMP-28 has the lowest catalytic efficiency for 8
and 9 among the tested cephalosporins (see Table 3 for
relative kcat/KM data). Comparison with the other major MBL
families also revealed that 8 and 9 were hydrolyzed more
efficiently by NDM-1 and VIM-2 than by IMP-28. We also
determined the IC50 of 8 and 9 against IMP-28 following
different incubation times (0−60 min). As shown in Figure S5,
maximum increase in potency is observed after 15 min
incubation, after which the potency stabilizes. This might
indicate that either active site recognition does not occur
instantly or that over 15 min time course, the more potent
hydrolysis products (8H and 9H) are produced, which might
be responsible for the sub-micromolar inhibition. These
findings indicate that conjugates 8 and 9 inhibit IMP-28
either by acting as slowly turned-over substrates and/or that
the hydrolyzed products 8H and 9H are more tightly bound
within the IMP active site than either the NDM or VIM active
sites.
To evaluate the inhibitory activity of the hydrolysis products
8H and 9H, the intact conjugates 8 and 9 were first fully
hydrolyzed by incubation with NDM-1, as described in the
Experimental Section (see Figure S8 for the LC−MS traces).
Following hydrolysis, the NDM-1 enzyme was completely
removed via spin-filtration, as confirmed by the lack of
nitrocefin activity in the filtrate. The partially hydrolyzed 8H
and 9H were then tested for their capacity to inhibit MBLs.
Table 3. Michaelis−Menten Parameters Determined for the
Cephalosporin Conjugates as Substrates of IMP-28, VIM-2,
a b
,
and NDM-1.
kcat/KM
relative
kcat/KM
enzyme substrate KM (μM) kcat (s−1
)
(μM−1·s−1
)
NDM-1
8
9
8
9
8
14.0
21.2
8.28
4.30
129 13
249
175 19
20.5
2
4
2
1
13.1
17.4
4.06
2.35
0.386
2.83
41.0
10.8
6.12
0.936
0.821
0.490
0.546
0.003
0.011
0.234
0.529
0.073
0.169
0.059
100
88
52
VIM-2
58
IMP-28
0.30
1.2
44
56
14
9
7
10
11
12
13
15
4
83.3 16
219 23
392 73
37.0
23.3
18
6.3
a
Experimental procedure has been described in detail in the
b
Experimental Section. See the Supporting Information for the
Michaelis−Menten graphs.
Figure 3. ITC thermograms resulting from titration of ZnSO4 into a solution containing: (A) compound 8; (B) purified NDM-1; (C) compound
8H; (D) compound 9; (E) purified NDM-1; (F) compound 9H.
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Figure 4. Modeling studies of compound 8 and 8H docked into the active site of IMP-1 (PDB ID: 1dd6)19 and NDM-1 (PDB ID: 4RL2).16 For
simplicity, only the diastereomer of 8 containing the R-thiomandelic acid moiety has been shown. (A) Compound 8 in the active site of IMP-1. The
carboxylates of both the thiomandelic acid moiety and at the cephem C-4 position are predicted to interact with the zinc ions in IMP-1. Also
notable is the π−π interaction of phenyl ring of 8 with Trp28. (B) Compound 8H in the active site of IMP-1. Compared to 8, the carboxylate
resulting from the β-lactam hydrolysis is predicted to replace the thiomandelic acid carboxylate in coordinating the zinc ions. In addition, the
phenyl ring of the thiomandelic acid moiety is extended toward the lipophilic pocket around Leu165. (C) Docking model of compound 8 shown in
an ensemble of overlaid X-ray structures of IMP-1. (D) Docking model of compound 8 shown in an ensemble of overlaid X-ray structures of NDM-
1.
Interestingly, both hydrolysis products were found to possess
potent activity against IMP-1 and IMP-28 with sub-micro-
molar IC50 values (Table S3). In addition, the hydrolysis
products 8H and 9H were evaluated for their zinc-binding
affinity using ITC. When zinc was titrated into the solution of
8 and 9 preincubated with NDM-1 to generate 8H and 9H in
situ, a binding interaction was observed with Kd values of 9.67
and 3.19 μM measured, respectively (Figure 3). By
comparison, the intact cephalosporins showed no zinc-binding
affinity. This affinity for zinc binding may therefore also
contribute to the inhibitory activity of 8H and 9H.
The IC50 data obtained for the various conjugates prepared
also provide some structure−activity insights (Table 2).
Specifically, elimination of the carboxylic acid (11), phenyl
group (13), or the entire thiomandelic acid fragment (15)
causes the activity against IMP-1 and IMP-28 to be decreased
by ca. 100-fold, suggesting that the thiomandelic acid fragment
introduces productive binding interactions with the IMP active
site. Among the derivatives, the moderate synergistic activity of
13 against IMP-28 producing Klebsiella pneumoniae could be
due to the fact that lower lipophilicity might contribute to
improved cellular accumulation of 13. Also interesting was the
observation that compound 9 was ca. 10-fold and 30-fold more
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potent than its diastereomer 10 against IMP-1 and IMP-28,
respectively.
Among them, cyclic boronates represented by taniborbac-
tam,20 indole carboxylic acids,21 and the thiazole carboxylic
acid ANT43122,23 hold promise for further clinical develop-
ment. As recently shown by our group and others, MBLs in the
B1 class (NDM, VIM, and IMP) exhibit different inhibitory
profiles when tested against known MBL inhibitors in vitro.24,25
Clearly, the development of selective and broad-spectrum
MBL inhibitors is not an easy task. In the case of ANT431 and
its related analogues, for instance, there is a promising
inhibitory activity against NDM-1 (IC50 = 2.67 μM) and
VIM-2 (IC50 = 6.7 μM). However, VIM-1 and IMP-1 remain
resistant. At this time, taniborbactam is the only β-lactamase
inhibitor in clinical trials that also exhibits activity against
MBLs (currently phase III, ClinicalTrials.gov Identifier:
NCT03840148).20 The unique cyclic boronate pharmaco-
phore26 of taniborbactam allows it to mimic the transition state
of the β-lactam hydrolysis reaction mediated by both serine
and MBLs. This explains the potent and broad-spectrum
activity of taniborbactam against β-lactamases of all four
Ambler classes (A−D). Interestingly, however, a closer look at
the MBL inhibition profile of taniborbactam and its related
analogues shows weak inhibition of the IMP family despite the
sub-micromolar IC50 against NDM-1 and VIM-2.20 Notably, a
recently published patent filing describes a library of indole-2-
carboxylic acids some of which demonstrate sub-micromolar
IC50 values against IMP-1, NDM-1, and VIM-2, as well as
promising synergistic activity in combination with merope-
nem.21 Interestingly, compound 8 described in our present
study demonstrates potent and selective inhibition of IMP-type
MBLs with comparatively little activity against NDM- and
VIM-type enzymes. In the absence of structural insights by X-
ray crystallography, computational approaches offer a means of
rationalizing this activity. In addition, the availability of crystal
structures for other MBLs from the B2 (e.g., CphA27) and B3
(e.g., L128 and AIM-129) classes also points to the opportunity
to conduct similar in silico studies aimed at designing inhibitors
that also cover such enzymes.
In an attempt to provide further insights into the binding
mode of the most potent compound (8), a computational
model was derived based on docking of this compound to the
published crystal structure of IMP-1.19 The docking hypothesis
is based on the high resemblance of 8H to the hydrolysis
product of cephalexin, a compound previously cocrystallized
with NDM-1.16 Thus, we overlaid the IMP-1 and NDM-1
structures and used the maximum common substructure
(MCS, see the Experimental Section) between 8H and the
hydrolysis product of cephalexin. The resulting docking pose
aligned well with 8H, as well as other representative hydrolysis
products presented in this work. Noteworthy is the fact that
both diastereomers of 8H, which could not be separated for
the assay, are accommodated in the binding site and in a
similar fashion (see Figure S9). We next studied the docking of
the parent compound 8, which revealed some interesting
findings in comparison with its hydrolysis product 8H: in the
docking model of compound 8 in IMP-1 (Figure 4A), the zinc
ions are anchored by the carboxylate on cephem C-4 together
with the carboxylate from the thiomandelic acid moiety. The
latter in 8H (Figure 4B) is replaced by the carboxylate
resulting from β-lactam hydrolysis. In NDM-1, however, the
carboxylate on cephem C-4 as well as β-lactam carbonyl of 8
are predicted to interact with the zinc centers (see the
Supporting Information for supplementary PDB files). This
difference in the mode of interaction of 8 with IMP-1 versus
NDM-1 may explain the importance of thiomandelic acid
carboxylate for the potency of 8 against IMPs. Also notable for
8H is the way the phenyl group of the thiomandelic acid
moiety interacts with the Trp28 and Leu165 residues of IMP-
1. The binding mode of 8 suggests that this phenyl group can
engage in a π−π interaction with Trp28 (Figure 4A). Upon
hydrolysis to 8H, however, this phenyl group is predicted to
move away from Trp28 and toward Leu165, where it can form
hydrophobic interactions in a pocket around that residue
(Figure 4B). Another interesting observation that may at least
partly explain the selectivity of 8 for IMP versus NDM-type
MBLs is a predicted interaction with Trp28. An analysis of all
available IMP-1 structures (Figure 4C) indicates that the
Trp28 side chain is part of a flexible loop region that can form
a cage around the binding site, optimally accommodating
compound 8. Interestingly, in NDM-1 this residue is replaced
by Phe70, where it is part of a more flexible region, as observed
in the analysis of all X-ray structures for NDM-1 (Figure 4D).
These differences may contribute to a mode of binding for
compound 8 in the NDM-1 active site that leads to hydrolysis
of the β-lactam ring, while in the case of IMP-1, a different
binding mode is adopted, leading instead to inhibition.
Specifically, the lower flexibility of the L3 loop in IMP-1,
and its higher proximity toward the side-chain phenyl of
compound 8, might therefore enforce binding interactions that
support the inhibitory activity of compound 8 rather than
accelerating its hydrolysis by the enzyme. This might explain
the relatively high KM value of 8 despite its potent inhibitory
activity against IMP-1. Molecular dynamics (MD) simulations
were also performed showing that the docking poses for each
diastereomer of 8 and 8H were stable along triplicate MD
simulations (3 × 10 ns, see Experimental Section for details).
In recent years, a number of groups have reported attempts
at designing inhibitors that offer a higher MBL selectivity than
zinc-chelating agents (e.g., DPA and EDTA) can provide.
CONCLUSIONS
■
We here describe a series of cephalosporin-based MBL
inhibitor prodrugs designed to release zinc-chelating small-
molecule thiols upon MBL-mediated hydrolysis. Notably,
while displaying potent inhibition of IMP-type MBLs, these
conjugates did not function as mechanistically predicted. While
MBL-mediated hydrolysis was observed, the release of the
small-molecule thiol fragments did not spontaneously occur for
the conjugates included in this study. This lack of release is
presumably due to the pKa of the corresponding thiols not
being low enough to enable them to behave as leaving groups.
Nonetheless, the finding that the cephalosporin conjugates (6,
8, and 9) selectively inhibit IMP enzymes is notable. Based on
kinetic analyses, the most potent conjugates 8 and 9 were
shown to be slowly turned-over substrates of IMP-28. In
addition, the hydrolysis products 8H and 9H were found to be
IMP-selective inhibitors. Our findings suggest that the IMP
inhibition observed with compounds 8 and 9 may be due to a
combination of effects, whereby the slowly turned-over
substrate and the resulting hydrolysis product both contribute
to enzyme inhibition. Furthermore, modeling studies indicate
that the interaction of 8/8H and 9/9H with the IMP active
site residues Trp28 and Leu165 may contribute to the
observed potency and IMP selectivity. These findings provide
new insights expected to be of value in future efforts aimed at
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white solid, which was filtered off and washed with minimum water
and ether. The crude material was purified by reversed-phase prep-
HPLC using a C18 column and an optimal gradient of buffer A (H2O
95%, ACN 5%, and TFA 0.1%) versus buffer B (ACN 95%, H2O 5%,
and TFA 0.1%). The quantities and yields below are reported based
on the purification of 100 mg of the crude product by prep-HPLC.
Compound 8. 40 mg (26%, over two steps). 1H NMR (400 MHz,
DMSO-d6): diastereomeric mixture δ 9.07 (apparent t, 1.8 H), 7.44−
7.21 (m, aromatic H, 9H), 5.61 (m, β-lactam C−H, 1.8H), 5.04 (d, J
= 4.8 Hz, β-lactam C−H, 0.8H), 4.88 (d, J = 4.7 Hz, β-lactam C−H,
1H), 4.65 (apparent d, aliphatic C−H, 1.8H), 3.69−3.32 (m, aliphatic
CH2, 10.8H), 13C NMR (101 MHz, DMSO-d6): δ 172.05, 171.98,
171.45, 171.43, 164.99, 163.52, 163.48, 137.83, 137.54, 136.29,
129.51, 129.05, 128.94, 128.89, 128.76, 128.71, 128.31, 127.24,
126.97, 125.65, 125.60, 59.38, 58.16, 52.63, 52.60, 42.06, 34.12, 33.76,
27.51, 27.44, HRMS (ESI): [M − H]− calcd, 497.0847; found,
497.0842.
improving the potency, as well as broadening the spectrum of
inhibition, of cephalosporin-based MBL inhibitor prodrugs.
EXPERIMENTAL SECTION
■
General. Compound 4 (GCLE), 7-ACA (7), and 7-ADCA (14)
were purchased from Combi-Blocks (US) and nitrocefin from
Cayman Chemical. The preparation of thiols 1−3 was performed as
previously described.11 Compound 16 was synthesized via the
acylation of 7-ACA following a previously reported procedure.30
Proton and carbon nuclear magnetic resonance spectra were recorded
on an AV400 NMR spectrometer (Bruker), and samples were
dissolved in CDCl3 or DMSO-d6. HRMS analyses were performed on
a Thermo Scientific Dionex UltiMate 3000 HPLC system with a
Phenomenex Kinetex C18 column (2.1 × 150 mm, 2.6 μm) at 35 °C
and equipped with a diode array detector. The samples were eluted
over a gradient of solution A (0.1% formic acid in water) versus
solution B (0.1% formic acid in ACN). This system was connected to
a Bruker micrOTOF-Q II mass spectrometer (ESI ionization)
calibrated internally with sodium formate. The purity of all the final
compounds was found to be >97%, as determined by NMR and
HPLC analyses.
Compound 9. 69 mg (47%, over two steps). 1H NMR (400 MHz,
DMSO-d6): δ 9.14 (d, J = 8.3 Hz, N−H, 1H), 7.39−7.13 (m,
aromatic H, 10H), 5.67 (dd, J = 8.3, 4.7 Hz, β-lactam C−H, 1H), 5.08
(d, J = 4.8 Hz, β-lactam C−H, 1H), 3.90−3.47 (m, aliphatic H, 7H),
3.04 (dd, J = 13.7, 9.8 Hz, aliphatic H, 1H), 2.93 (dd, J = 13.7, 5.8 Hz,
aliphatic H, 1H), 13C NMR (101 MHz, DMSO-d6): δ 172.61, 170.99,
164.66, 163.08, 138.14, 135.84, 129.03, 129.01, 128.27, 128.23,
127.45, 126.55, 126.50, 124.95, 58.96, 57.75, 48.23, 41.58, 38.16,
33.38, 26.97, HRMS (ESI): [M − H]− calcd, 511.1003; found,
511.1000.
Compound 6. GCLE (4, 1.0 g, 2.1 mmol) and NaI (314 mg, 2.1
mmol) were stirred in DMF (10 mL) for 30 min at room
temperature. Then, mercaptoacetophenone (479 mg, 3.15 mmol)
and sodium bicarbonate (200 mg, 2.38 mmol) were added
successively, and the mixture was stirred overnight. The reaction
mixture was then partitioned between water and DCM, followed by
washing the organic layer with brine (3 × 20 mL). The concentration
of the organic layer and purification of the residue on silica using ethyl
acetate and the DCM mixture as the eluent furnished 5 as a pale-
yellow solid (854 mg, 68%). 1H NMR (400 MHz, CDCl3): δ 7.89 (d,
J = 8.3 Hz, aromatic H, 1H), 7.58 (t, J = 8.0 Hz, aromatic H, 1H),
7.45 (t, J = 8.0 Hz, aromatic H, 2H), 7.37−7.25 (m, aromatic H, 7H),
6.85 (dd, J = 8.6 Hz, J = 1.8 Hz, aromatic H, 2H), 5.99 (d, J = 9.2 Hz,
1H), 5.77 (m, β-lactam C−H, 1H), 5.14 (s, benzyloxy CH2, 2H), 4.90
(d, J = 4.9 Hz, 1H), 3.99−3.45 (m, aliphatic H, 11H), 13C NMR (101
MHz, DMSO-d6): δ 194.41, 171.14, 164.50, 161.52, 159.83, 135.37,
133.72, 133.46, 130.67, 129.40, 129.10, 128.69, 128.53, 128.49,
127.64, 126.78, 124.58, 113.91, 67.93, 59.03, 57.74, 55.23, 43.26,
37.81, 33.81, 27.72. HRMS (ESI): [M + H]+ calcd, 603.1624; found,
603.1620. To 5 (600 mg, 1.0 mmol) was added TFA/anisole (15 mL/
3 mL), and the mixture was stirred at 0 °C for 1 h. It was then
concentrated under vacuum, and the residue was precipitated by a 1:1
mixture of diethyl ether and petroleum ether. The solid was isolated
by centrifugation and purified by reversed-phase prep-HPLC using a
C18 column and an optimal gradient of buffer A (H2O 95%, ACN
5%, and TFA 0.1%) versus buffer B (ACN 95%, H2O 5%, and TFA
0.1%) to afford 6 (51 mg, 35%, based on the purification of ∼100 mg
of the crude product by prep-HPLC). 1H NMR (400 MHz, CDCl3):
δ 7.85 (d, J = 7.3 Hz, aromatic H, 1H), 7.53−7.22 (m, aromatic H,
8H), 6.50 (d, J = 8.8 Hz, 1H), 5.72 (dd, J = 8.9 Hz, J = 4.7 Hz, β-
lactam C−H, 1H), 4.90 (d, J = 4.7 Hz, β-lactam C−H, 1H), 3.97−
3.44 (m, aliphatic H, 8H), 13C NMR (101 MHz, DMSO-d6): δ
195.06, 171.36, 165.01, 163.45, 136.24, 135.91, 133.78, 129.44,
129.15, 128.79, 128.63, 127.36, 126.90, 125.49, 59.37, 58.22, 42.03,
38.20, 33.70, 27.45. HRMS (ESI): [M − H]− calcd, 481.0897; found,
481.0863.
Compound 10. 33 mg (43%, over two steps). 1H NMR (400 MHz,
DMSO-d6): δ 9.12 (d, J = 8.4 Hz, N−H, 1H), 7.37−7.17 (m,
aromatic H, 10H), 5.65 (dd, J = 8.4, 4.7 Hz, β-lactam C−H, 1H), 4.98
(d, J = 4.8 Hz, β-lactam C−H, 1H), 3.73−3.33 (m, aliphatic H, 7H),
3.07 (dd, J = 13.8, 8.6 Hz, aliphatic H, 1H), 2.89 (dd, J = 13.8, 7.1 Hz,
aliphatic H, 1H), 13C NMR (101 MHz, DMSO-d6): δ 173.36, 171.43,
165.03, 163.48, 138.76, 136.29, 129.61, 129.50, 128.71, 128.67,
126.98, 126.62, 125.77, 59.38, 58.19, 47.85, 42.07, 37.81, 33.57, 27.10,
HRMS (ESI): [M + H]+ calcd, 513.1154; found, 513.1151.
Compound 11. 82 mg (74%, over two steps). 1H NMR (400 MHz,
DMSO-d6): δ 9.13 (d, J = 8.3 Hz, NH, 1H), 7.35−7.22 (m, aromatic
H, 5H), 5.65 (dd, J = 8.3 Hz, J = 4.7 Hz, β-lactam C−H, 1H), 5.06 (d,
J = 4.7 Hz, β-lactam C−H, 1H), 3.79−3.47 (m, aliphatic H, 8H), 13C
NMR (101 MHz, DMSO-d6): δ 171.41, 165.11, 163.62, 138.81,
136.29, 129.49, 129.34, 128.89, 128.69, 128.22, 127.37, 126.96,
125.25, 59.40, 58.36, 42.06, 35.81, 33.78, 27.44. HRMS (ESI): [M +
H]+ calcd, 455.1099; found, 455.1098.
Compound 12. 79 mg (68%, over two steps). 1H NMR (400 MHz,
DMSO-d6): δ 9.15 (d, J = 8.3 Hz, N−H, 1H), 7.33−7.19 (m,
aromatic H, 10H), 5.65 (dd, J = 8.4, 4.7 Hz, β-lactam C−H, 1H), 5.13
(d, J = 4.7, β-lactam C−H, 1H), 3.82−3.49 (m, aliphatic H, 6H),
2.85−2.65 (m, aliphatic H, 4H). 13C NMR (101 MHz, DMSO-d6): δ
171.45, 165.20, 163.73, 140.89, 136.30, 129.50, 129.29, 129.01,
128.78, 128.70, 126.97, 126.64, 125.11, 59.40, 58.51, 42.07, 36.30,
32.95, 32.44, 27.39, HRMS (ESI): [M + H]+ calcd, 469.1256; found,
469.1256.
Compound 13. 88 mg (27%, over two steps). 1H NMR (400 MHz,
DMSO-d6): δ 9.14 (d, J = 8.3 Hz, N−H, 1H), 7.34−7.21 (m,
aromatic H, 5H), 5.65 (dd, J = 8.3 Hz, J = 4.7 Hz, β-lactam C−H,
1H), 5.11 (d, J = 4.8 Hz, β-lactam C−H, 1H), 3.73−3.20 (m,
aliphatic H, 8H), 13C NMR (101 MHz, DMSO-d6): δ 170.55, 170.44,
164.10, 162.47, 135.31, 128.51, 127.72, 126.48, 125.99, 124.48, 58.43,
57.31, 41.10, 32.94, 32.74, 26.42. HRMS (ESI): [M + H]+ calcd,
423.0685; found, 423.0702.
Compound 15. 7-ADCA (14, 2.14 g, 10 mmol) was dissolved in
saturated bicarbonate solution (20 mL), to which phenylacetyl
chloride (1.5 mL, 11.3 mmol) dissolved in acetone (10 mL) was
added in several portions. The mixture was stirred overnight at room
temperature and then acidified to pH 2.0 using 1 M HCl. The
precipitate was filtered off and washed with a minimum amount of
cold water. The crude was purified by reversed-phase prep-HPLC
using a C18 column and an optimal gradient of buffer A (H2O 95%,
ACN 5%, TFA 0.1%) versus buffer B (ACN 95%, H2O 5%, TFA
General Procedure for the Synthesis of Compounds 8−13. To a
solution of BF3·OEt2 (2.6 mL, 21.3 mmol, 3.0 equiv) in ACN (10
mL) were added the corresponding thiols (10.7 mmol, 1.5 equiv) and
7-ACA (1.9 g, 7.1 mmol, 1.0 equiv.) successively. The mixture was
stirred at 45−50° for 2 h, after which it was diluted with water and pH
was adjusted to 4 by adding 28% ammonium hydroxide solution. The
precipitate was filtered off and washed with cold water and acetone.
The crude product (1.0 g) was added to a mixture of saturated
bicarbonate solution (6 mL) and acetone (9 mL). Then, phenylacetyl
chloride (2.0 equiv) was added dropwise and the mixture was stirred
overnight at room temperature. Diluting the mixture with water
followed by acidification to pH 2.0 using 1.0 M HCl resulted in a
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0.1%). (85 mg, 75%, based on the purification of ∼100 mg of the
compounds and zinc sulfate were dissolved in 20 mM Tris−HCl
buffer (pH 7.0). The experiments consisted of titrating 2 mM zinc
sulfate through 19 × 2.0 μL aliquots (except the first aliquot which
was 0.4 μL) into 200 μM solutions of the cephalosporin conjugates
incubated with NDM-1 (187 nM) for 2 h at room temperature. The
experiments were performed at 25 °C with 150 s interval between
titrations, and reference power was set at 10.0 μcal/s. The data were
analyzed using Microcal PEAQ-ITC analysis software. In separate
experiments, upon the titration of zinc sulfate into the solutions of
cephalosporin conjugates or NDM-1, no binding interaction was
observed.
NMR-Based Monitoring of the Enzymatic Hydrolysis. The
cephalosporin conjugates were dissolved in DMSO-d6 and diluted
in deuterated PBS (pH 7.4) or deuterated 50 mM HEPES (pH 7.4),
each supplemented with 1 μM ZnSO4. IMP-28 was added to the
solution, and the final concentration of the enzyme, test compounds,
and DMSO were 320 nM, 1 mg/mL, and 1%, respectively. Following
incubation at 25 °C, the 1H NMR spectra were measured on a Bruker
400 MHz spectrometer at various time points.
LCMS-Based Monitoring of the Enzymatic Hydrolysis. The
cephalosporin conjugates were dissolved in 50 mM HEPES buffer
(pH 7.2) supplemented with 1 μM ZnSO4 and 0.01% Triton X-100.
IMP-28 was added to the solution, and the final concentration of the
enzyme, test compounds, and DMSO were 320 nM, 1 mg/mL, and
1% respectively. Following incubation at 25 °C and at different time
points, the solution was diluted in ACN (1:2 v/v) and centrifuged at
12,000 rpm for 5 min. The supernatant was analyzed on an LCMS-
8040 triple-quadrupole liquid chromatograph mass spectrometer
(LC−MS/MS, Shimadzu) using a C18 column (3 μm, 3.0 × 150 mm,
Shimadzu) and a gradient of 5−100% pure ACN against 0.1% formic
acid.
In Silico Studies. All computational modeling was performed in the
Schrodinger Suite version 2019-4.32 Figures were generated using
PyMOL. 3D coordinates of the ligands were generated using
LigPrep33 with the OPLS3e forcefield.34 Protein structures for IMP-
1 (PDB ID: 1dd6)19 and NDM-1 (PDB ID: 4RL2)16 were prepared
using the Protein Preparation Wizard.35,36 Thereafter, compounds
were docked using GLIDE-SP.37 A maximum common substructure
constraint for all product compounds was used, which was derived
from cephalexin coordinates in PDB ID 4RL2. The substrate
compounds were docked without using constraints. The best pose
was maintained according to GLIDE docking score and visual
inspection of the poses. MD simulations were performed using
Desmond38 in the Schrodinger Suite using the standard protocol for
system setup, using the OPLS3 forcefield.34 Three replicate-
independent 10 ns simulations were run for each of the reported
protein−ligand systems.
1
crude product by prep-HPLC). H NMR (400 MHz, DMSO-d6): δ
9.09 (d, J = 8.2 Hz, NH, 1H), 7.33−7.21 (m, aromatic H, 5H), 5.60
(dd, J = 8.2 Hz, J = 4.6 Hz, β-lactam C−H, 1H), 5.03 (d, J = 4.7 Hz,
β-lactam C−H, 1H), 3.61−3.35 (m, aliphatic H, 4H), 2.03 (s, methyl,
3H), 13C NMR (101 MHz, DMSO-d6): δ 171.44, 164.82, 163.98,
136.33, 130.21, 129.48, 128.68, 126.93, 123.21, 59.33, 57.56, 42.03,
29.40, 19.87. HRMS (ESI): [M + H]+ calcd, 333.0909; found,
333.0917.
Enzyme Production and Purification. The procedures for the
overexpression and purification of IMP-1, IMP-28, NDM-1, and VIM-
2 have been described in detail in the previous publication.12,31
Enzymatic Preparation of 8H and 9H. Compounds 8 and 9 (2
mM each) were incubated with NDM-1 (187 nM) at room
temperature in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES)−NaOH, pH 7.2, supplemented with 1 μM ZnSO4 and
0.01% Triton X-100. The progress of hydrolysis was monitored by
LC−MS (Figure S8). After 2 h, the conversion was complete, and
compounds 8H and 9H were separated from the enzyme by spin
filtration (3 kDa filter cutoff, Amicon) at 12,000 rpm for 5 min.
Enzyme Inhibition Assay. The cephalosporin derivatives were
tested for their inhibitory activity against NDM-1, VIM-2, and IMP-
28 using the chromogenic substrate nitrocefin. The assay buffer was
50 mM HEPES, pH 7.2, supplemented with 1 μM ZnSO4 and 0.01%
Triton X-100. In brief, on a flat-bottom polystyrene 96-well
microplate NDM-1 (6 nM), VIM-2 (8 nM), or IMP-28 (1 nM)
was incubated with various concentrations of the test compounds for
15−60 min at 25 °C. Nitrocefin (10 μM, ∼2 × KM) was added to the
wells, and absorption at 492 nm was immediately monitored on a
TECAN Spark microplate reader over 30 scan cycles. For measuring
time 0 in the time-dependent IC50 assay, the enzyme was added last,
after which the absorbance was immediately measured. The initial
velocity data were used for IC50 curve-fitting using GraphPad Prism 7.
All the compounds were tested in three independent replicates.
Determination of the Kinetic Parameters of Cephalosporin
Conjugates. Hydrolysis of the cephalosporin conjugates was
monitored on a Tecan Spark microplate reader using UV-transparent
96-well plates (UV-Star, Greiner). Various concentrations of the test
compounds were dissolved in 50 mM HEPES−NaOH, pH 7.2,
supplemented with 1 μM ZnSO4 and 0.01% Triton X-100. Followed
by the addition of MBLs dissolved in the same buffer, absorption at
260 nm was measured immediately over 30−40 scan cycles at 25 °C.
The obtained initial velocity data were plotted against the substrate
concentration, and KM and Vmax were determined using the
Michaelis−Menten fitting model on GraphPad Prism 7.
MIC Determination and Synergy Assays. The MIC of the test
compounds was determined following the guidelines published by
Clinical and Laboratory Standards Institute (CLSI) and as described
earlier.11 Synergy between the cephalosporin derivatives and β-lactam
antibiotics was evaluated by the following protocol: β-lactam
antibiotics dissolved in MHB with the concentration corresponding
to 4× MIC was added to polypropylene 96-well microplates and
serially diluted (25 μL/well). Then, all three columns received a fixed
concentration of the test compounds dissolved in MHB (25 μL/well).
Multiple concentrations of the test compounds were evaluated in this
way. Finally, bacterial suspensions grown to the OD600 of 0.5 were
diluted 100× in MHB before adding to the plate (50 μL/well). The
microplates were then covered with breathable seals and incubated
overnight with shaking at 37 °C for 15−20 h. Dipicolinic acid was
used as the positive control.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00362.
■
sı
Molecular formula strings, analytical-spectral data
1
including H NMR, 13C NMR, and HPLC traces, IC50
curves, stability data, enzymatic degradation monitored
1
by H NMR and LCMS, docking figures, and PDB
coordinates for compounds 8 and 8H docked into IMP-
1 and NDM-1 active sites (PDF)
Molecular formula strings (CSV)
Stability Analysis in MHB. The solutions of the test compounds (1
mM) in MHB were incubated at 37 °C for 15 h. Then, 100 μL of the
MHB solution was precipitated by adding to ACN (200 μL)
supplemented with 2 mM benzocaine, vortexed, and centrifuged
(12,000 rpm, 5 min). The supernatant was analyzed by reversed-phase
analytical HPLC using a C18 column and an optimal gradient of buffer
A (H2O 95%, ACN 5%, and TFA 0.1%) versus buffer B (ACN 95%,
H2O 5%, and TFA 0.1%). The detector wavelength was set at 254 nm.
Isothermal Titration Calorimetry. The ITC titrations were
performed on a PEAQ-ITC calorimeter (Malvern). All the test
PDB coordinates (ZIP)
AUTHOR INFORMATION
Corresponding Author
■
Nathaniel I. Martin − Biological Chemistry Group, Institute of
Biology Leiden, Leiden University, 2333 BE Leiden, The
Netherlands; orcid.org/0000-0001-8246-3006;
Email: n.i.martin@biology.leidenuniv.nl
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Article
(4) Bonomo, R. A.; Burd, E. M.; Conly, J.; Limbago, B. M.; Poirel,
L.; Segre, J. A.; Westblade, L. F. Carbapenemase-producing
organisms: a global scourge. Clin. Infect. Dis. 2017, 66, 1290−1297.
(5) McGeary, R. P.; Tan, D. T.; Schenk, G. Progress toward
inhibitors of metallo-β-lactamases. Future Med. Chem. 2017, 9, 673−
691.
(6) Groundwater, P. W.; Xu, S.; Lai, F.; Váradi, L.; Tan, J.; Perry, J.
D.; Hibbs, D. E. New Delhi metallo-β-lactamase-1: structure,
inhibitors and detection of producers. Future Med. Chem. 2016, 8,
993−1012.
(7) Tehrani, K. H. M. E.; Martin, N. I. β-Lactam/β-lactamase
inhibitor combinations: an update. Medchemcomm 2018, 9, 1439−
1456.
Authors
Kamaleddin H. M. E. Tehrani − Biological Chemistry Group,
Institute of Biology Leiden, Leiden University, 2333 BE
Leiden, The Netherlands
Nicola Wade − Biological Chemistry Group, Institute of
Biology Leiden, Leiden University, 2333 BE Leiden, The
Netherlands
Vida Mashayekhi − Division of Cell Biology, Department of
Biology, Faculty of Science, Utrecht University, 3584 CH
Utrecht, The Netherlands
Nora C. Bruchle − Biological Chemistry Group, Institute of
̈
Biology Leiden, Leiden University, 2333 BE Leiden, The
Netherlands
(8) Fast, W.; Sutton, L. D. Metallo-β-lactamase: inhibitors and
reporter substrates. Biochim. Biophys. Acta, Proteins Proteomics 2013,
1834, 1648−1659.
Willem Jespers − Department of Cell and Molecular Biology,
Biomedical Center, Uppsala University, SE-751 24 Uppsala,
Sweden; Division of Drug Discovery & Safety, Leiden
Academic Centre for Drug Research, Leiden University, 2333
CC Leiden, The Netherlands
Koen Voskuil − Biological Chemistry Group, Institute of
Biology Leiden, Leiden University, 2333 BE Leiden, The
Netherlands
Diego Pesce − Laboratory of Genetics, Wageningen University
and Research, 6700 AA Wageningen, The Netherlands;
Department of Evolutionary Biology and Environmental
Studies, University of Zurich, 8057 Zurich, Switzerland
Matthijs J. van Haren − Biological Chemistry Group, Institute
of Biology Leiden, Leiden University, 2333 BE Leiden, The
Netherlands
Gerard J. P. van Westen − Division of Drug Discovery &
Safety, Leiden Academic Centre for Drug Research, Leiden
University, 2333 CC Leiden, The Netherlands; orcid.org/
0000-0003-0717-1817
(9) Ju, L.-C.; Cheng, Z.; Fast, W.; Bonomo, R. A.; Crowder, M. W.
The continuing Challenge of metallo-β-lactamase inhibition: mech-
anism matters. Trends Pharmacol. Sci. 2018, 39, 635−647.
(10) Rotondo, C. M.; Wright, G. D. Inhibitors of metallo-β-
lactamases. Curr. Opin. Microbiol. 2017, 39, 96−105.
(11) Tehrani, K. H. M. E.; Martin, N. I. Thiol-containing metallo-β-
lactamase inhibitors resensitize resistant gram-negative bacteria to
meropenem. ACS Infect. Dis. 2017, 3, 711−717.
(12) van Haren, M. J.; Tehrani, K. H. M. E.; Kotsogianni, I.; Wade,
̈
N.; Bruchle, N.; Mashayekhi, V.; Martin, N. I. Cephalosporin prodrug
inhibitors overcome metallo-β-lactamase driven antibiotic resistance.
Chem.Eur. J. 2020, 27, 3806−3811.
(13) Strijtveen, B.; Kellogg, R. M. Synthesis of (racemization prone)
optically active thiols by SN2 substitution using cesium thiocarbox-
ylates. J. Org. Chem. 1986, 51, 3664.
(14) Pérez-Llarena, F. J.; Fernández, A.; Zamorano, L.; Kerff, F.;
Beceiro, A.; Aracil, B.; Cercenado, E.; Miro, E.; Oliver, A.; Oteo, J.;
Navarro, F.; Bou, G. Characterization of a novel IMP-28 metallo-β-
lactamase from a Spanish Klebsiella oxytoca clinical isolate.
Antimicrob. Agents Chemother. 2012, 56, 4540−4543.
(15) Hanessian, S.; Wang, J. Design and synthesis of a
cephalosporin−carboplatinum prodrug activatable by a β-lactamase.
Can. J. Chem. 1993, 71, 896−906.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00362
Notes
(16) Feng, H.; Ding, J.; Zhu, D.; Liu, X.; Xu, X.; Zhang, Y.; Zang, S.;
Wang, D.-C.; Liu, W. Structural and mechanistic insights into NDM-1
catalyzed hydrolysis of cephalosporins. J. Am. Chem. Soc. 2014, 136,
14694−14697.
(17) Evans, L. E.; Krishna, A.; Ma, Y.; Webb, T. E.; Marshall, D. C.;
Tooke, C. L.; Spencer, J.; Clarke, T. B.; Armstrong, A.; Edwards, A.
M. Exploitation of antibiotic resistance as a novel drug target:
development of a β-lactamase-activated antibacterial prodrug. J. Med.
Chem. 2019, 62, 4411−4425.
(18) Jackson, A. C.; Zaengle-Barone, J. M.; Puccio, E. A.; Franz, K. J.
A Cephalosporin prochelator inhibits New Delhi metallo-β-lactamase
1 without removing zinc. ACS Infect. Dis. 2020, 6, 1264−1272.
(19) Concha, N. O.; Janson, C. A.; Rowling, P.; Pearson, S.;
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding was provided by the European Research Council
(ERC consolidator grant to NIM, grant agreement no.
725523).
ABBREVIATIONS
■
7-ACA, 7-aminocephalosporanic acid; 7-ADCA, 7-amino-
desacetoxycephalosporanic acid; DPA, dipicolinic acid; IMP,
imipenemase; ITC, isothermal titration calorimetry; NDM,
New Delhi metallo-β-lactamase; MCS, maximum common
substructure; MBL, metallo-β-lactamase; MHB, Mueller−
Hinton broth; VIM, Verona integron-encoded metallo-β-
lactamase
̀
Cheever, C. A.; Clarke, B. P.; Lewis, C.; Galleni, M.; Frere, J.-M.;
Payne, D. J.; Bateson, J. H.; Abdel-Meguid, S. S. Crystal structure of
the IMP-1 metallo-β-lactamase from Pseudomonas Aeruginosa and its
complex with a mercaptocarboxylate inhibitor: binding determinants
of a potent, broad-spectrum inhibitor. Biochemistry 2000, 39, 4288−
4298.
REFERENCES
■
(20) Liu, B.; Trout, R. E. L.; Chu, G.-H.; McGarry, D.; Jackson, R.
W.; Hamrick, J. C.; Daigle, D. M.; Cusick, S. M.; Pozzi, C.; De Luca,
F.; Benvenuti, M.; Mangani, S.; Docquier, J.-D.; Weiss, W. J.; Pevear,
D. C.; Xerri, L.; Burns, C. J. Discovery of taniborbactam (VNRX-
5133): a broad-spectrum serine- and metallo-β-lactamase inhibitor for
carbapenem-resistant bacterial infections. J. Med. Chem. 2020, 63,
2789−2801.
(21) Brem, J.; Rydzik, A. M.; Mcdonough, M. A.; Schofield, C. J.;
Morrison, A.; Hewitt, J.; Pannifer, A.; Jones, P. Inhibitors of metallo-
beta-lactamases. WO 2017093727 A1, 2017.
(1) Spencer, J.; Read, J.; Sessions, R. B.; Howell, S.; Blackburn, G.
M.; Gamblin, S. J. Antibiotic recognition by binuclear metallo-β-
lactamases revealed by X-ray crystallography. J. Am. Chem. Soc. 2005,
127, 14439−14444.
(2) King, D. T.; Worrall, L. J.; Gruninger, R.; Strynadka, N. C. J.
New Delhi metallo-β-lactamase: structural insights into β-Lactam
recognition and inhibition. J. Am. Chem. Soc. 2012, 134, 11362−
11365.
(3) Drawz, S. M.; Bonomo, R. A. Three decades of beta-lactamase
inhibitors. Clin. Microbiol. Rev. 2010, 23, 160−201.
9150
https://doi.org/10.1021/acs.jmedchem.1c00362
J. Med. Chem. 2021, 64, 9141−9151

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(22) Everett, M.; Sprynski, N.; Coelho, A.; Castandet, J.; Bayet, M.;
Bougnon, J.; Lozano, C.; Davies, D. T.; Leiris, S.; Zalacain, M.;
Morrissey, I.; Magnet, S.; Holden, K.; Warn, P.; De Luca, F.;
Docquier, J. D.; Lemonnier, M. Discovery of a novel metallo-β-
lactamase inhibitor that potentiates meropenem activity against
carbapenem-resistant Enterobacteriaceae. Antimicrob. Agents Chemo-
ther. 2018, 62, No. e00074.
(23) Leiris, S.; Coelho, A.; Castandet, J.; Bayet, M.; Lozano, C.;
Bougnon, J.; Bousquet, J.; Everett, M.; Lemonnier, M.; Sprynski, N.;
Zalacain, M.; Pallin, T. D.; Cramp, M. C.; Jennings, N.; Raphy, G.;
Jones, M. W.; Pattipati, R.; Shankar, B.; Sivasubrahmanyam, R.;
Soodhagani, A. K.; Juventhala, R. R.; Pottabathini, N.; Pothukanuri,
S.; Benvenuti, M.; Pozzi, C.; Mangani, S.; De Luca, F.; Cerboni, G.;
Docquier, J.-D.; Davies, D. T. SAR studies leading to the
identification of a novel series of metallo-β-lactamase inhibitors for
the treatment of carbapenem-resistant Enterobacteriaceae infections
that display efficacy in an animal infection model. ACS Infect. Dis.
2019, 5, 131−140.
(37) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;
Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.;
Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new
approach for rapid, accurate docking and scoring. 1. method and
assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739−1749.
(38) SC’06: Proceedings of the 2006 ACM/IEEE Conference on
Supercomputing; Association for Computing Machinery: New York,
NY, USA, 2006.
(24) Thomas, C. A.; Cheng, Z.; Yang, K.; Hellwarth, E.; Yurkiewicz,
C. J.; Baxter, F. M.; Fullington, S. A.; Klinsky, S. A.; Otto, J. L.; Chen,
A. Y.; Cohen, S. M.; Crowder, M. W. Probing the mechanisms of
inhibition for various inhibitors of metallo-β-lactamases VIM-2 and
NDM-1. J. Inorg. Biochem. 2020, 210, 111123.
(25) Wade, N.; Tehrani, K. H. M. E.; Bruchle, N. C.; Haren, M. J.;
̈
Mashayekhi, V.; Martin, N. I. Mechanistic Investigations of Metallo-β-
Lactamase Inhibitors: Strong zinc binding is not required for potent
enzyme inhibition. ChemMedChem 2021, 16, 1651−1659.
(26) Cahill, S. T.; Cain, R.; Wang, D. Y.; Lohans, C. T.; Wareham,
D. W.; Oswin, H. P.; Mohammed, J.; Spencer, J.; Fishwick, C. W. G.;
McDonough, M. A.; Schofield, C. J.; Brem, J. Cyclic boronates inhibit
all classes of β-lactamases. Antimicrob. Agents Chemother. 2017, 61,
No. e02260.
̀
(27) Garau, G.; Bebrone, C.; Anne, C.; Galleni, M.; Frere, J.-M.;
Dideberg, O. A metallo-β-lactamase enzyme in action: crystal
structures of the monozinc carbapenemase CphA and its complex
with biapenem. J. Mol. Biol. 2005, 345, 785−795.
(28) Ullah, J. H.; Walsh, T. R.; Taylor, I. A.; Emery, D. C.; Verma,
C. S.; Gamblin, S. J.; Spencer, J. The Crystal structure of the L1
metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A
resolution. J. Mol. Biol. 1998, 284, 125−136.
(29) Leiros, H.-K. S.; Borra, P. S.; Brandsdal, B. O.; Edvardsen, K. S.
W.; Spencer, J.; Walsh, T. R.; Samuelsen, Ø. Crystal structure of the
mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa:
insights into antibiotic binding and the role of Gln157. Antimicrob.
Agents Chemother. 2012, 56, 4341−4353.
(30) Keltjens, R.; Vadivel, S. K.; De Vroom, E.; Klunder, A. J. H.;
Zwanenburg, B. A New Convenient synthesis of 3-carboxycephems
starting from 7-aminocephalosporanic acid (7-ACA). Eur. J. Org.
Chem. 2001, 2001, 2529−2534.
(31) Tehrani, K. H. M. E.; Bruchle, N. C.; Wade, N.; Mashayekhi,
̈
V.; Pesce, D.; van Haren, M. J.; Martin, N. I. Small molecule
carboxylates inhibit metallo-β-lactamases and resensitize carbapenem-
resistant bacteria to meropenem. ACS Infect. Dis. 2020, 6, 1366−1371.
̈
̈
(32) Schrodinger Release 2019-4: Maestro, Schrodinger, LLC, New
York, NY, 2019.
̈
̈
(33) Schrodinger Release 2019-4: LigPrep, Schrodinger, LLC, New
York, NY, 2019.
(34) Roos, K.; Wu, C.; Damm, W.; Reboul, M.; Stevenson, J. M.; Lu,
C.; Dahlgren, M. K.; Mondal, S.; Chen, W.; Wang, L.; Abel, R.;
Friesner, R. A.; Harder, E. D. OPLS3e: extending force field coverage
for drug-like small molecules. J. Chem. Theory Comput. 2019, 15,
1863−1874.
(35) Madhavi Sastry, G.; Adzhigirey, M.; Day, T.; Annabhimoju, R.;
Sherman, W. Protein and ligand preparation: parameters, protocols,
and influence on virtual screening enrichments. J. Comput.-Aided Mol.
Des. 2013, 27, 221−234.
̈
(36) Schrodinger Release 2020-4: Protein Preparation Wizard; Epik,
̈
̈
Schrodinger, LLC, New York, NY, 2016; Impact, Schrodinger, LLC,
̈
New York, NY, 2016; Prime, Schrodinger, LLC, New York, NY, 2020.
9151
https://doi.org/10.1021/acs.jmedchem.1c00362
J. Med. Chem. 2021, 64, 9141−9151