Cephalosporin Prodrug Inhibitors Overcome Metallo-β-Lactamase Driven Antibiotic Resistance

Article abstract of DOI:10.1002/chem.202004694

The increasing prevalence of metallo-β-lactamase (MBL)-expressing bacteria presents a worrying trend in antibiotic resistance. MBLs rely on active site zinc ions for their hydrolytic activity and the pursuit of MBL-inhibitors has therefore involved the investigation of zinc chelators. To ensure that such chelators specifically target MBLs, a series of cephalosporin prodrugs of two potent zinc-binders: dipicolinic acid (DPA) and 8-thioquinoline (8-TQ) was prepared. Although both DPA and 8-TQ bind free zinc very tightly (K_d values in the low nm range), the corresponding cephalosporin conjugates do not. The cephalosporin conjugates are efficiently hydrolyzed by MBLs to release DPA or 8-TQ, as confirmed by using both NMR and LC-MS studies. Notably, the cephalosporin prodrugs of DPA and 8-TQ show potent inhibitory activity against NDM, VIM, and IMP classes of MBLs and display potent synergy with meropenem against MBL-expressing clinical isolates of K. pneumoniae and E. coli.

Full text of DOI:10.1002/chem.202004694

Accepted Article
Accepted Article
Title: Cephalosporin prodrug inhibitors overcome metallo-b-lactamase
driven antibiotic resistance
Authors: Matthijs J. van Haren, Kamaleddin H.M.E. Tehrani, Ioli
Kotsogianni, Nicola Wade, Nora Brüchle, Vida Mashayekhi,
and Nathaniel I. Martin
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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published online in Early View as soon as possible and may be different
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to ensure accuracy of information. The authors are responsible for the
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To be cited as: Chem. Eur. J. 10.1002/chem.202004694
Link to VoR: https://doi.org/10.1002/chem.202004694
01/2020

10.1002/chem.202004694
Chemistry - A European Journal
RESEARCH ARTICLE
Cephalosporin prodrug inhibitors overcome metallo-β-lactamase
driven antibiotic resistance
Matthijs J. van Haren[a], Kamaleddin H.M.E. Tehrani[a], Ioli Kotsogianni[a], Nicola Wade[a], Nora
Brüchle[a], Vida Mashayekhi[b], and Nathaniel I. Martin*[a]
[a]
[b]
M.J. van Haren, K.H.M.E. Tehrani, I. Kotsogianni, N. Wade, N, Brüchle, N.I. Martin
Biological Chemistry Group, Institute of Biology Leiden
Leiden University
Sylviusweg 72, 2333 BE, Leiden (NL)
E-mail: n.i.martin@biology.leidenuniv.nl
V. Mashayekhi
Department of Biology
Utrecht University
Universiteitsweg 99, 3584 CG, Utrecht (NL)
Supporting information for this article is given via a link at the end of the document.
Abstract: The increasing prevalence of metallo-β-lactamase (MBL)
currently no MBL inhibitors approved for use in humans making
expressing bacteria presents a worrying trend in antibiotic resistance. it very difficult for physicians to treat patients infected with MBL-
MBLs rely on active site zinc ions for their hydrolytic activity and the
pursuit of MBL-inhibitors has therefore involved the investigation of
zinc chelators. To ensure that such chelators specifically target
MBLs, we prepared a series of cephalosporin prodrugs of two potent
zinc-binders: dipicolinic acid (DPA) and 8-thioquinoline (8-TQ). While
both DPA and 8-TQ bind free zinc very tightly (Kd values in the low
nM range), the corresponding cephalosporin conjugates do not. The
cephalosporin conjugates are efficiently hydrolyzed by MBLs to
release DPA or 8-TQ, as confirmed using both NMR and LC-MS
studies. Notably, the cephalosporin prodrugs of DPA and 8-TQ show
potent inhibitory activity against NDM, VIM, and IMP classes of
MBLs and display potent synergy with meropenem against MBL-
expressing clinical isolates of K. pneumoniae and E. coli.
producing pathogens.[7–9]
The common feature shared by all MBLs is the presence of
essential zinc ions in their active site that play a key role in the
hydrolytic mechanism. The chelation or stripping of zinc from the
active site has been demonstrated to be an effective approach
for deactivating MBLs. Ethylene-diaminetetraacetic acid (EDTA),
dipicolinic acid (DPA), aspergillomarasmine A (AMA), and a
variety of aminocarboxylic acids effectively bind zinc and have
been shown to inhibit MBLs, including the clinically relevant New
Delhi
Metallo-β-lactamase
(NDM-1).[10–12]
While
such
compounds are effective at inhibiting MBLs under optimized in
vitro assay conditions, their potent metal binding properties
would be expected to result in severe off-target effects in more
complex biological settings. Metalloproteins are estimated to
comprise about a third of the human proteome and, more
specifically, zinc-dependent enzymes account for almost 10% of
the human proteome.[13,14] For this reason, the direct use of
strong zinc binding MBL inhibitors is likely to be of limited
therapeutic application. In addition to the chelators described
above, a number of thiol-based compounds have also been
Introduction
Antimicrobial resistance presents a growing challenge to global
health in the treatment of bacterial infections and carries with it a
significant economic burden.[1–3] Among the numerous antibiotic
resistance mechanisms known to be of clinical relevance, the
activity of β-lactamase enzymes is of increasing concern. These
enzymes can hydrolyze the β-lactam ring of penicillins,
cephalosporins, and carbapenems, rendering them inactive. The
family of β-lactamase enzymes can be divided into two main
classes based on their mechanism of action: serine β-
lactamases (SBLs) that utilize an active site serine for the
hydrolysis of the β-lactam ring, and metallo-β-lactamases
(MBLs) that rely on active site zinc ions to activate a water
molecule for the hydrolysis.[4,5] A variety of clinically approved
SBL inhibitors are available, which, when combined with a
conventional β-lactam antibiotic, can be used to effectively treat
infections due to SBL-producing strains.[6] In contrast, there are
investigated
bisthiazolidines, and various thiol carboxylates.[15–17] Recent
reports describing 8-thioquinoline (8-TQ) as potent zinc
as
MBL
inhibitors,
including
captopril,
a
binding motif also prompted us to investigate its capacity to
inhibit MBLs.[18–20] While we were able to show that 8-TQ is a
potent zinc binder with the capacity to inhibit MBLs, we also
found that it has the tendency to rapidly oxidize to the
corresponding disulfide, eliminating its zinc binding and MBL
inhibiting properties. This problem is common to many thiol-
based MBL inhibitors and thus limits their potential for
therapeutic use.[17]
1
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10.1002/chem.202004694
Chemistry - A European Journal
RESEARCH ARTICLE
A
methoxybenzyl 3-(chloromethyl)-8-oxo-7-[(phenylacetyl)amino]-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (GCLE) was
converted into the corresponding iodide in situ by treatment with
sodium iodide followed by addition of freshly reduced 8-TQ.HCl
in the presence of sodium bicarbonate. Deprotection of the p-
methoxybenzyl (PMB) protecting group using a mixture of TFA
and anisole (5:1) followed by HPLC purification provided
compound 6 in excellent yield. For the synthesis of the
corresponding DPA-conjugate 13, one of the carboxylate
moieties of DPA was first PMB-protected by treatment of DPA
with the PMB-isourea (generated from diisopropylcarbodiimide
and p-methoxybenzyl alcohol) and 4-dimethylaminopyridine
(DMAP) in DMF, affording acid 9. GCLE was again converted to
its corresponding iodide using sodium iodide in situ, followed by
coupling of the mono-PMB-protected DPA 9 to yield protected
conjugate 10 in good yield. Attempted deprotection of the ester
linked conjugate with the same conditions used in the
preparation of compound 6, however, led to the cleavage of the
ester bond between DPA and the cephalosporin core.
Gratifyingly, milder conditions (TFA and anisole (2.5:1) in DCM
at 0°C) allowed for PMB group removal without cleavage of the
ester. Subsequent purification by preparative HPLC yielded
compound 13 in good yield. Notably, in the synthesis of both 6
and 13 no indication of Δ2-Δ3 double bond isomerization, which
is known to occur in cephalosporins under basic coupling
conditions, was observed. To further expand upon conjugates 6
and 13, the corresponding sulfoxides and sulfones were also
synthesized to investigate the impact of the oxidation state of the
cephalosporin sulfur atom on the activity and stability of the
conjugates. Oxidation was achieved using 1 or 2.5 equivalent of
meta-chloroperoxybenzoic acid (mCPBA) to provide the
sulfoxide or sulfone, respectively. For 8-TQ conjugates 7 and 8,
mCPBA oxidation was first performed on GCLE to avoid
oxidation of the thioether linkage present in the 8TQ conjugates.
In generating the DPA conjugates 14 and 15, oxidation was
performed on the PMB-protected intermediate 10.
R
NH
O
R
O
R
O
O
N
O
NH
NH
MBL
inhibitor
(active)
HO
HO
O
MBL
H
S
H
S
+
H
N
N
S
Zn2+ Zn2+
O
O
O
O
H
H
OH
OH
OH
MBL
inhibitor
(inactive)
MBL
inhibitor
(inactive)
hydrolyzed cephalosporin
B
H
N
R =
S
H
O
O
S
N
N
O
O
O
OH
N
O
R
or
OH
8-Thioquinoline
(8-TQ)
Dipicolinic acid
(DPA)
Figure 1. A) Proposed mechanism of MBL-mediated hydrolysis of the beta-
lactam ring in cephalosporin prodrugs with subsequent release of the zinc
binding MBL inhibitor. B) Structures of the cephalosporin prodrug conjugates
of 8-thioquinoline (8-TQ) and dipicolinic acid (DPA).
To address the aforementioned shortcomings of zinc binding
amino-carboxylates and small molecule thiols as MBL inhibitors
we here describe a cephalosporin prodrug strategy that allows
for the active inhibitor to be released only in the presence of the
target MBL. Specifically, this prodrug strategy is based on the
mechanism of the β-lactamase-mediated hydrolysis of
cephalosporins (Figure 1A). It is well established that for
cephalosporins containing a suitable leaving group at the 3-
position, hydrolysis of the β-lactam ring leads to the subsequent
release of the leaving group. This unique reactivity has been
previously exploited in the design of cephalosporin-based
probes for β-lactamase activity[21] as well as in the development
of other prodrugs capable of delivering toxic payloads to cells
expressing β-lactamases.[22,23] In our design, the incorporation of
the strong zinc binding compound is achieved by covalently
linking either the carboxylate or thiol moiety involved in zinc
binding to the cephalosporin 3-position. In this way zinc binding
is temporarily shut off and, in the case of thiols, disulfide
formation is also blocked. Only upon hydrolysis of the β-lactam
ring are the strong zinc binders released from the 3-position in
their active form. To investigate this prodrug strategy as an
approach to developing MBLs inhibitors, DPA and 8-TQ were
selected as representative zinc binders and linked to a 7-
phenacyl-cephalosporin core (Figure 1B). The zinc binding
properties of both the free chelators and the corresponding
cephalosporin prodrugs were assessed using isothermal titration
calorimetry (ITC). In addition, the mechanism of MBL-mediated
prodrug cleavage was confirmed using both NMR and LC-MS
technique. Furthermore, the inhibitory activity of these prodrugs
was evaluated against different members of the MBL family and
synergy assays used to measure their capacity to resensitize
MBL-expressing clinical isolates to the carbapenem antibiotic
meropenem.
A
O
S
O
S
n
H
N
H
N
n
H
N
O
H
N
O
a
N
O
O
Cl
S
O
O
O
O
R
PMB
3, n = 0, R = PMB
4, n = 1, R = PMB
5, n = 2, R = PMB
6, n = 0, R = H
7, n = 1, R = H
8, n = 2, R = H
GCLE, n=0
1, n=1
d
b
c
2, n=2
B
O
O
O
O
N
e
N
PMB
HO
OH
HO
O
DPA
9
O
H
H
N
H
N
n
H
S
S
f
O
O
N
Cl
N
O
O
O
N
R
O
O
O
O
Results and Discussion
O
O
O
R
PMB
13, n = 0, R = H
14, n = 1, R = H
15, n = 2, R = H
10, n = 0, R = PMB
11, n = 1, R = PMB
12, n = 2, R = PMB
g
b
c
The cephalosporin prodrug conjugates of 8-TQ and DPA were
prepared by linking both compounds to the 7-phenacyl-
cephalosporin scaffold as indicated in Scheme 1. For the
preparation of the 8-TQ conjugates, 8-TQ was freshly reduced
with tris(2-carboxyethyl)phosphine (TCEP) using either resin-
bound TCEP or a solution of TCEP in 0.1 M HCl followed by
extraction in chloroform. Next, commercially available 4-
Scheme 1. Synthesis of A) 8-thioquinoline conjugates 6-8 and B) dipicolinic
acid conjugates 13-15. Reagents and conditions: a) 1. NaI, DMF, rt, 1h, 2. 8-
thioquinoline hydrochloride, NaHCO3, DMF, rt, 4h; b) mCPBA (1 eq), DCM,
0ºC, 3h; c) m-CPBA (2.5 eq), DCM, 0ºC to rt, overnight; d) TFA/anisole (5:1),
0ºC to rt, 1h; e) O-PMB-N,N'-diisopropylisourea DMAP, DMF, DCM, rt,
overnight; f) 9, NaHCO3, DMF, rt, overnight, g) TFA/anisole (2.5:1), DCM, 0ºC,
2h.
2
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10.1002/chem.202004694
Chemistry - A European Journal
RESEARCH ARTICLE
To quantify the zinc binding properties of DPA and 8-TQ, and
evaluate the masking efficiency of the prodrug approach,
dissociation constants (Kd values) for Zn2+ were determined
using ITC. Titration of zinc sulfate into DPA revealed a Kd value
of 389 ± 45 nM with a 2:1 stoichiometry of DPA to Zn2+. This Kd
value is somewhat different (ca. 6-fold lower) than that
We next set out to confirm the mechanism of release of 8-TQ
and DPA from the corresponding cephalosporin conjugates. To
investigate the MBL-mediated release of 8-TQ and DPA via
hydrolysis of the β-lactam ring we devised an in situ NMR assay
wherein compounds 6 and 13 were incubated with NDM-1 (Fig.
2). Briefly, NDM-1 was added at a final concentration of 374 nM
to an NMR tube containing cephalosporin conjugate 6 or 13 in
20 mM HEPES with 500 µM zinc sulfate in D2O. The 1H NMR
data presented in Figure 2 clearly show the appearance of the
vinylic cephalosporin by-product formed upon elimination of 8-
TQ (compound 6) and DPA (compound 13). As can also be
seen, the hydrolysis of DPA conjugate 13 proceeds more rapidly
than hydrolysis of 8TQ conjugate 6. Notably, no indication of
hydrolysis of either 6 or 13 were found in control experiments
where no enzyme was present. These results support the
proposed mechanism of action wherein the zinc binding 8-TQ or
DPA are released in an MBL-dependent manner. It is also worth
noting that in order to achieve complete hydrolysis of the
cephalosporin conjugates a relatively high 374 nM concentration
of NDM-1 was required. When working with lower
concentrations of enzyme the hydrolysis of 6 and 13 would
proceed only partially before stalling, presumably due to
inhibition of the enzyme by the newly released 8-TQ or DPA
(data not shown). The formation of the vinylic by-product and
release of 8-TQ and DPA was further confirmed using LC-MS by
incubation of compounds 6 and 13 with NDM-1 (see figures S6
and S7 in the supporting information).
previously reported by our group[24]
, a difference likely
attributable to the optimized lower concentration of TRIS buffer
used in the current study (20 mM versus 100 mM). Prior to its
use in the ITC experiment, 8-TQ was freshly reduced using
resin-bound TCEP followed by titration with zinc sulfate. This
revealed a very strong zinc-binding interaction with a low
nanomolar Kd value close to the ITC’s limit of detection. In order
to fully quantify the 8-TQ/Zn2+ interaction,
a competition
experiment was established wherein freshly reduced 8-TQ was
titrated into a premixed solution of DPA and zinc sulfate. The
results of this analysis confirmed 8-TQ to be a highly potent zinc
chelator with a Kd value of 1.87 ± 0.15 nM and a 2:1
stoichiometry of 8-TQ to Zn2+. Next, the zinc binding capacities
of the cephalosporin prodrugs of DPA and 8-TQ were evaluated
to assess how effectively zinc binding is masked. All of the 8-TQ
conjugates were found to be comparably stable and among the
DPA conjugates sulfoxide analogue 14 demonstrated the best
stability in the buffer conditions used for the zinc binding assay
and was therefore used in the ITC experiment. No appreciable
zinc binding was observed for compounds 6 and 14, confirming
that the zinc binding properties of 8-TQ and DPA are completely
blocked upon conjugation to the cephalosporin (thermograms
provided in Fig. S4 in the supporting information).
O
O
NH
5
5
4
4
H
H
N
O
H
O
S
1
H
2
H
O
S
1
H
2
H
H
N
H
N
S
6
6
CH2
OH
S
CH2
N
O
O
N
O
OH
N
S
+
+
N
OH
O
OH
3
O
3
O
O
N
O
OH
O
OH
HS
HO
OH
Compound 13
Compound 6
N
8-TQ
O
O
DPA
Figure 2. 1H NMR spectra showing the MBL-mediated hydrolysis of compounds 6 (left panel) and 13 (right panel) over time. For both compounds, the
hydrolysis by NDM-1 leads to the clean formation of the vinylic product and release of the zinc binding compound. For clarity, the region of the spectra including
the suppressed water signal has been removed. In the case of compound 6, the signals for the proton at position 2 and one of the protons at position 3 overlap
with the suppressed water signal and are therefore not visible (full NMR spectra provided in supporting information).
3
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Chemistry - A European Journal
RESEARCH ARTICLE
The MBL inhibitory activity of 8-TQ and DPA and their
corresponding cephalosporin prodrugs was next assessed
against a panel of purified MBLs, including members of the
clinically most relevant NDM, VIM and IMP families (Table
1). Enzyme inhibition assays were performed as previously
complex with zinc still bound in the MBL active site, the zinc
dependence of the inhibition observed for 8-TQ, DPA, and
conjugates 6 and 14 was next evaluated. The compounds were
tested for their inhibitory activity against NDM-1 in the presence
of 0.1, 1.0, and 10 µM zinc sulfate. Interestingly, while the
activity of 8-TQ and DPA showed a strong dependence on the
concentration of zinc in the media, this effect was not observed
for conjugates 6 and 14 (see Fig. S3 in the supporting
information). An explanation for this observation may be that
while 8-TQ and DPA function as simple chelators in solution, in
prodrug form they are only released in the active site of an MBL
where they may then be capable of functioning in a more
localized manner. This localized release of 8-TQ or DPA may
allow for formation of a ternary complex with the zinc bound in
described using
a fluorescent cephalosporin substrate for
assessing MBL activity, added following a 10 minute pre-
incubation of the enzyme with the inhibitor.[21] Effective inhibition
was observed for 8-TQ as well as its conjugates 6-8 against all
MBLs tested with IC50 values in the low micromolar range.
Interestingly, the conjugates were generally more potent
inhibitors of NDM-1 and VIM-2 than against the IMP enzymes
tested. By comparison, 8-TQ showed similar activity against all
MBLs evaluated suggesting that cephalosporin conjugates 6-8
may be better substrates for NDM-1 and VIM-2 than for IMP-
type MBLs. Similar inhibitory potency was measured for DPA
and prodrug conjugates 13-15, however in this case the
oxidation state of the cephalosporin sulfur atom had a more
pronounced effect on activity. While DPA and conjugate 13
showed similar inhibition against all enzymes tested with IC50
values in the low micromolar range, sulfoxide 14 was nearly 10-
fold more active with an IC50 value of 0.299 µM against NDM-1
and 0.756 µM against VIM-2. Interestingly, the increased
potency of sulfoxide 14 did not extend to the IMP type enzymes
tested. By comparison sulfone analogue 15 was found to be
significantly less active with an IC50 of 18.78 µM for NDM-1 and
10.83 µM for VIM-2. Against IMP-1 and IMP-28, however, both
sulfoxide 14 and sulfone 15 showed a similarly reduced activity
relative to DPA or conjugate 13.
the MBL active site, in
a manner independent of the
concentration of free zinc in solution.
The ability of the 8-TQ and DPA cephalosporin prodrugs to
resensitize MBL-expressing bacteria to a clinically used β-lactam
antibiotic was next investigated. The stability of the compounds
in the assay buffers was first evaluated to ensure that any
observed activity could be fairly attributed to MBL-mediated
hydrolysis of the conjugates rather than degradation or auto-
hydrolysis under the assay conditions used. In the HEPES buffer
used for the NMR hydrolysis experiments and the enzyme
inhibition assays, the conjugates showed good stability over the
time course of the experiment (no significant hydrolysis after 6
hours at room temperature). However, for bacterial growth
assays, standard conditions involve the use of Mueller-Hinton
Broth (MHB) at 37ºC. The stability of the 8-TQ and DPA
conjugates under these conditions revealed that the DPA
conjugates 13-15 showed appreciable degradation over time
with stability depending on the oxidation state of the
cephalosporin sulfur. Specifically, DPA-conjugate 13 (sulfide)
showed 85% degradation after 2 hours, while sulfone 15 showed
a half-life of about 5 hours and sulfoxide 14 showed a half-life of
about 14 hours. By comparison, 8-TQ conjugates were found to
be much more stable. Compound 6 and 7 showed very little sign
of degradation with >90% remaining intact after 24 hours.
Table 1. Inhibitory activity 8TQ, DPA and conjugates 6-8 and 13-15 against
selected MBLs
NDM-1
VIM-2
IC50 (µM)
IMP-1
IC50 (µM)
IMP-28
IC50 (µM)
IC50 (µM)[a]
8-TQ
6
2.99 ± 0.58
2.31 ± 0.25
2.18 ± 0.14
8.47 ± 1.02
3.13 ± 0.12
2.13 ± 0.07
0.299 ± 0.005
18.78 ± 1.22
4.20 ± 1.00
3.64 ± 0.27
6.35 ± 0.56
4.17 ± 0.65
4.34 ± 0.67
1.87 ± 0.10
0.756 ± 0.056
10.83 ± 0.60
4.42 ± 0.14
12.21 ± 0.25
33.57 ± 1.72
28.92 ± 1.29
15.16 ± 0.17
5.22 ± 0.25
15.09 ± 1.09
18.71 ± 2.43
4.20 ± 0.11
10.32 ± 0.22
71.87 ± 3.05
46.24 ± 3.13
9.65 ± 0.62
7.39 ± 0.10
46.70 ± 2.76
61.87 ± 1.82
7
8
Sulfone
8 showed some degradation over time, with an
DPA
13
14
15
estimated half-life of about 24 hours (see Fig. S5 in the
supporting information). These stability data indicate that the
ester linkage in the DPA-cephalosporin conjugates is not
sufficiently stable under the conditions used for the bacterial
growth inhibition experiments. Therefore, for the purpose of the
subsequent antibacterial synergy assays we focused on the 8-
TQ conjugates 6-8. The compounds were first tested for inherent
activity against a series of MBL-expressing clinical isolates of E.
coli, K. pneumoniae and P. aeruginosa (see supplemental Table
S3). Standard minimum inhibitory concentration (MIC) assays
revealed that none of the compounds were active on their own
(MIC values >256 µg/mL). Next, the capacity for 8-TQ and
prodrugs 6-8 to synergize with meropenem against MBL-
expressing strains was evaluated. In an initial screen two
different NDM-1 expressing K. Pneumoniae strains were used to
assess the synergy of the compounds at 25% of the maximum
concentration tested in the previous MIC assays (64 µg/mL) to
exclude additive effects. The results of the synergy studies
conducted with 8-TQ itself were clouded by formation of the
insoluble 8-TQ disulfide (the disulfide species precipitates and
impairs the visual readout of the bacterial growth assay). In the
case of prodrugs 6-8 however, a clear and significant level of
[a] Half-maximal inhibitory concentration of the compounds tested against
NDM-1, VIM-2, IMP-1, and IMP-28. Inhibitors pre-incubated with the MBL for
10 min. followed by addition of the FC5 substrate (full assay details provided in
the supporting information). Values reported based on triplicate experiments.
As the activation of these prodrugs is governed by hydrolysis of
the cephalosporin β-lactam ring, the time-dependent activity of
inhibitors 6 and 14 was next evaluated. The compounds were
incubated with NDM-1 for 0, 10, 20, 30, 45 or 60 minutes prior to
the addition of the fluorescent enzyme substrate. For compound
6 no significant change in IC50 was observed with longer
incubation times. However, for compound 14, a clear decrease
in IC50 from 560 nM to 120 nM was observed with longer
incubation times (see Fig. S2 in the supporting information). The
observation that a decrease in IC50 is only observed for DPA
and not for 8-TQ might be explained by the parallel formation of
the inactive disulfide balancing out any decrease in IC50. To
further investigate whether these inhibitors work via stripping
zinc from the MBL active site or through formation of a ternary
4
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10.1002/chem.202004694
Chemistry - A European Journal
RESEARCH ARTICLE
synergy with meropenem was observed free of disulfide Conclusion
precipitation. These findings suggest that the prodrug strategy
enables effective delivery of otherwise reactive thiols as MBL
inhibitors. In a preliminary screen with two highly resistant NDM-
1 expressing isolates, compounds 6-8 all showed effective
synergy. Notably, the most stable 8-TQ-cephalosporin conjugate
6 was also found to be the most effective synergist, lowering the
MIC of meropenem from 16-32 µg/mL (resistant) to 1-4 µg/mL
(sensitive) against the two strains tested. Compound 6 was
therefore selected for further evaluation against an expanded
panel of MBL-producing clinical isolates of E. coli, K.
pneumoniae and P. aeruginosa (Table 2 and Fig. S8-S9 in the
supporting information). Potent synergy with meropenem was
consistently observed against all K. pneumoniae and E. coli
strains tested expressing the main MBL classes (NDM, VIM,
IMP) with measured enhancements ranging from 8- to 128-fold.
It is it notable that compound 6 exhibited the most effective
synergy when applied at 64 µg/mL (corresponding to 130 µM), a
concentration ca. 60x times higher than the IC50 value measured
for NDM-1 in the inhibition assays with purified enzyme (Table 1).
The higher concentration of compound 6 required to elicit a
notable effect in the bacterial assays might be explained by the
need for the compound to pass the outer membrane in order to
engage the target MBL. From the synergy data obtained, the
fractional inhibitory concentration (FIC) values were also
determined with values below 0.5 indicating a synergistic effect
between meropenem and the MBL inhibitor. Notably,
significantly less synergy was found against the P. aeruginosa
strains tested, an observation in keeping with the known low-
permeability of the P. aeruginosa outer membrane to antibiotics
and other antiseptic agents.[25]
The emergence of MBL-driven antibiotic resistance and a lack of
clinically approved MBL inhibitors pose a serious threat to global
health. We here describe
a new series of cephalosporin
prodrugs designed to deliver zinc binding MBL inhibitors in a
targeted manner. Recent reports have demonstrated that such
strategies can be effective for delivering antibacterial metal
chelators such as pyrithione.[23,26] In the present study we
focused on new cephalosporin prodrugs of 8-thioquinoline (8-
TQ) and dipicolinic acid (DPA). This approach allows for the
controlled activation of the zinc-binding properties of the inhibitor
only in the presence of the target MBL and, in the case of 8-TQ,
also protects the active species from oxidative deactivation prior
to target engagement. The 8-TQ and DPA prodrugs here studied
effectively inhibit purified NDM-, VIM-, and IMP-type enzymes
via an activation mechanism shown to be depended on MBL-
mediated β-lactam ring hydrolysis. Notably, while elevated
concentrations of free zinc were found to diminish the inhibitory
potency of 8-TQ and DPA alone, the same effect was not
observed for the corresponding cephalosporin conjugates.
These findings point to a proximity effect for the prodrugs,
wherein the zinc-binding MBL inhibitor is released within the
MBL active site itself, serving to enhance its selectively. In
bacterial growth assays the 8-TQ conjugates demonstrated
potent synergy with meropenem against a number of clinical
isolates of E. coli and K. pneumoniae expressing NDM-, VIM-,
and IMP- type MBLs. Taken together these results indicate that
such prodrug strategies can effectively deliver zinc-binding MBL
inhibitors to resistant bacteria in a selective manner. In the
present study the conjugates prepared contained the phenacyl
substituent at the cephalosporin C-7 position. Among 3rd and 4th
generation cephalosporins a range of optimized substituents are
found at this position imparting increased activity and stability.
Ongoing efforts are therefore aimed at similarly enhancing the
activity of the MBL inhibitor-cephalosporin prodrugs here
described.
Table 2. Bacterial growth inhibition by meropenem in the presence of
cephalosporin prodrug 6 against clinical isolates of MBL-producing Gram-
negative bacteria.
Strain
MBL
MIC (µg/mL)[a]
FIC[b]
meropenem
+ 6
K. pneumoniae
(strain JS022)
NDM-1
NDM-1
VIM-1
16
32
64
16
4
1 (16)
≤0.188
≤0.250
≤0.188
≤0.156
≤0.156
≤0.156
≤0.156
≤0.188
≤0.188
≤0.133
NS[c]
K. pneumoniae
(strain N11-2218)
4 (8)
4 (16)
K. pneumoniae
(strain RC0048)
Acknowledgements
K. pneumoniae
(strain RC0021)
VIM-1
0.5 (32)
0.13 (32)
0.13 (32)
1 (32)
This work was funded by the European Research Council (ERC
consolidator grant to NIM, grant agreement no. 725523).
K. pneumoniae
(strain NRZ-293)
VIM-2
K. pneumoniae
(strain JS265)
IMP-28
NDM-1
NDM-1
VIM-2
4
Keywords: antibiotic resistance • metallo-β-lactamase • inhibition
E. coli
(strain RC0089)
32
8
• zinc chelator • synergy
E. coli
(strain 2018-015)
0.5 (16)
0.25 (16)
0.063 (128)
32 (2)
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6
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10.1002/chem.202004694
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RESEARCH ARTICLE
Entry for the Table of Contents
Cephalosporin prodrug inhibitors overcome metallo-β-lactamase driven antibiotic resistance
Matthijs J. van Haren,
Metallo-β-lactamases (MBLs) are bacterial
resistance enzymes that degrade β-lactam
antibiotics including last line of defense
carbapenems like meropenem. We here
Kamaleddin H.M.E. Tehrani, Ioli
Kotsogianni, Nicola Wade, Nora
Brüchle, Vida Mashayekhi, and
Nathaniel I. Martin*
report
a new series of cephalosporin
prodrug inhibitors that exploit the hydrolytic
action of the target MBL itself to activate
the inhibitor. The prodrugs display potent
MBL inhibition and effectively synergize
with meropenem against highly resistant
MBL-expressing isolates.
7
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