Small molecule suppression of carbapenem resistance in ndm-1 producing klebsiella pneumoniae

Article abstract of DOI:10.1021/ml200290p

The already considerable global public health threat of multidrug-resistant Gram-negative bacteria has become even more of a concern following the emergence of New Delhi metallo-β-lactamase (NDM-1) producing strains of Klebsiella pneumoniae and other Gram-negative bacteria. As an alternative approach to the traditional development of new bactericidal entities, we have identified a 2-aminoimidazole-derived small molecule that acts as an antibiotic adjuvant and is able to suppress resistance of a NDM-1 producing strain of K. pneumoniae to imipenem and meropenem, in addition to suppressing resistance of other β-lactam nonsusceptible K. pneumoniae strains. The small molecule is able to lower carbapenem minimum inhibitory concentrations by up to 16-fold, while exhibiting little bactericidal activity itself.

Full text of DOI:10.1021/ml200290p

Letter
pubs.acs.org/acsmedchemlett
Small Molecule Suppression of Carbapenem Resistance in NDM-1
Producing Klebsiella pneumoniae
Roberta J. Worthington, Cynthia A. Bunders, Catherine S. Reed, and Christian Melander*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States
S
* Supporting Information
ABSTRACT: The already considerable global public health
threat of multidrug-resistant Gram-negative bacteria has
become even more of a concern following the emergence of
New Delhi metallo-β-lactamase (NDM-1) producing strains of
Klebsiella pneumoniae and other Gram-negative bacteria. As an
alternative approach to the traditional development of new
bactericidal entities, we have identified a 2-aminoimidazole-
derived small molecule that acts as an antibiotic adjuvant and is
able to suppress resistance of a NDM-1 producing strain of K.
pneumoniae to imipenem and meropenem, in addition to suppressing resistance of other β-lactam nonsusceptible K. pneumoniae
strains. The small molecule is able to lower carbapenem minimum inhibitory concentrations by up to 16-fold, while exhibiting
little bactericidal activity itself.
KEYWORDS: NDM-1, Klebsiella pneumoniae, antibiotic adjuvant, resistance suppression, 2-aminoimidazole
he emergence of resistance to multiple antimicrobial
agents in pathogenic bacteria has become a significant
past with carbapenem antibiotics, typically one of the
treatments of last resort for MDR K. pneumoniae.7 However,
isolation of carbapenemase producing strains, along with strains
possessing porin deletions, has been increasingly reported, thus
rendering these newer MDR K. pneumoniae strains recalcitrant
to carbapenem therapy.5
T
global public health threat. Drug-resistant bacterial infections
cause considerable patient mortality and morbidity, and rising
antibiotic resistance is seriously threatening the vast medical
advancements made possible by antibiotics over the past 70
years.1 This situation is so dire that the Infectious Diseases
Society (ISDA) has issued a call to action from the biomedical
community to deal with the multidrug-resistant (MDR)
bacterial threat.2
In 2008, a new plasmid-encoded metallo-β-lactamase was
identified from a K. pneumoniae clinical isolate recovered from a
patient hospitalized in New Delhi and named New Delhi
metallo-β-lactamase (NDM-1).8 NDM-1 is not inhibited by
current β-lactamase inhibitors and is able to inactivate all β-
lactams except aztreonam; however, NDM-1 encoding plasmids
coharbor a number of widely varying resistance determinants,
rendering bacteria carrying this plasmid resistant to almost all
current antibiotics. Of significant concern is that NDM-1
producers are already becoming highly prevalent, and NDM-1
has been observed in clinical isolates of the opportunistic
bacterium Acinetobacter baumannii and in strains of Escherichia
coli and other Enterobacteriaceae. Current therapy for
carbapenem-resistant K. pneumoniae clinical isolates is limited
to tigecycline or polymyxins such as colistin; however,
development of resistance to both of these antibiotics has
been encountered during treatment regimens.9
The majority of recent efforts have focused on strategies to
deal with MDR Gram-positive bacteria such as methicillin-
resistant Staphylococcus aureus (MRSA). In fact, the only two
new antibiotic classes introduced into the clinic in the last two
decades (daptomycin and linezolid) are Gram-positive
selective. There have not been the same research efforts and
successes directed toward the development of strategies to deal
with the increasing prevalence of MDR Gram-negative bacterial
infections.3 Klebsiella pneumoniae is among the most frequently
observed nosocomial Gram-negative bacteria worldwide and
causes numerous infections including urinary tract infections,
pneumonia, and intra-abdominal infections.4 K. pneumoniae has
caused increasing concern in recent years, as strains have
become resistant to virtually all antibiotics, through both
mutations in chromosomally encoded genes and acquisition of
genes from mobile plasmids and integrons.5 Extended-spectrum
β-lactamase (ESBL) producing K. pneumoniae strains have
become endemic in hospitals across many geographical regions.
ESBLs confer resistance to third generation cephalosporins
such as ceftriaxone, cefotaxime, and ceftazidime, in addition to
monobactams such as aztreonam.6 Infections caused by ESBL
producing K. pneumoniae have been successfully treated in the
While the development of new antibiotics is one approach
for the treatment of MDR K. pneumoniae, the fact remains that
only two new classes of antibiotics have been introduced into
the clinic over the last two decades.10 Furthermore, bacteria
invariably develop resistance to any introduced therapy that
Received: December 15, 2011
Accepted: February 4, 2012
Published: February 4, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
relies solely upon a bacteriostatic/bactericidal mechanism. For
example, daptomycin was introduced into the clinic in 2003,
and daptomycin-resistant strains were identified less than a year
later.11 Alternative approaches to controlling bacterial in-
fections are therefore sorely needed.12
Analogues with straight chain alkyl substituents of
intermediate length (five or six carbons) on the phenyl ring
were found to be considerably more active than those
possessing a butyl or heptyl chain. The more bulky t-butyl
and phenyl substituents exhibited very little activity (Table 1).
We recently reported that 2-aminoimidazole (2-AI) 1
(Figure 1) has the ability to lower the minimum inhibitory
Table 1. NDM-1 K. pneumoniae Carbapenem MICs in the
Presence of Compounds 3a−f
MIC (μg/mL)
compd (MIC/concn tested)
imipenem
meropenem
no compd
64
64
32
8
256
128
128
32
Figure 1. Small molecule 1 is able to suppress MDRAB and MRSA
antibiotic resistance.
3a (200/50 μM)
3b (200/50 μM)
3c (100/30 μM)
3d (100/30 μM)
3e (200/50 μM)
3f (>200/50 μM)
concentration (MIC) of certain antibiotics against MDR A.
baumannii (MDRAB) and MRSA.13 Given the unmet medical
need associated with NDM-1 K. pneumoniae, we initially
screened compound 1 for antibiotic resistance suppression
activity against the NDM-1 producing strain of K. pneumoniae
available from the ATCC (BAA-2146). Unfortunately, this
compound was found to possess only marginal activity.
Subsequently, we screened ∼50 compounds with known
biological activities from our in-house 2-AI library for the ability
to suppress resistance of K. pneumoniae to imipenem and
meropenem using a modified broth microdilution protocol.14
In this assay, the MIC of each 2-AI derivative is initially
determined in cation-adjusted Mueller Hinton broth
(CAMHB). The MICs of imipenem and meropenem are
then subsequently determined in CAMHB supplemented with
the 2-AI at a concentration of ≤30% of the MIC. It is our
experience that our 2-AI derivatives, at a concentration of 30%
of the MIC, exhibit very little microbicidal activity, thus
allowing us to attribute suppression of antibiotic resistance to
nonmicrobicidal effects of the compounds. From this screen,
we identified a series of analogues of 2-AI-aryl compounds15
that are members of our library (Scheme 1) and were shown to
4
16
32
64
64
128
The lead compound of this series, compound 3d, was able to
reduce the MICs of both imipenem and meropenem against the
NDM-1 producing K. pneumoniae strain by 16-fold (from 64 to
4 μg/mL for imipenem and from 256 to 16 μg/mL for
meropenem) at a concentration (30 μM) that was shown to
exhibit very little microbicidal activity by colony count analysis
(Figure 2). Increasing the concentration of compound 3d to 40
a
Scheme 1. Synthesis of 2-AI Aryl Series 3a−f
Figure 2. Time−kill curves for combinations of imipenem (IMP) and
compound 3d against K. pneumoniae BAA-2146. Solid lines indicate
the absence of compound 3d, and broken lines indicate the presence
of compound 3d (30 μM). Black = control, red = 32 μg/mL IMP, blue
= 8 μg/mL IMP, and green = 2 μg/mL IMP.
and 50 μM resulted in MIC reductions of 64- and 256-fold,
respectively, for imipenem, taking the MIC below the new
CLSI breakpoint for imipenem susceptibility (≤1 μg/mL).16
Checkerboard assays to determine whether compound 3d is
acting synergistically (ΣFIC ≤ 0.5) with carbapenem antibiotics
were performed as described previously.17 The ΣFIC calculated
for compound 3d with both imipenem and meropenem was
0.375, indicating that the combinations are synergistic.
We then further validated the adjuvant activity of the lead
compound 3d by constructing time−kill curves from colony-
forming unit (CFU) data at time points up to 16 h (Figure 2).
This serves as another method to confirm that the combination
of compound 3d and the carbapenem is acting synergistically
(≥2 log10 decrease in the number of CFUs relative to the count
obtained with the antibiotic alone indicates synergy), in
addition to allowing us to monitor the effect of compound
a
Reagents and conditions: (i) CH2N2, Et2O, 0 °C, 1 h. (ii)
Concentrated HBr, 15 min. (iii) BocHNC(NH)NH2, DMF, room
temperature, 72 h. (iv) TFA, CH2Cl2, room temperature, 2 h.
lower the MICs of imipenem and meropenem against the
NDM-1 producing strain of K. pneumoniae. This synthesis of
this series is depicted in Scheme 1 and commenced with
conversion of the commercially available para-substituted
benzoyl chlorides to the corresponding α-bromoketones, by
treatment with diazomethane followed by HBr. The α-
bromoketones were then cyclized with Boc-protected guanidine
to form the 2-AI heterocycle. Subsequent TFA-mediated
deprotection and counterion exchange afforded 2-AI HCl
salts 3a−f, which were used for biological testing.
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ACS Medicinal Chemistry Letters
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3d as a function of time. K. pneumoniae cultures were grown in
various concentrations of imipenem in the presence and
absence of compound 3d (30 μM) in CAMHB. Bacteria were
plated at various time points, and the number of viable CFUs
was enumerated. The reduction in CFUs as compared to
control bacteria is displayed in Table 2.
varying lengths, branched alkyl groups, and aromatic groups of
varying size. The synthetic approach to these second-generation
aryl 2-AI analogues is outlined in Scheme 2. A variety of readily
Scheme 2. Synthesis of 4,5-Disubstituted Analogues of
a
Compound 3d
Table 2. Log Reduction in K. pneumoniae CFU by Imipenem
in the Presence and Absence of Compound 3d
Log reduction in CFU/mL as compared
to untreated control
time
(h)
imipenem concn (μg/
imipenem
alone
imipenem + 30 μM
mL)
3d
32
16
8
4.36 0.06
3.16 0.05
2.53 0.12
0.86 0.06
0.79 0.02
0.92 0.03
0.39 0.05
0.62 0.07
0.53 0.01
0.38 0.03
>5.0
>5.0
4
8
>5.0
4
>5.0
2
3.84 0.12
>5.0
32
16
8
>5.0
>5.0
4
3.71 0.20
0.88 0.17
>5.0
a
2
Reagents and conditions: (i) HN(OMe)Me·HCl, BOP, Et3N,
32
16
8
<0.1
CH2Cl2, room temperature, 16 h. (ii) 4-Hexylphenyl magnesium
bromide, 2-methyltetrahydrofuran (0.5 M), −20 °C−room temper-
ature, 16 h. (iii) TFA, CH2Cl2, room temperature, 2 h. (iv) EtOH/
H2O, H2NCN, pH 4.3, 95 °C, 3 h.
<0.1
<0.1
<0.1
<0.1
>5.0
16
2.40 0.19
0.30 0.08
<0.1
4
2
available Boc-protected amino acids with aromatic and straight
chain and branched alkyl substituents were used as the starting
point for this synthesis.
Conversion of the starting carboxylic acids to the
corresponding Weinreb amides 4h−n was followed by addition
of 4-hexylphenyl magnesium bromide to form a series of α-
amino ketones 5h−n. Amine deprotection and subsequent
condensation with cyanamide afforded 4,5-disubstituted 2-AIs
6h−n. After purification, each compound was converted to the
corresponding HCl salt for biological testing.
As can be seen, we observe more marked reduction in CFUs
for combination treatments as compared to treatment with
imipenem alone. The effect of compound 3d, when compared
to imipenem alone, is most apparent from 4 h of incubation
onward (after bacteria leave lag phase); for example, at an
imipenem concentration of 16 μg/mL (1/4 MIC), after 4 h, the
antibiotic alone reduces the number of CFUs by only 3.16 log
units in comparison to untreated control, while the addition of
compound 3d results in a >5.0 log unit reduction. After 8 h, this
effect is even more marked with only a 0.39 log unit reduction
for the antibiotic alone as compared to a >5.0 log unit
reduction in the presence of compound 3d. In addition, a
considerable reduction in the number of CFUs can still be
observed at lower imipenem concentrations; for example, at 4
μg/mL (1/16 MIC), the addition of compound 3d results in a
>5.0 log reduction, as compared to just 0.86 log reduction for
imipenem alone after 4 h of incubation. The growth of bacteria
cultured in the presence of imipenem alone recovers very
quickly (after 2 h), possibly due to degradation of the antibiotic
by the NDM-1 producing bacteria. The addition of compound
3d slows this recovery, although a rebound in growth is still
observed for combination treatments at all imipenem
concentrations lower than 32 μg/mL. Regrowth, however, is
typical of bacterial cultures (both resistant and sensitive)
treated with β-lactam antibiotics.
The second-generation compounds were then tested for their
ability to lower the imipenem and meropenem MICs of the
NDM-1 producing strain of K. pneumoniae (Table 3).
Table 3. NDM-1 K. pneumoniae Carbapenem MICs in the
Presence of Compounds 6h−n
MIC (μg/mL)
compd (MIC/concn tested)
imipenem
meropenem
no compd
64
8
256
32
6h (100/30 μM)
6i (100/30 μM)
6j (>200/50 μM)
6k (200/50 μM)
6l (>200/50 μM)
6m (>200/50 μM)
6n (>200/50 μM)
16
64
32
32
32
64
64
128
64
128
128
128
In an effort to improve the efficacy of compound 3d and to
explore the effects of further substitution of the 2-AI
heterocycle, we synthesized a second generation of 2-AI-aryl
analogues that possessed a 4,5-disubstitution pattern. We have
previously observed that the introduction of substituents at the
5-position of compound 1 resulted in increased suppression of
oxacillin resistance in MRSA.17 Substituents were chosen to
allow comparison of the effect of straight chain alkyl groups of
Unfortunately, however, unlike our previous MRSA study, we
found that introduction of substituents at the 5-position of
compound 3d did not result in increased activity; nonetheless,
compounds 6h and 6i, in which the 5-position substituent is a
methyl and propyl group, respectively, were able to lower MIC
values. Compound 6h effected an 8-fold drop in the MICs of
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ACS Medicinal Chemistry Letters
Letter
both imipenem and meropenem at a concentration of 30 μM.
Increasing the chain length of the new substituent to six
carbons resulted in almost complete loss of activity, as did the
introduction of branched (iso-butyl) and aromatic (phenyl,
benzyl, and naphthyl) groups.
We next determined whether the resistance suppression
activity of compound 3d was specific to the NDM-1 producing
strain of K. pneumoniae or if compound 3d was able to suppress
carbapenem resistance in other MDR K. pneumoniae. To
address this issue, we investigated the ability of compound 3d
to suppress resistance in two other β-lactam-resistant strains of
K. pneumoniae. Against the KPC-2 producing strain (ATCC
BAA-1705) at 30 μM, compound 3d was able to reduce the
imipenem MIC by 16-fold (from 32 to 2 μg/mL), and against
an ESBL producing strain (ATCC 700603), compound 3d, at
30 μM, reduced the cefotaxime MIC by 8-fold (from 8 to 1 μg/
mL).
Finally, preliminary investigations into the mechanism of
action of the lead compound were carried out. Given the
amphiphilic nature of the lead compound 3d, we examined the
effect that it has on the permeability of the K. pneumoniae cell
membrane. Bacterial membrane damage was examined using
the Baclight assay as described by Hilliard et al.18 After
exposure to compound 3d for 1 h, the permeability of the K.
pneumoniae membrane (NDM-1 producing strain) was
considerably increased, with an intact/permeabilized membrane
ratio of 8.5% of the control (DMSO only treated) bacteria at its
active resistance suppression concentration of 30 μM. To
investigate whether this is the sole mechanism by which
compound 3d is able to suppress antibiotic resistance, we also
examined the effect of two compounds that were much less
active as suppressors of carbapenem resistance than compound
3d. Compound 3e, which differs from compound 3d only by
one extra methylene unit in the tail, and effected a reduction in
carbapenem MIC of just 2−4-fold, resulted in an intact/
permeabilized membrane ratio of 21.2% of the control bacteria
at a concentration of 50 μM. From the second generation of
analogues, compound 6j, which possesses a hexyl chain at the
4-position of the 2-AI ring and did not reduce the imipenem
MIC at all (reduction of meropenem MIC was 2-fold), had a
comparable effect on membrane permeability to compound 3d,
with an intact/permeabilized membrane ratio of 8.13% of the
control bacteria at a concentration of 30 μM. The fact that two
much less active compounds affected bacterial membrane
permeability considerably suggests that this is not the sole
mechanism by which compound 3d is able to lower antibiotic
resistance.
meropenem by 8−16-fold at a concentration (30 μM) at which
the lead compound itself displays little microbicidal activity.
Assaying at a slightly elevated concentration (50 μM) resulted
in a 256-fold reduction in imipenem MIC. Furthermore, the
lead compound of this series is also able to suppress resistance
in other carbapenem- and cephalosporin-resistant K. pneumo-
niae strains. The lead compound, along with two much less
active compounds, was shown to considerably affect permea-
blilization of the K. pneumoniae cell membrane. The lead
compound was shown to have very little hemolytic activity at
the concentration at which it affected the bacterial membrane.
Studies are currently underway to determine the mechanism of
action of these compounds and to identify the molecular
targets, along with further analogue synthesis to identify more
efficacious compounds. Given the urgent need for new
strategies to deal with the problem of MDR Gram-negative
bacteria, the identification of a novel small molecule that is able
to effect such a marked suppression of carbapenem resistance
represents an opportunity not only for the development of new
therapeutic entities but also for the identification of new targets
for future medicinal chemistry efforts.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic methods and compound characterization for all new
compounds and protocols for MIC determination and time−
1
kill curves, along with representative H NMR and 13C NMR
spectra and membrane permeability assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ccmeland@ncsu.edu.
Author Contributions
R.J.W., C.A.B., and C.S.R. performed the experiments outlined
in the manuscript, while R.J.W., C.A.B., and C.M. contributed
to writing the manuscript.
Funding
We thank the NIH (GM055769) and the V foundation for
partial support of this work.
Notes
The authors declare the following competing financial interest-
(s):CM has significant financial interest in a corporate entity
seeking to commercialize 2-AI derivatives as antibiotic
adjuvants.
Because of the effect that compound 3d has on the bacterial
membrane, we were interested in the effect that the compound
has on eukaryotic cell membranes, as an indicator of the
potential of this scaffold to be used as an antibiotic adjuvant.
We therefore investigated the hemolytic activity of compound
3d using mechanically difibrinated sheep blood as described
previously.15 Compound 3d had a much smaller effect on blood
cell membranes as compared to bacterial membranes. The
HD50 (the concentration at which the compound lyses 50%
cells as compared to the positive control) of compound 3d was
>500 μM, and the % lysis at the active concentration (30 μM)
was 2.5%.
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