Inhibitors of aminoglycoside resistance activated in cells

Article abstract of DOI:10.1021/cb200366u

The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside N-6′-acetyltransferase (AAC(6′)). Aminoglycoside-CoA bisubstrates are highly

Full text of DOI:10.1021/cb200366u

Letters
pubs.acs.org/acschemicalbiology
Inhibitors of Aminoglycoside Resistance Activated in Cells
Kenward Vong, Ingrid S. Tam, Xuxu Yan, and Karine Auclair*
́ ́
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6
S
* Supporting Information
ABSTRACT: The most common mechanism of resistance to
aminoglycoside antibiotics entails bacterial expression of drug-
metabolizing enzymes, such as the clinically widespread aminoglyco-
side N-6′-acetyltransferase (AAC(6′)). Aminoglycoside-CoA bisub-
strates are highly potent AAC(6′) inhibitors; however, their inability to
penetrate cells precludes in vivo studies. Some truncated bisubstrates
are known to cross cell membranes, yet their activities against AAC(6′)
are in the micromolar range at best. We report here the synthesis and
biological activity of aminoglycoside-pantetheine derivatives that,
although devoid of AAC(6′) inhibitory activity, can potentiate the
antibacterial activity of kanamycin A against an aminoglycoside-
resistant strain of Enterococcus faecium. Biological studies demonstrate that these molecules are potentially extended to their
corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-
concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize
resistant strains of bacteria to aminoglycoside antibiotics.
he World Health Organization (WHO) has recently
classified antibiotic resistance as one of the three greatest
Results and Discussion. Design. CoA is an essential
cofactor in many biological processes, most notably in the
synthesis and oxidation of fatty acids.12 In almost all
prokaryotes and eukaryotes, the common precursor to CoA is
pantothenic acid, which is obtained either through de novo
biosynthesis or from active transport from the environ-
ment.13,14 Bacteria are also known to actively import exogenous
amounts of pantetheine for transformation into CoA.15 This
biosynthetic system has been shown to transform pantothena-
mides16 and other derivatives for use in protein labeling.17,18
Because of its promiscuity, we envisaged to take advantage of
the CoA biosynthetic pathway to generate the potent AAC(6′)
inhibitors 2a−e in cells. Compounds 3a−e were designed to be
membrane-permeable substrates of the CoA biosynthetic
enzymes (Figure 2). Activation of 3a−e to the bisubstrate
inhibitors 2a−e was expected to proceed via the action of
pantothenate kinase (PanK),19 phosphopantetheine adenylyl-
transferase (PPAT),20 and dephosphocoenzyme A kinase
(DPCK).21 Based on their known AAC(6′) inhibitory activity,7
compounds 2a−e produced in vivo would then be expected to
block aminoglycoside resistance caused by this enzyme. This is
an unexplored approach to generate molecules that resensitize
bacteria to aminoglycoside antibiotics.
T
threats to human health. Among antibacterial agents, amino-
glycosides occupy a special niche due to their effectiveness
against aerobic, Gram-negative, and Gram-positive bacteria.1,2
Inevitably, however, their clinical use has been compromised by
the rapid emergence of bacterial resistance.3 The most common
mechanism of resistance to aminoglycosides entails bacterial
expression of drug-metabolizing enzymes, such as the clinically
widespread aminoglycoside N-6′-acetyltransferase (AAC-
(6′)).4−6 Using acetyl coenzyme A (AcCoA), AAC(6′)
acetylates the 6′-amino group of aminoglycosides.
Previous work in our group focused on aminoglycoside-CoA
bisubstrate inhibitors of AAC(6′).7−9 Many of these were
nanomolar inhibitors, such as 2a−c (Figure 1, panel a), and
also proved to be useful structural probes for different AAC(6′)
isoforms.7,10 Due to their negatively charged phosphate groups,
however, these bisubstrate inhibitors were not expected to
permeate cellular membranes. We subsequently reported
truncated derivatives able to penetrate bacterial cells.11 One
of these inhibitors, compound 1, was the first molecule shown
to block aminoglycoside resistance in cells. Its potency,
however, was greatly lower (Ki = 11 μM) than that of its
corresponding bisubstrate (Ki = 0.076 μM). With the aim of
generating more potent cell-permeable AAC(6′) inhibitors, we
designed molecules that would be extended into bisubstrates
once inside bacterial cells. We report here the synthesis and
biological studies of compounds 3a−e (Figure 1, panel a) that
have no AAC(6′) inhibitory activity of their own yet have the
ability to block aminoglycoside resistance in cells. We also
report evidence to support activation of 3a−e to 2a−e in vivo
by enzymes of the coenzyme A (CoA) biosynthetic pathway.
Synthesis. The synthesis and biological activity of com-
pounds 2a−d were previously reported.7 An additional
bisubstrate containing a longer linker, compound 2e, was
synthesized here (see Supporting Information). Target
compounds 3a−e were prepared in 2 steps (Figure 1, panel
Received: September 14, 2011
Accepted: January 4, 2012
Published: January 4, 2012
© 2012 American Chemical Society
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Figure 1. Structure, synthesis, and AAC(6′) inhibition constants of the compounds discussed. (a) Structure of compounds 1, 2a−e, and 3a−e as well
as the AAC(6′)-Ii inhibition constants (Ki) for 2a−e and 3a−e. (b) Synthetic scheme used to generate compounds 3a−e. Abbreviations used: DCC,
1,3-dicyclohexylcarbodimide; DIPEA (Hunig’s base), N,N-diisopropylethylamine; DMAP, N,N-dimethyl-4-aminopyridine; DTT, 1,4-dithiothreitol;
THF, tetrahydrofuran; RT, room temperature.
Figure 2. Proposed mechanism for the activation of 3a−e to 2a−e and the potentiation effect of 2a−e on the activity of kanamycin A against
resistant E. faecium. Following cell membrane penetration, 3a−e are expected to undergo phosphorylation by PanK to produce 6a−e, followed by
PPAT-catalyzed adenylylation to 7a−e and phosphorylation by DPCK to generate 2a−e. Compounds 2a−e are known inhibitors of AAC(6′)-Ii. The
inactivation of this resistance-causing enzyme allows aminoglycosides such as kanamycin A to disrupt protein synthesis and cause cell death.
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Figure 3. Transformation of 3a−e by the CoA biosynthetic enzymes. (a) Michaelis−Menten constants (Km), turnover rates (kcat) and catalytic
efficiencies (kcat/Km) calculated for the transformation of compounds 3a−e by PanK. (b) Approximate ratio of starting material and the different
products (expressed as a relative percentage 5%) measured by HPLC for the in vitro transformation of 3a−e by a combination of PanK, PPAT and
DPCK. (c) HPLC chromatograms evaluating the in vitro biosynthetic transformation of compounds 3a−e by a combination of PanK, PPAT, and
DPCK. Reaction mixtures were incubated with (I) water, (II) 3a, (III) 3b, (IV) 3c, (V) 3d, and (VI) 3e.
b). This pathway was adapted from a methodology reported by
us.11 All final compounds were purified twice by HPLC to
ensure purity (>95%), and the low yields reported reflect losses
due to extensive sample manipulation.
A biosynthetic in vitro assay was designed to determine the
potential of compounds 3a−e to be fully extended to
compounds 2a−e by the enzymes PanK, PPAT, and DPCK.
LC−MS analysis of the reaction mixtures was used to monitor
the transformation of 3a−e by these 3 enzymes in one-pot
(Figure 3, panel b and c). A product of a mass corresponding to
2a−e is clearly observed for all but the reaction of 3a.
Moreover, the biosynthetic intermediates 6a−c and 7a−e are
also identified. The absence of detectable 6d and 6e (Figure 3,
panel c, V and VI) is attributed to a more complete
transformation of 3d and 3e to 2d and 2e, respectively. The
approximate percent conversions of 3a−e to 2a−e, 6a−e, and
7a−e observed (Figure 3, panel b) are consistent with an
increased efficiency of these enzymes with increasing chain
length up to n = 4. Overall, these results suggest that 3b−e may
be extended by bacteria to the full bisubstrates 2b−e, which
may in turn inhibit AAC(6′) and block resistance.
These in vitro results encouraged us to test 3a−e in cells. As
mentioned above, E. faecium, chromosomally encoded with
AAC(6′)-Ii, is one of the leading causes of hospital-acquired
infections.22 Using a standard checkerboard assay, the ability of
3a−e to resensitize E. faecium ATCC 19434 to the amino-
glycoside kanamycin A was investigated. Intrinsically, com-
pounds 3a−e were found to lack any antibacterial activity
against E. faecium. This was also the case when tested against
Staphylococcus aureus ATCC 29213 and 43300, Acinetobacter
Biological Studies. Figure 1 summarizes the AAC(6′)-Ii
inhibition constants of 2a−e. This AAC(6′) isoform was
selected because it is naturally expressed in Enterococcus faecium,
one of the leading causes of hospital-acquired infections.22 In
general, the shorter the linker, the more potent is the inhibitor.
As expected from the lack of inhibition previously reported for
3a,11 none of 3a−e (tested up to 500 μM) show inhibitory
activity against purified AAC(6′)-Ii. To investigate the potential
of compounds 3a−e to be extended to 2a−e by the CoA
biosynthetic enzymes in cells, kinetic studies were performed
with purified PanK, the rate-limiting enzyme of this pathway,23
and the Michaelis−Menten constants (Km), turnover rates
(kcat), and catalytic efficiencies (kcat/Km) for the PanK
transformation of 3a−e were determined (Figure 3, panel a).
The observed trend shows a correlation between the linker
length and catalytic efficiency. Derivatives with longer linkers
are generally better PanK substrates, whereas those with shorter
linkers are poorer substrates (as judged from kcat/Km).
Although none are as good as the natural substrate pantothenic
acid, 3d and 3e are comparable to the alternative substrate,
pantetheine.
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baumannii ATCC 19606, Pseudomonas aeruginosa ATCC
27853, Klebsiella penumoniae ATCC 13883, and Escherichia
coli ATCC 25922 and 11775 (data not shown). In the absence
of compounds 3a−e, the minimum concentration of kanamycin
A causing a 50% growth inhibition (MIC50) of E. faecium is
∼125 μg mL−1 (Figure 4, panel a). As expected from the in vitro
most common mechanisms of resistance. Numerous resistance
mechanisms have been reported for penicillins (efflux,
decreased cell permeability, and expression of various β-
lactamases), yet the combination of amoxicillin with the β-
lactamase inhibitor clavulinate has generated sales of more than
a billion dollars in one year only.24 Expression of AAC(6′)
enzymes is one of the most common determinants of
aminoglycoside resistance in the clinics. We have previously
reported that aminoglycoside-CoA bisubstrates are potent
AAC(6′) inhibitors.7 Although these were useful molecules to
interrogate enzyme behavior, their use is limited by their
inability to penetrate cells membranes. On the other hand, a
truncated bisubstrate inhibitor, compound 1, was found to
block resistance in cells, yet its potentiation effect on the
antibacterial activity of kanamycin A was modest. We report
here a new strategy to improve the potency while maintaining
cell-permeability of aminoglycoside resistance inhibitors.
Taking advantage of the promiscuity of the CoA biosynthetic
pathway, we have designed cell-permeable molecules that are
extended to potent bisubstrate inhibitors in cells.
In summary, compounds with longer linkers (e.g., 3d and 3e)
are better substrates of PanK in vitro and also better
potentiators of the antibacterial activity of kanamycin A in E.
faecium. Interestingly, this is opposite to the trend observed for
in vitro AAC(6′) inhibition by the corresponding bisubstrates
2a−e. Since none of 3a−e show intrinsic antibacterial activity
of their own, we attribute the trend observed in cells to the rate-
limiting role of prodrug activation. Although compounds with
longer linkers (e.g., 2c−e) are poorer AAC(6′) inhibitors, their
formation from the corresponding prodrugs 3c−e in vivo is
likely more efficient as suggested by the in vitro biosynthetic
studies (Figure 3, panel c, IV, V, and VI). Figure 2 shows the
proposed activation path of 3a−e and explains how the
antibacterial activity of kanamycin is rescued by these
molecules. Of note is that our previous studies with AAC(6′)-
Ii11 suggest that intermediates 6b−e and 7b−e may also inhibit
the enzyme to some extent. To our knowledge, compounds
3b−e show the largest potentiation effect reported to date for
the antibacterial activity of kanamycin A against a bacterial
strain resistant to aminoglycosides. This work presents a new
approach for generating molecules that resensitize bacteria to
aminoglycoside antibiotics. Moreover, it is envisioned that the
prodrug activation strategy used here has the potential to find
use with other biologically active CoA derivatives.
Figure 4. Results from checkerboard assays performed with a resistant
strain of E. faecium expressing AAC(6′)-Ii. (a) Potentiation effect of
compounds 3a−e (512 μg mL−1) on the antibacterial activity of
kanamycin A. (b) Dose-dependent potentiation effect of compound 3c
on the antibacterial activity of kanamycin A. The data was fitted with a
sigmoidal dose−response (variable slope) equation. All data points are
from quadruplicate experiments.
METHODS
■
Synthesis. General Procedure for Synthesis of Compounds
3a−e. Neamine free base (145 mg, 0.45 mmol) was dissolved in water
(3 mL), and the corresponding NBD ester (5a−e, 0.23 mmol) was
dissolved in acetone (2 mL). The two solutions were mixed and stirred
for 10 min (5a) or 20 min (5b−e). In another vial, D-pantethine (66
mg, 0.12 mmol), DIPEA (1 mL, 5.7 mmol), and DTT (20 mg, 0.13
mmol) were mixed in acetone (2 mL) and sonicated until a
homogeneous mixture is obtained. This solution was then transferred
to the neamine/NBD ester reaction mixture and then allowed to stir
overnight. The solvent was evaporated under vacuum, and the residue
was dissolved in water (10 mL). TFA was added to pH ∼3, which was
then followed by three successive ethyl acetate washes. The aqueous
layer was evaporated to dryness by lyophilization. HPLC purification
was achieved through method 3 followed by method 4 (see
Supporting Information). Compounds 3a−e all appear as white fluffy
powders after lyophilization (yields 20−50%).
data, addition of 3a has a negligible effect on the MIC50 of
kanamycin A. Compounds 3b−e on the other hand decrease
the MIC50 of kanamycin A considerably, with 3d and 3e
causing the MIC50 to drop by half. The potentiation effects
observed here for 3b−e are much superior to that previously
reported for compound 1.11 It is noteworthy that for each of
3b−e, a dose-dependent behavior is observed, as exemplified
for 3c (Figure 4, panel b). Finally, LC−MS analysis of the
cellular mixture obtained when exposing E. faecium lysate to 3d
shows a peak corresponding to bisubstrate 2d, which is absent
in the negative control (see Supporting Information).
Aminoglycosides were the first antibacterial agents effective
against tuberculosis and many Gram-negative pathogens. As
with other antibiotics, bacterial resistance to aminoglycosides is
threatening their clinical use. One demonstrated strategy to
overcome antibiotic resistance is to design inhibitors of the
General Procedure for Synthesis of Compounds 5a−e from
Carboxylic Acids 4a−e. endo-N-Hydroxy-5-nornornene-2,3-dicarbox-
imide (NBD) (1.07 g, 6 mmol) and the corresponding carboxylic acid
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(6 mmol, 4a−e) were dissolved in dichloromethane (50 mL). DCC
(1.24 g, 6 mmol) was added to the mixture, followed by a catalytic
amount of DMAP (∼20 mg). A few minutes after addition of DCC, a
white solid (DCU) precipitated out. The reaction was stirred at RT
overnight. The presence of the desired NBD ester (5a−e) was
monitored by TLC (1:1 ethyl acetate/hexanes, Rf = 0.45−0.50). The
solid DCU was removed by filtration and the filtrate was evaporated to
approximately 1 mL. Flash column chromatography (ethyl acetate/
hexanes 1:2) was used to purify the desired compounds, which
typically appeared as white, flakey solids upon evaporation of all
solvents (yields 64−82%).
Biological Assays. AAC(6′)-Ii Inhibition Assay. AAC(6′)-Ii was
expressed and purified as previously described.7 The AAC(6′)-Ii
inhibition assay was conducted with a BioLogic SFM 400 stopped-flow
mixing chamber controlled by an MPS-60 unit. The data was collected
on a MOS 250 UV−Vis spectrophotometer at 412 nm. The TC-100
cuvette used had an observation path length of 1 cm. Data was
processed with Biokine32 ver 4.2 (BioLogic) to determine initial rates,
which were next fitted in GraphPad Prism 4.0 to determine kinetic
parameters. The total reaction volume was 440 μL, from 4 reagent-
containing chambers each containing an equal volume of 110 μL.
Measurements were taken at a wavelength of 412 nm. All solutions
were in MES Buffer (25 mM, pH 6.0). The kinetic activity of AAC(6′)-
Ii was first determined with varying concentrations of AcCoA and a
fixed concentration of neamine, using the following solutions in 4
separate chambers: (1) MES buffer solution (25 mM); (2) various
concentrations of AcCoA (50, 100, 150, and 200 μM); (3) neamine
(1.6 mM) and DTNB (4 mM); and (4) AAC(6′)-Ii (4 μM). The
enzymatic activity of AAC(6′)-Ii was next determined in the presence
of the molecules of interest to measure inhibition constants (Ki). To
this end, the following solutions were separated in the instrument into
4 chambers: (1) MES buffer solution (25 mM); (2) varying
concentrations of inhibitor (1, 2, 4, and 8 μM) mixed with various
concentrations of AcCoA (50, 100, 150, and 200 μM); (3) neamine
(1.6 mM) and DTNB (4 mM); and (4) AAC(6′)-Ii (4 μM). All data
points are from triplicate experiments.
PanK Enzyme Assay. The E. coli pantothenate kinase (PanK or
coaA enzyme) was expressed and purified as previously described.25
Enzyme activity was measured as described in the literature.16 This
assay couples the production of ADP to the consumption of NADH
through the activity of pyruvate kinase and lactic dehydrogenase. The
decrease of NADH concentration was monitored at 340 nm. Reactions
were performed at 25°C in an Agilent 8453 UV−Vis spectropho-
tometer coupled to an Agilent 89090A Peltier temperature controller.
Kinetic parameters were determined by fitting the rate data into the
Michaelis−Menten equation using GraphPad Prism 4.0. Each reaction
mixture (500 μL) contained ATP (1.5 mM), NADH (0.3 mM),
phosphoenolpyruvate (0.5 mM), MgCl2 (10 mM), KCl (20 mM),
pyruvate kinase (5 units), lactic dehydrogenase (5 units), and PanK (5
μg, 278 nM) in Tris-HCl buffer (50 mM, pH 7.6). The reaction was
initiated by addition of the desired substrate (10−160 μM). All data
points are from triplicate experiments.
Biosynthetic in Vitro Assay with a Mixture of PanK, PPAT, and
DPCK Enzymes. The E. coli enzymes pantothenate kinase (PanK or
coaA enzyme), phosphopantetheine adenylyltransferase (PPAT or
coaD enzyme), and dephosphocoenzyme A kinase (DPCK or coaE
enzyme) were expressed and purified as previously described.25 Each
reaction mixture (500 μL) contained ATP (5.0 mM), KCl (20 mM),
MgCl2 (10 mM), DTT (2.0 mM), PanK (5 μg, 278 nM), PPAT (5 μg,
500 nM), and DPCK (5 μg, 454 nM) in Tris-Cl Buffer (50 mM, pH
7.6). The reaction was initiated with the addition of pantetheine (5.0
mM) for comparison, water as a negative control, or compounds 3a−e
(5.0 mM). Reactions were incubated for 3 h at RT and then stopped
by heating the mixture to 95 °C for 5 min. The precipitated protein
was removed by centrifugation (13,000 rpm for 5 min), and the
supernatant was analyzed by LC−MS. Reversed-phase analytical
HPLC was performed with an analytical 4.60 × 250 mm, SYNERGI 4
μm Hydro-RP 80A (Phenomenex) column coupled to an Agilent 6120
Quadrupole LC−MS system for ESI-MS analysis. The HPLC
conditions had the sample eluted at a flow rate of 0.5 mL min−1
using a combination of mobile phase A (H2O) and mobile phase B
(acetonitrile). Elution conditions are as follows: isocratic 1% phase B
from 0 to 3 min; followed by the following linear gradients of phase B:
1−10% from 3−5 min; 10−15% from 5−10 min; 15−30% from 10−
13 min; and finally isocratic phase B at 30% from 13−26 min. The
detector was set to 214 nm. These experiments were run in duplicates.
Checkerboard Assay To Determine the Potentiation Effect of
Compounds 3a−e on the Antibacterial Activity of Kanamycin A on
a Resistant Strain. A two-dimensional checkerboard MIC assay was
carried out as previously described11 to observe the potentiation
activity of compounds 3a−e toward the antibacterial activity of
kanamycin A against Enterococcus faecium ATCC 19434 that expresses
AAC(6′)-Ii. Compounds 3a−e were diluted by 2-fold sequential
dilutions to create a gradient from 32 to 512 μg mL−1. The
concentrations used for kanamycin A were 0, 10, 20, 40, 50, 100, 150,
200, 250, and 300 μg mL−1. The cultures were grown at 37 °C for 16 h
in 96-well plates, and then monitored for optical density at 600 nm
using a Spectramax 190 microplate reader (Molecular Devices). Data
was normalized against a positive growth control and reported as a
percentage of bacterial cell growth. All data points are from
quadruplicate experiments.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed descriptions of experimental procedures, character-
ization of all compounds as well as selected HPLC traces and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: karine.auclair@mcgill.ca.
ACKNOWLEDGMENTS
■
This work was supported by research grants from the Canadian
Institute of Health Research (CIHR) and the National Science
and Engineering Research Council of Canada (NSERC) to
K.A. K.V. was supported by the CIHR Strategic Training
́ ́
Initiative in Chemical Biology and Fonds quebecois de la
recherche sur la nature et les technologies (FQRNT)
scholarships. The authors are grateful to G. D. Wright at
McMaster University for sharing his AAC(6′)-Ii, PanK, PPAT,
and DPCK expression plasmids.
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