Thiol-Containing Metallo-β-Lactamase Inhibitors Resensitize Resistant Gram-Negative Bacteria to Meropenem

Article abstract of DOI:10.1021/acsinfecdis.7b00094

The prevalence of infections caused by metallo-β-lactamase (MBL) expressing Gram-negative bacteria has grown at an alarming rate in recent years. Despite the fact that MBLs can deactivate virtually all β-lactam antibiotics, there are as of yet no approved

Full text of DOI:10.1021/acsinfecdis.7b00094

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Letter
Thiol-Containing Metallo-#-Lactamase Inhibitors Resensitize
Resistant Gram-Negative Bacteria to Meropenem
Kamaleddin Haj Mohammad Ebrahim Tehrani, and Nathaniel I. Martin
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00094 • Publication Date (Web): 18 Aug 2017
Downloaded from http://pubs.acs.org on August 21, 2017
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Thiol-Containing Metallo-β-Lactamase Inhibitors Resensitize
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Resistant Gram-Negative Bacteria to Meropenem
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Kamaleddin Haj Mohammad Ebrahim Tehrania and Nathaniel I. Martina ,*
aDepartment of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands;
Tel: +31 (0)6 1878 5274; Fax: +31 (0)30 - 253 6655; E-mail: n.i.martin@uu.nl
ABSTRACT
The prevalence of infections caused by metallo-β-lactamase (MBL) expressing Gram-
negative bacteria has grown at an alarming rate in recent years. Despite the fact that MBLs
can deactivate virtually all β-lactam antibiotics, there are as of yet no approved drugs
available that inhibit their activity. We here examine the ability of previously-reported thiol-
based MBL inhibitors to synergize with meropenem and cefoperazone against a panel of
Gram-negative carbapenem-resistant isolates expressing different β-lactamases. Among the
compounds tested, thiomandelic acid 3 and 2-mercapto-3-phenylpropionic acid 4 were found
to efficiently potentiate the activity of meropenem, especially against an IMP producing
strain of K. pneumoniae. In light of the zinc-dependent hydrolytic mechanism employed by
MBLs, biophysical studies using isothermal titration calorimetry were also performed
revealing a correlation between the synergistic activity of thiols 3 and 4 and their zinc-
binding ability with measured Kd values of 9.8 and 20 µM respectively.
KEYWORDS:
Antibiotic Resistance, metallo-β-lactamase, MBL inhibitor, zinc binding, synergy
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INTRODUCTION
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Resistance to β-lactam antibiotics poses a serious threat to human health. The
enzymes responsible for such resistance are the β-lactamases and are especially prevalent
among Gram-negative bacteria.1,2 These enzymes are divided in two classes: the serine β-
lactamases (SBLs) which hydrolyze beta lactam ring by a serine nucleophile in their active
site; and the metallo-β-lactamases (MBLs) whose mechanism relies upon the presence of one
or two active site zinc ions.3 These zinc ions stabilize a nucleophilic hydroxide species that is
believed to be the active agent in the hydrolysis the β-lactam ring leading to antibiotic
inactivation. The best-studied MBLs include the NDM (New Delhi metallo-beta-lactamase),
VIM (Verona integron-encoded metallo-β-lactamase) and IMP (imipenemase) enzymes
which collectively exhibit a broad substrate specificity and hydrolyze antibiotics from all
known beta-lactam classes.4
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The global concern relating to antibiotic resistance has led to a number of different
strategies aimed at addressing the problem. One such approach involves the co-administration
of antibiotic adjuvants capable of maintaining the activity of existing antibiotics.5 This
strategy is widely effective in treating infections due to bacteria that express SBLs. Clinically
relevant SBL inhibitors include clavulanic acid, sulbactam, tazobactam and avibactam which
effectively protect β-lactam antibiotics from inactivation when administered as combination
therapies. By comparison, there are no clinically used MBL inhibitors available for use in
addressing the growing threat posed to the β-lactam arsenal by these enzymes.
Attempts to identify inhibitors of the MBLs have revealed a number of compound
classes that display promising activities when tested using in vitro enzyme inhibition assays.6
Such compounds include small molecules that contain functionalities often associated with
zinc binding such as: thiols, dicarboxylates, hydroxamates, aryl sulfonamides, N-arylsulfonyl
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hydrazones and tetrazole-based compounds.3,7,8 Among the known MBL inhibitors, sulfur-
containing small molecules containing either a free thiol or a sulfur atom masked as a
heterocycle9,10 are among the best characterized.3,7,11–15 Structures as simple as
mercaptoacetic acid and mercaptopropionic acid have been shown to be effective inhibitors
of the IMP-1 enzyme (Ki = 0.23 µM and 0.19 µM respectively).16 Follow-up studies
identified higher analogs of mercapto-carboxylic acids including 2-arylmethyl-2-
mercaptoacetic acids and their thioesters as potent MBL inhibitors with IC50 values in the
low-nanomolar range.17 Similarly, thiomandelic acid was found to be a broad-spectrum
inhibitor of different MBLs including BCII (Ki = 0.34 µM), IMP-1 (Ki = 0.029 µM), IMP-2
(Ki = 0.059 µM) and VIM-1 (Ki = 0.230 µM)18 and 2-ω-phenylpropyl-3-mercaptopropionic
acid has been reported as potent inhibitor of VIM-2 (Ki = 0.220 µM).19,20 Other examples of
structurally similar MBL inhibitors include compounds containing a free thiol with a
neighboring carbonyl functionality such as α-mercaptoacetophenone,21 thiosalicylic acid,18
and interestingly, the dipeptide drug captopril which is used in the treatment of
hypertension.22 The inhibitory activity of these molecules is attributed to the ability of the
thiol group to chelate the zinc ion present in the MBL active site as supported by several X-
ray crystallography studies.21,23–26
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The inhibitory activity of thiol-containing small molecules against clinically relevant
MBLs in vitro prompted us to conduct a series of antibacterial assays to evaluate the
synergistic activity of such compounds with the representative β-lactams meropenem and
cefoperazone. To do so, the sensitizing effects of thiols 1-5 (Fig. 1) on the activity of
meropenem and cefoperazone were assessed against a panel of Gram-negative bacteria
expressing various β-lactamases. The stability of the thiols was also assessed under the assay
condition employed and isothermal titration calorimetry (ITC) was used to measure the zinc-
binding affinity of the most synergistically-active compounds.
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Figure 1. Thiol-based MBL inhibitors and disulfides evaluated
for synergy with meropenem and cefoperazone in the current
study.
RESULTS AND DISCUSSION
Thiols 1-5 were initially tested alone for antibacterial activity against a panel of
carbapenem-resistant Gram-negative pathogens expressing MBLs including NDM, VIM and
IMP or SBLs such as KPC-2 and OXA-48. These studies revealed that none of the thiols
inhibited bacterial growth at the highest concentration tested of 64 µg/mL. With the
exception of P. aeruginosa M-120 which was susceptible to meropenem, all of the MBL-
expressing strains used our study exhibited resistance to both meropenem and cefoperazone
with MIC values ranging from 8 to >256 µg/mL. For use as a reference MBL inhibitor known
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to synergize with β-lactam antibiotics, we turned to the work of Migliavacca and coworkers
who reported a zinc chelating mixture of EDTA and 1,10-phenanthroline as being synergistic
with imipenem in preventing growth of MBL-expressing strains of Pseudomonas
aeruginosa.27 We found that the EDTA/1,10-phenanthroline mixture was similarly effective
in lowering the MIC of meropenem and cefoperazone against the MBL-expressing strains
used in our study (Table 1). Also, and as expected, the EDTA/1,10-phenanthroline mixture
showed no synergistic effect with meropenem against the two SBL-expressing strains also
evaluated.
Table 1. MIC (µg/mL) of meropenem (Mer) and Ceferazone (Cef) tested alone or in combination with thiol MBL-inhibitors 3 and 4
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Isolates
Mer
Cef
Mer + 3a
Cef + 3
Mer + 4
Cef + 4
Mer + EPb Cef + EPb
K. pneumoniae
RC10 (KPC-2)
>128
>256
128
>256
>128
>256
>128c
>256c
>256d
8c
K. pneumoniae
RC 45 (OXA-48)
64
64/32
16/8
>256
>256
256
64
>256
256
≤2
64
>256
>256
≤2
16d
K. pneumoniae
RC51 (VIM-1)
0.5
1
≤0.5c
≤0.125g
≤1c
(≥128e, ≤0.27f)
(32e, ≤0.38f)
K. pneumoniae
JS265 (IMP-28)
0.125
0.125
≤2g
(128e, ≤0.07f)
(≥128e, ≤0.07f) (64e, ≤0.13f) (≥128e, ≤0.07f)
E. coli
RC89 (NDM-1)
128/64
32
>256
128
16
>256
16
>256
>256c
8c
(8e)
(8e)
P. aeruginosa
RC60 (VIM)
4
16
8
16
0.5c
(8e, ≤0.38f)
(8e, ≤0.5f)
(4e)
(8e, ≤0.5f)
P. aeruginosa
JS80 (IMP)
64
256
8
8
8
16
4h
4g
(8e)
(32e, ≤0.38f)
(8e, ≤0.5f)
(16e, ≤0.25f)
K. pneumoniae
RC21 (VIM-1)
64/32
32/16
>128/128
32/16
16/8
>256
>256
>256
>256
>256
>256
128/64
0.5
>256
>256
>256
>256
>256
>256
1
>256
>256
>256
>256
>256
>256
≤0.5g
≤0.25g
≤1g
≤2g
(64e, ≤0.19f)
(32e, ≤0.38f)
E. aerogenes
RC22 (VIM-1)
0.5
1
64g
(64e, ≤0.25f)
(16e, ≤0.38f)
K. pneumoniae
RC48 (VIM-1)
4
8
≤2g
(>32e, ≤0.38f)
(16e, ≤0.5f)
K. pneumoniae
JS22 (NDM-1)
8
2
≤0.5g
≤0.25g
≤1g
>256g
>256g
>256g
4 (32)d
(4e)
(16e, ≤0.38f)
K. pneumoniae
JS177 (NDM-1)
4
1
(4e)
(8e)
K. pneumoniae
JS37 (NDM-1)
64/32
<1
16
16
(4e)
(4e)
P. aeruginosa
M-120 (IMP)
n.d.
2
n.d.
4
n.d.
(32e, ≤0.25f)
(16e, ≤0.38f)
a Thiols 3 and 4 added at 64 µg/mL. None of the thiols inhibited growth at this concentration when tested alone.
b EP = EDTA/1,10-phenanthroline mixture
c EP mixture applied at 16 µg/mL EDTA and 1 µg/mL 1,10-phenanthroline.
d EP mixture applied at 64 µg/mL EDTA and 4 µg/mL 1,10-phenanthroline.
e Fold reduction of MIC. Reduction of MIC greater than 8-fold shown in bold.
f Lowest FIC measured (FIC<0.5 regarded as an indication of synergy).
g EP mixture applied at 32 µg/mL EDTA and 2 µg/mL 1,10-phenanthroline.
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With a reliable reference system in hand, the capacity for thiols 1-5 to synergize with
meropenem was next investigated. Table 1 shows the synergy data of the two most potent
thiols 3 and 4 (see supplemental information Table S2 for complete activity data for
compounds 1-5). Captopril 1 exhibited moderate to weak synergy at a concentration of 64
µg/mL while thiosalicylic acid 2 displayed no appreciable synergy when tested at the same
concentration. By comparison, when administered at 64 µg/mL thiols 3 and 4 significantly
lowered the MIC of meropenem against all the MBL-producing isolates tested. The activity
of compound 5 was also promising but interestingly was limited to the only two Klebsiella
strains tested. Thiols 3-5 have previously been shown to be more potent MBL inhibitors than
compounds 1 and 2 in biochemical enzyme inhibition assays17,18,21,23 and our MIC synergy
results follow the same trend. Notably, for compounds 3 and 4 we observed broad-spectrum,
and in some cases, potent synergistic activity with meropenem against the MBL-producing
isolates evaluated. Building on the encouraging results of the preliminary synergy assays
(carried out at fixed thiol concentration of 64 µg/mL) we next performed a series of
checkerboard synergy assays in which the MIC of meropenem was determined at varying
concentrations of inhibitors 1-5. Such an approach provides for a better picture of the
synergistic relationship between the two combined agents and allows for determination of the
fractional inhibitory concentration (FIC) index. Briefly, FIC values are calculated by adding
the following two fractional values: (MIC of compound A in combination/MIC of compound
A alone) + (MIC of compound B in combination/MIC of compound B alone). In general, an
FIC index value <0.5 is regarded as an indication of synergy.28 A complete overview of all
the checkerboard assays performed as well as the corresponding FIC index values is provided
in the supplemental information. Among the MBL-expressing strains used the two Klebsiella
isolates were most effectively resensitized to the meropenem when administered in
combination with thiols 3-5. Of particular note, compounds 3 and 4 were both found to
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significantly potentiate meropenem against the IMP-28 producing Klebsiella strain tested
with FIC values ≤0.07 and ≤0.13 respectively (based on the concentrations tested, see table 1
for checkerboard FIC data of thiols 3 and 4 and supplemental information for graphical
representation of checkerboard assays).
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Thiols are well known for their tendency to form homo- or heterodisulfides in
biological systems. Such reactivity is of special importance in the case of thiol-based MBL
inhibitors such as compounds 1-5 as it has been reported that in their disulfide form their
activity is significantly reduced.18 In this regard we selected compounds 3-5 as the three most
active thiols from our synergy assays and monitored their conversion to the corresponding
disulfides under the assay conditions used. Thiols 3-5 were thus incubated in Mueller-Hinton
broth at 37 °C and sample aliquots analyzed at time points ranging from 0 to 8 hours. As
shown in figure 2, thiols 3 and 4 were found to form their corresponding disulfides (6 and 7
respectively) with half-lives of ca. 5 hours. By comparison, thiol 5 was oxidized to 8 more
rapidly with a half-life in the range of minutes which may also explain its lower level of
synergy relative to 3 and 4. Disulfides 6-8 were synthesized for use as reference compounds
in the stability assays and were evaluated for their synergy with meropenem against the two
most susceptible Klebsiella isolates identified (Supplemental Table S20). The three disulfides
exhibited very low levels of synergy relative that of the corresponding free thiols. The slight
synergy observed for these disulfides may in fact be attributable to a reductive process carried
out by the bacteria themselves to release a small amount of the more active thiol. Many
bacteria contains redox active enzymes capable of disulfide reduction both in cytoplasm and
periplasmic space.29
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Disulfide 6
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Thiol 3
Thiol 4
Disulfide 7
Disulfide 8
Thiol 5
Figure 2. Time-dependent oxidation of thiols 3-5 to corresponding
disulfides 6-8 by incubation in Mueller-Hinton broth at 37 °C.
The superior synergistic activity and relative stability of thiols 3 and 4 prompted us to
further characterize their zinc binding abilities using isothermal titration calorimetry (ITC).
To do so a solution of zinc chloride was titrated into the sample well containing either 3 or 4
(both 3 and 4 were both to be found stable in the buffer conditions used for the ITC
experiments) and the heat of binding monitored. In this way a number of thermodynamic
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binding parameters are revealed including Kd (dissociation constant), ΔH (enthalpy), ΔG
(Gibbs free energy) and ΔS (entropy). As shown in figure 3, compounds 3 and 4 exhibited
high affinities for Zn2+ with Kd values of 9.8 and 20.0 µM respectively. Also of note was the
lack of any measurable binding interaction when zinc chloride was titrated into solutions of
disulfides 6 and 7. The zinc binding abilities of the reference compounds EDTA and 1,10-
phenanthroline were also assessed using ITC showing a strong interaction (Supplemental
figures S3 and S4). The results of these ITC studies correlate well with the synergy data
obtained and suggest that zinc binding may be a useful predictor for a compound’s ability to
resensitize MBL-expressing organisms to beta-lactam antibiotics. Furthermore, the relative
ease with which ITC can be used to assess zinc binding by small molecules may make it a
complimentary technique for identifying new lead compounds capable of effectively
inhibiting MBLs.
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(B)
(A)
Kd = 9.81 ± 1.12 µM
Kd = 19.99 ± 1.30 µM
ΔH = -48.14 ± 2.17 kJ.mol-1
ΔS = -65.66 ± 7.35 J.mol-1.K-1
ΔG = -28.57 ± 0.28 kJ.mol-1
ΔH = -37.72 ± 0.87 kJ.mol-1
ΔS = -36.60 ± 2.97 J.mol-1.K-1
ΔG = -26.81 ± 0.16 kJ.mol-1
Figure 3. ITC Thermograms for binding of Zn2+ by thiols 3 (A) and 4 (B). A solution of zinc chloride (2.0 mM) was titrated
into the sample cell containing thiol 3 or 4 (0.2 mM). Thermodynamic parameters shown based on triplicate binding assays
and the reported as mean ± SE. Errors estimated via Monte Carlo simulations using the error of individual experiments.
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CONCLUSION
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While small molecule thio-carbonyl compounds have previously been shown to inhibit
various MBLs, their ability to synergize with β-lactam antibiotics in overcoming MBL-
associated resistance has not been extensively studied. We here demonstrate a significant
level of synergism between meropenem and a series of thiols, most notably thiomandelic acid
3 and 2-merpto-3-phenylpropionic acid 4. Combinations of meropenem with 3 or 4 exhibit
antibacterial activity against a number of Gram-negative bacteria expressing different MBLs
including IMP, NDM and VIM. Given the high degree of active site heterogeneity among the
different types of MBL enzymes,11 designing an inhibitor with potent inhibitory activity
towards several types of MBL is challenging. In this light, thiomandelic acid 3 is unique
given its ability to inhibit a range of MBLs and, as shown in the present study, its capacity to
resensitize MBL-expressing Gram-negative isolates to meropenem, an important β-lactam
antibiotic of last resort. In addition, ITC studies showed thiols 3 and 4 to be effective zinc
chelators with low-micromolar Kd values supporting the proposed mechanism of action for
these compounds. In this regard ITC may provide a useful means of (pre)screening for zinc-
binding MBL inhibitors. While compounds 3 and 4 exhibit potent synergy with meropenem,
their propensity to oxidize and likely ability to interact with free zinc and other metallo
proteins precludes their use as clinical MBL inhibitors. In this regard, MBL inhibitors
employing free thiols as zinc chelating groups are more likely to be of value as tool
compounds for biochemical studies involving MBLs. Optimized analogues or other classes of
MBL inhibitors capable of overcoming such pharmacokinetic hurdles present a key objective
in the continued fight against antibiotic resistance.
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MATERIALS AND METHODS
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Materials. Potassium thioacetate was purchased from Combi-Blocks Inc. (San Diego, CA
USA). Thiosalicylic acid was purchased from Fisher Scientific (Waltham, MA USA). Other
reagents including captopril, 2-bromoacetophenone, S-mandelic acid, L-phenylalanine, were
purchased from Sigma-Aldrich company.
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Synthesis. Among the thiols selected for investigation, captopril 1 and thiosalicylic acid 2
were commercially available while compounds 3-5 required preparation via previously
reported synthetic routes.21,30–32 Briefly, compound 3 was synthesized via esterification of S-
mandelic acid which was followed by mesylation of the hydroxyl group. Substitution of the
tosylate ester with thioacetate anion followed by acidic hydrolysis furnished 3.30 For the
synthesis of compound 4, L-phenylalanine was converted to its corresponding α-
bromocarboxylic acid through a sodium nitrite mediated halo-deamination reaction, which
was subsequently reacted with potassium thioacetate to afford S-acetyl derivative of 4. Basic
hydrolysis of the latter intermediate led to the final product 4.31,32 Thioacetophenone 5 was
prepared via a two-step procedure involving thiolation of α-bromoacetophenone with
potassium thioacetate followed by a basic hydrolysis.21 In addition, disulfides 6-833–35 were
readily prepared by reacting the corresponding thiol with iodine in water/acetonitrile.36
MIC determinations and synergy assays. The antibacterial activity of compounds 1-5 was
evaluated alone and in combination with meropenem against a panel of beta-lactamase
producing Gram-negative bacteria including K. pneumoniae RC10 (KPC-2), K. pneumoniae
RC 45 (OXA-48), K. pneumoniae RC51 (VIM-1), K. pneumoniae JS265 (IMP-28), E. coli
RC89 (NDM-1), P. aeruginosa RC60 (VIM), P. aeruginosa JS80 (IMP), K. pneumoniae
RC21 (VIM-1), E. aerogenes RC22 (VIM-1), K. pneumoniae RC48 (VIM-1), K. pneumoniae
JS22 (NDM-1), K. pneumoniae JS177 (NDM-1), K. pneumoniae JS37 (NDM-1) and P.
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aeruginosa M-120 (IMP). The CLSI guidelines were used to determine minimum inhibitory
concentrations (MICs). Starting from glycerol stocks, bacterial strains were cultured on blood
agar plates and incubated at 37 °C. A single colony was then transferred to tryptic soy broth
(TSB) and incubated with shaking at 37 °C until the optical density of the bacterial
suspension reached a level equivalent to the 0.5 McFarland standard. The suspension was
then diluted to 106 CFU/mL in Mueller-Hinton broth (MHB). Using polypropylene microtiter
plates, the wells of the first row received 50 µL of the test compounds dissolved in MHB and
were subjected to serial dilution. Finally, 50 µL of the bacterial suspension was added and the
plates were sealed and incubated at 37 °C with constant shaking (at 600 RPM). The next
morning, the plates were inspected for visible bacterial growth.
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Synergy between meropenem (or cefoperazone) and thiols 1-5 was evaluated as follows: To
the top row of a 96 well plate, 100 µL of a solution of meropenem at 4x MIC was added. 50
µL aliquots of this solution was serially diluted down each row to achieve a range of
decreasing meropenem concentrations. Next, thiols 1-5 (50 µL aliquots) were added to the
wells to provide a concentration range of 128 to 16 µg/mL. The relevant bacterial suspension
(100 µL/well) was finally added to each well to give a final concentration of thiols 1-
5 ranging from 64 to 8 µg/mL. The plates were sealed and incubated overnight at 37 °C.
MICs were determined the next morning by visual inspection and used in calculating
fractional inhibitory concentration index (FICI) values. As a standard MBL inhibitor cocktail,
a 16:1 (w/w) mixture of ethylenediaminetetraacetic acid disodium salt-phenanthroline (EP)
was also used. The MIC of the EP mixture was first determined against each strain so that a
sub-MIC concentrations could be used for synergy assays. All the assays were performed in
duplicates. Supplemental tables S3-S17 provide a graphical representation of the
checkerboard assays.
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Stability analysis. To prepare calibration curves for thiols 3-5 and the corresponding
disulfides 6-8 each compound was dissolved in DMSO to prepare a stock solution (10
mg/mL) that was then immediately diluted in Mueller-Hinton broth (MHB) to reach
concentrations ranging from 1 µg/mL to 256 µg/mL. The samples were then processed as
follows: To precipitate undesired media components, the sample solutions were diluted with
acetonitrile (1:3 v/v), vortexed for 10 seconds, and centrifuged at 10000 rpm for 10 min. The
supernatant was retained and stored at -20 °C until HPLC analysis. Samples were analyzed
by analytical RP-HPLC using a Phenomenex Gemini C-18 110A column (250×4.60 mm, 5
micron) at a flow rate of 1 mL/min and their UV absorbance were detected at 214 nm. For the
analysis of compounds 4, 5, 7, and 8, the gradient started with 0% of buffer B (95%
acetonitrile, 0.1% TFA) and 100% buffer A (5% acetonitrile, 0.1% TFA) increasing to 50%
buffer B over 5 min followed by an increase to 100% buffer B over 10 min and maintenance
at 100% buffer B for 3 min. The buffer gradient was then returned to 0% buffer B in 2 min
and maintained at 0% for an additional 5 min to re-equilibriate the system. For the analysis of
compounds 3 and 6 the gradient started with 0% of buffer B, increased to 50% buffer B over
5 min, then to 90% buffer B over 8 min followed by a final increase to 100% buffer B over 1
min. After 1 min at 100% buffer B the gradient returned to 0% buffer B over 2 min and was
maintained at 0% for 3 min to re-equilibriate the system. The calibration curves were linear
from 2-256 µg/mL for compounds 3-6 and from 1.6-200 µg/mL for compounds 7 and 8 with
r² > 0.990 in all the cases. Due to the relatively short half-life of compound 5 in MHB, the
medium needed to be supplemented with 5.0 mM TCEP to obtain a suitable calibration curve.
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To assess the half-lives of thiols 3-5 in MHB, each compound was dissolved in MHB (200
µg/mL) and incubated at 37 °C. At time points of 0, 0.5, 1, 2, 4 and 8 hours, 100 µL aliquots
of each samples were taken and subjected to the same processing described above for the
standards prior to HPLC analysis.
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Table 2. The concentrations of the metal/ligands used for the ITC experiments
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Experiment
(metal:ligand)
ZnCl2 : compound 3/4/6/7
Zinc chloride
concentration (mM) concentration (mM)
Ligand
2.0
0.4
1.0
0.2
0.04
0.1
ZnCl2 : EDTA
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ZnCl2 : 1,10-phenanthroline
Isothermal titration calorimetry. ITC experiments were performed using a MicroCal Auto-
ITC200 instrument (Malvern). The test compounds and zinc chloride were dissolved in
Tris.HCl buffer (20 mM, pH 7.0) and degassed using a sonication bath (10 min) before
running the experiments. The zinc chloride solution was titrated into a solution of 3, 4, 6, 7,
EDTA, or 1,10-phenanthroline (see Table 3 for specific concentrations used) over 26 aliquots
of 1.5 µL (except the first injection which was 0.5 µL) with 120 seconds spacing between
injections. All the experiments were performed at 25 °C in triplicate with reference power set
at 2.0 µcal/sec. The generated peaks were integrated using Origin 7.0 software. The error for
all the reported thermodynamic parameters was estimated through Monte Carlo simulation
the standard errors of three experiments.
ASSOCIATED CONTENT
Supporting Information
1
Spectral characterization of compounds 3-8 including H-NMR, 13C-NMR and high-
resolution mass spectrometry data. Tables of antibiotic susceptibility and checkerboard
synergy assays using meropenem or cefoperazone combined with thiols 1-5 or disulfides 6-8
(tables S1-S20). ITC thermograms of binding interactions between Zn2+ and thiols 3 and 4,
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disulfides 6 and 7, 1,10-phenanthroline and EDTA (figures S1-S9). This material is available
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free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
n.i.martin@uu.nl
Funding Sources
No competing financial interests have been declared. Financial support provided by Utrecht
University and the Netherlands Organization for Scientific Research (VIDI grant to NIM).
ACKNOWLEDGMENT
The authors thank Dr. Ad Fluit (Utrecht University Medical Center) and Dr. Sandra
Bernhards (Leiden University Medical Center) for kindly supplying the carbapenem-resistant
clinical isolates. We also thank Laurens Kleijn for useful discussions and assistance with ITC
measurements and Javier Sastre-Torano for assistance in developing the LC-MS assays and
performing high-resolution mass measurements.
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FOR TABLE OF CONTENTS USE ONLY
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Thiol-Containing Metallo-β-Lactamase Inhibitors Resensitize Resistant
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Gram-Negative Bacteria to Meropenem
Kamaleddin Haj Mohammad Ebrahim Tehrania and Nathaniel I. Martina ,*
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