Carbamylmethyl Mercaptoacetate Thioether: A Novel Scaffold for the Development of L1 Metallo-β-lactamase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00058

Given the clinical importance of metallo-β-lactamases (MβLs), a new scaffold, N-substituted carbamylmethyl mercaptoacetate thioether, was constructed. The obtained molecules 1-16 inhibited MβLs from all three subclasses, but preferentially L1 from subclass B3. Compound 9 with a p-carboxyphenyl substituent exhibited the broadest spectrum with at least 70% inhibition of enzymes from all subclasses at 100 μM, while compound 5 with a p-methylphenyl substituent was the most potent inhibitor of any individual enzyme, with 97% inhibition at 100 μM and an IC₅₀ value of 0.41 μM against L1. Isothermal titration calorimetry assays corroborate findings from UV-vis spectrophotometric assays that the inhibition of L1 by 5 is dose-dependent. Docking studies suggest that the carboxyl group, the sulfide atom, and the carbonyl group of the carbamyl coordinate Zn2 in a chelating fashion. Using E. coli cells expressing L1, 6 and 8 were able to decrease cefazolin minimum inhibitory concentration 8-fold.

Full text of DOI:10.1021/acsmedchemlett.7b00058

Letter
pubs.acs.org/acsmedchemlett
Carbamylmethyl Mercaptoacetate Thioether: A Novel Scaffold for
the Development of L1 Metallo-β-lactamase Inhibitors
Ya-Nan Chang,† Yang Xiang,† Yue-Juan Zhang,† Wen-Ming Wang,† Cheng Chen,† Peter Oelschlaeger,‡
and Ke-Wu Yang*,†
†Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biology Innovation
Laboratory, College of Chemistry and Materials Science, Northwest University, Xi’an 710127, P. R. China
‡Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, 309 East Second Street,
Pomona, California 91766, United States
S
* Supporting Information
ABSTRACT: Given the clinical importance of metallo-β-
lactamases (MβLs), a new scaffold, N-substituted carbamyl-
methyl mercaptoacetate thioether, was constructed. The
obtained molecules 1−16 inhibited MβLs from all three
subclasses, but preferentially L1 from subclass B3. Compound
9 with a p-carboxyphenyl substituent exhibited the broadest
spectrum with at least 70% inhibition of enzymes from all
subclasses at 100 μM, while compound 5 with a p-
methylphenyl substituent was the most potent inhibitor of
any individual enzyme, with 97% inhibition at 100 μM and an
IC50 value of 0.41 μM against L1. Isothermal titration calorimetry assays corroborate findings from UV−vis spectrophotometric
assays that the inhibition of L1 by 5 is dose-dependent. Docking studies suggest that the carboxyl group, the sulfide atom, and the
carbonyl group of the carbamyl coordinate Zn2 in a chelating fashion. Using E. coli cells expressing L1, 6 and 8 were able to
decrease cefazolin minimum inhibitory concentration 8-fold.
KEYWORDS: Antibiotic resistance, metallo-β-lactamase, L1, inhibitor, mercaptoacetate thioether
MβL inhibitors.21 Accordingly, the development of new MβL
ntibiotic resistance has become a concerning global health
inhibitor scaffolds is urgently needed.22
1
Aproblem, with the World Health Organization (WHO)
It has been reported that a series of mercaptoacetic acid
thioester derivatives inhibited MβLs.23 Recently, our studies
indicated that the amino acid mercaptoacetic acid thioester is a
highly promising scaffold for the development of MβL L1
(subclass B3) inhibitors, exhibiting IC50 values in the
submicromolar range using cefazolin as substrate, and partial
thioesters also showed effective inhibitory activities against the
MβLs NDM-1 (subclass B1) and ImiS (subclass B2).24 Docking
studies revealed potential binding modes of the amino acid
thioesters to the active site of L1 in which the carboxylate group
interacts with both Zn(II) ions and Ser221, and the acylamino
group forms hydrogen bonds with Tyr32.24 That scaffold was
subsequently optimized to yield more potent L1 inhibitors.25
Also, our studies revealed that azolylthioacetamides with an
acylamino group are potential broad-spectrum inhibitors of
MβLs.26,27
reporting “that several million people are infected with antibiotic-
resistant bacteria per annum.”2 β-Lactam antibiotics are the most
broadly prescribed drugs for the treatment of bacterial infections,
but their effectiveness has been threatened by the emergence of
pathogenic bacteria producing β-lactamases,3 which catalyze β-
lactam hydrolysis.4 β-Lactamases have been categorized as
serine-β-lactamases (SβLs, Ambler classes A,5 C, and D) and
metallo-β-lactamases (MβLs, Ambler class B), according to their
mechanisms of action. MβLs are further divided into three
subclasses B1, B2, and B3, based on their amino acid sequence
and metal occupancies.6,7
Since MβLs can deactivate most β-lactam antibiotics,
including widely used drug families, such as penicillins,
cephalosporins, and carbapenems,8,9 the development of
inhibitors of MβLs is an essential strategy for maintaining the
usefulness of existing β-lactam antibiotics. In combination with
appropriate antibiotics, clavulanic acid, tazobactam, sulbactam,
and avibactam10,11 have been widely approved for clinical
treatment of antibiotic-resistant bacteria containing SβLs.
Meanwhile, various MβL inhibitors have been reported,12
including trifluoromethyl alcohol and ketone,13 dicarboxylic
acids,14,15 thiols,16 sulfates,17 hydroxamates,18 tetrazoles,19 and
sulfonamides.20 However, there are no reports of clinically useful
The earlier mentioned amino acid mercaptoacetic acid
thioesters were potent inhibitors of L1, a subclass B3MβL, but
were less potent against other, more clinically important MβLs
Received: February 14, 2017
Accepted: April 24, 2017
Published: April 24, 2017
© XXXX American Chemical Society
A
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Letter
tested. In addition, there is a possibility that thioesters could be
hydrolyzed and yield mercaptoacetate, which could inhibit other
metalloenzymes and thus cause toxicities. In this work, we
therefore constructed a novel series of compounds containing a
thioether rather than a thioester, the N-substituted carbamyl-
methyl mercaptoacetate thioethers. The novel molecules were
found to be significantly more stable when incubated in aqueous
solutions of pH 5.0, 7.0, and 9.0 over a period of 4 days than the
previously reported thioesters, as monitored by UV−vis spectra
(Figure S1). These new molecules were tested against various
MβLs, indicating preference for L1. Inhibition of L1 was
investigated in more detail by steady-state kinetics and
isothermal titration calorimetry (ITC). Cytotoxicity of the
compounds against L-929 mouse fibroblastic cells was tested,
their potential interactions with the enzyme were investigated by
molecular docking, and their ability to inhibit L1 in E. coli cells
was tested by determining cefazolin minimum inhibitory
concentrations (MICs).
Table 1. Percent Inhibition of IMP-1, ImiS, and L1 by 100 μM
N-Substituted Carbamylmethyl Mercaptoacetate Thioether
a
% inhibition
ImiS
% inhibition
ImiS
compd
IMP-1
L1
compd
IMP-1
L1
1
2
3
4
5
6
7
8
43
40
50
80
37
64
44
45
18
74
24
21
52
29
21
85
92
95
95
92
97
97
77
71
9
71
53
48
48
51
55
59
69
87
39
34
25
21
20
23
21
75
80
79
73
97
89
78
91
10
11
12
13
14
15
16
a
The substrate for IMP-1 and L1 was cefazolin, and for ImiS, it was
imipenem.
Table 2. IC50 of N-Substituted Carbamylmethyl
a
Mercaptoacetate Thioethers against L1
Sixteen N-substituted carbamylmethyl mercaptoacetate thio-
ethers were synthesized by a synthetic route shown in Scheme S1
in the Supporting Information. In brief, amine was added to a
mixture of dichloromethane and triethylamine with stirring on an
ice−water bath, chloroacetyl chloride was dropwise added, and
the resulting mixture was stirred at room temperature for 2 h.
The solvent was removed under vacuum, and the resulting
residue was recrystallized with anhydrous ethanol to offer 2-
chloro-N-substituted carbamylmethyl chloride. Mercaptoacetic
acid was dripped into a solution of acetone with KOH, the
resulting mixture was added into a solution of the intermediate
N-substituted carbamylmethyl chloride dissolved in acetone and
refluxed for 2 h at 60 °C. After cooling to room temperature, the
precipitate formed was filtered off, washed with anhydrous
ethanol, and dried under vacuum to give the target compounds.
The structures of the N-substituted carbamylmethyl mercaptoa-
cetate thioethers are shown in Figure 1. All compounds were
compd
IC50 (μM)
compd
IC50 (μM)
1
2
3
4
5
6
7
8
0.98 0.04
0.58 0.02
0.46 0.01
0.59 0.02
0.41 0.02
0.43 0.01
4.21 0.05
>20
9
7.5 0.6
10
11
12
13
14
15
16
1.04 0.03
2.01 0.04
∼20
0.46 0.05
1.23 0.02
3.81 0.02
0.75 0.02
a
The substrate used was cefazolin and inhibitor concentrations were
varied between 0.1 and 20 μM.
1
characterized by H and 13C NMR and confirmed by MS (see
Supporting Information).
To test whether these compounds were inhibitors of MβLs,
several enzymes were overexpressed and purified as previously
described28 and are detailed in the Supporting Information. The
inhibitory activities of the prepared carbamylmethyl mercaptoa-
cetate thioethers against purified MβLs from different subclasses
were tested on an Agilent UV8453 UV−vis spectrophotometer
using 50 μM cefazolin as substrate for B1 and B3 enzymes and 40
μM imipenem for ImiS (B2) and 100 μM inhibitor in the
enzyme-specific buffer. Enzyme and inhibitor were preincubated
Figure 2. Michaelis−Menten plots of L1-catalyzed hydrolysis of
cefazolin in the absence and presence of 4 (top) and 11 (bottom).
●
○
Concentrations of inhibitor 4/11 were 0/0 μM ( ), 0.25/2.5 μM ( ),
■
▼
▽
0.5/5.0 μM ( ), 1.0/10 μM ( ), and 2.0/20 μM ( ).
Figure 1. Structures of the synthesized N-substituted carbamylmethyl
mercaptoacetate thioethers.
for 30 min before adding cefazolin or imipenem, which were then
monitored at 262 or 300 nm, respectively, to determine the initial
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Table 3. MIC Values (μg/mL) of Cefazolin for E. coli-DH10B/pBC SK(+)-L1 in the Presence of the N-Substituted
Carbamylmethyl Mercaptoacetate Thioethers 1−16 at a Concentration of 1024 μg/mL (A) and 6 and 8 at Concentrations from 4
a
to 1024 μg/mL (B)
A
compd
MIC
1
16
9
2
8
3
4
5
6
2
7
8
2
16
11
16
16
12
16
16
13
16
16
15
16
compd
MIC
10
16
14
16
16
16
16
B
compd\conc.
4
8
16
16
16
32
16
16
64
16
16
128
16
8
256
8
512
1024
6
8
16
16
16
16
8
8
2
2
8
a
In the absence of inhibitor, the MIC of cefazolin for E. coli-DH10B/pBC SK(+)-L1 was 16 μg/mL, and for E. coli not expressing L1, it was 2 μg/
mL.
velocity v0. We tested different preincubation times (3−60 min)
and did not find any difference in residual activity, concluding
that these are time-independent inhibitors. Percent inhibition,
defined as enzyme activity without inhibitor (100%) minus
residual activity with 100 μM inhibitor, is reported for all
enzymes tested in Figure S2 in the Supporting Information. The
results for IMP-1, ImiS, and L1 as representatives of subclasses
B1, B2, and B3, respectively, are summarized in Table 1.
All compounds showed varying degrees of inhibition of the
different enzymes. Between IMP-1, ImiS, and L1, compound 9
showed the broadest spectrum of inhibition with more than 70%
inhibition. In terms of specific inhibition of a particular enzyme,
the compounds were most potent against L1 with compounds 5,
6, and 13 reaching the highest percent inhibition with 97%.
While we propose that derivatives of this series of thioethers
could be developed as more potent broad-spectrum inhibitors or
specific inhibitors of the various individual enzymes in future
studies, the present series clearly has a preference for L1.
Therefore, for the remainder of this report, we will focus on
inhibition studies of L1.
10) than when in meta (11) or para (8 and 12) position. Among
13−16 with aliphatic substituents, 13 with a benzyl substituent
was the most potent inhibitor, indicating that an aromatic group
is also favorable if not directly linked to the carbamyl nitrogen.
In order to identify the inhibition mode of the carbamylmethyl
mercaptoacetate thioethers, we studied inhibition kinetics of L1
with different substrate concentrations and different concen-
trations of 4 and 11 as representatives of compounds with
aromatic substituents with either electron-donating or electron-
accepting groups. The concentrations of 4 and 11 were varied
from 0 to 2.0 and from 0 to 20 μM, respectively, and substrate
(cefazolin) concentrations were varied between 8 and 48 μM.
Enzyme and inhibitors were preincubated for 30 min before
starting the kinetic assays. The mode of inhibition was
determined by generating Michaelis−Menten plots, and Ki
values were determined by fitting initial velocity versus substrate
concentration at each inhibitor concentration using GraphPad
Prism 7. The Michaelis−Menten plots of L1-catalyzed hydrolysis
of cefazolin in the absence and presence of compounds 4 and 11
are shown in Figure 2. They indicate that both 4 and 11 exhibit a
mixed inhibition mode against L1. The Ki values were
determined to be 3.7 0.1 μM for 4 and 31.8 0.3 μM for
11, respectively (average standard deviation of triplicates).
Isothermal titration calorimetry (ITC) has an increasing
significance in enzyme kinetic studies owing to its general
applicability and sensitivity. In the present work, enzyme-
catalyzed progress of hydrolysis of substrate in the absence and
presence of inhibitor was monitored on a Malvern MicroCal iTC
200 instrument at 25 °C. The concentration of enzyme was 5
nM, the concentration of substrate (cefazolin) was 160 μM, and
the concentration of inhibitor was varied from 0 to 100 μM. The
L1-catalyzed hydrolysis progress curves of cefazolin in the
absence and presence of 5 at different concentrations are shown
in Figure S2.
The inhibitor concentrations causing 50% decrease of enzyme
activity (IC50) of compounds 1−16 against L1 were determined
in 30 mM Tris, pH 8.5. These kinetic experiments were done in
triplicate, and the average values
standard deviations are
reported in Table 2. The kinetic parameters for uninhibited
cefazolin hydrolysis by L1 were determined as Km = 36 2 μM
and kcat = 197 1 s−1.
The IC50 data indicate that all of the carbamylmethyl
mercaptoacetate thioethers inhibit L1 with an IC50 value range
from 0.4 to 20 μM except for 8 (>20 μM). Compounds 3, 5, 6,
and 13 showed the lowest IC50 values between 0.41 and 0.46 μM,
consistent with their high % inhibition (95−97%) at 100 μM
inhibitor concentration. An assessment of the structure−activity
relationship (SAR) of these N-substituted carbamylmethyl
mercaptoacetate thioethers and their activities reveals some
interesting trends. Compounds 1−12 all contain an aromatic
substituent. Using 1 with a phenyl group (IC50 = 0.98 μM) as a
reference, 2−6 with an electron-donating group on the phenyl
ring were more potent inhibitors of L1, with IC50 values ranging
from 0.41 to 0.59 μM, while 7−12 with an electron-accepting
group on the phenyl were equally or less potent inhibitors with
IC50 values ranging from 1.04 to >20 μM. Compounds 5 and 6
with an electron-donating group in para-position of the phenyl
were slightly more potent (IC50 = 0.41 and 0.43 μM) than 2−4
with a substituent in ortho-position of the phenyl (IC50 = 0.46−
0.59 μM). The electron-accepting nitro and chloro substituents
result in more potent inhibitors when in ortho-position (7 and
It is clearly observed that the heat flow decreased gradually
with the increase in concentration of 5 from 0 to 100 μM,
demonstrating the inhibitory effect of the N-substituted
carbamylmethyl mercaptoacetate thioether on thermodynamics.
Based on ITC experiments like the one shown in Figure S3 (top)
but at different substrate concentrations, reaction rates were
calculated and plotted against substrate concentrations for
different inhibitor concentrations.29 The reaction rates from
ITC at different substrate concentrations in the presence of 0, 50,
and 100 μM 5 are shown in Figure S3 (bottom) and confirm the
spectrophotometric results.
The ability of compounds 1−16 to restore the antimicrobial
activity of cefazolin against bacteria expressing L1 was
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To explore potential binding modes, compounds 1, 5, and 12
as typical representatives of the aromatic N-substituted
carbamylmethyl mercaptoacetate thioethers without substituent,
with an electron-donating substituent and with an electron-
accepting substituent on the phenyl ring, respectively, were
docked into the active site of the L1 crystal structure.30 The
lowest-energy docking conformations of the selected clusters of
1, 5, and 12 (see Supporting Information for details) are shown
in Figure 3A, B, and C, respectively. The carboxylate of all three
compounds acts as a bridging ligand of the two Zn(II) ions and
forms a hydrogen bond with Ser221, tightly anchoring these
inhibitors in the active site, as seen previously with amino acid
thioesters.24,25 The hydrogen of the carbamyl group also forms a
hydrogen bond with Tyr32 in all three complexes. The
substituent on the phenyl ring seems to affect the orientation
of the ring and the adjacent carbamyl group, orienting the
carbonyl oxygen toward the two Zn(II) ions in the L1/5 complex
at distances of 3.2 and 3.3 Å (enlarged view in panel D), but not
in the other two complexes, providing a rationale for the lower
IC50 value observed with this compound. With the carbamyl
oxygen acting as an additional ligand, 5 is effectively a chelating
agent of the two Zn(II) ions, especially Zn2. Such a chelating
effect was not observed previously with the amino acid
thioesters,24,25 possibly due to the closer proximity of the
thioester oxygen to the carboxylate group in those compounds,
not allowing for enough conformational freedom. The addition
of a methylene group in the thioethers presented here seems to
provide the proper geometry for chelation.
For biomedical applications, the potential toxicity of inhibitors
is a major concern. Although no hydrolysis was previously
observed in vitro, the previously reported thioesters23−25 can
potentially be subject to hydrolysis upon administration in a
patient, which would result in mercaptoacetate. Thiol com-
pounds have a high potential to inhibit other zinc enzymes; e.g.,
L-captopril, a potent MβL inhibitor, also inhibits angiotensin
converting enzyme.31 In fact, while some of the most potent MβL
inhibitors reported to date are thiols,32,33 none of them have been
developed into clinically useful drugs. One rationale for
designing thioethers as opposed to the previously reported
thioesters24,25 was to avoid the potential hydrolysis resulting in
mercaptoacetate and cytotoxicity. Upon incubation in aqueous
solution over an extended period of time, especially at lower pH,
a representative thioether was clearly more stable than a thioester
(Figure S1), suggesting that the thioethers are less prone to
hydrolysis. A selection of the newly designed N-substituted
carbamylmethyl mercaptoacetate thioethers, 1, 5, 7, and 16, was
subjected to a cytotoxicity assay with mouse fibroblast cells
(L929) with different working concentrations (12.5, 25, 50, 100,
200, 400 μM). As shown in Figure S4, none of them affected
viability of the L-929 mouse fibroblastic cells at concentrations
up to 400 μM, indicating that these thioethers have low
cytotoxicity.
Figure 3. Low-energy docking conformations of compounds 1 (A), 5
(B), and 12 (C) docked into the active site of L1 (PDB code 2AIO30).
The enzyme backbone is shown as a cartoon in green, and selected
residues are shown as sticks colored by element (H, white; C, cyan; N,
blue; O, red; S, yellow). Zn(II) ions are shown as magenta spheres; the
lower (front) one is Zn2 and the upper (back) Zn1. Compounds 1, 5,
and 12 are also shown as sticks with the same color code as amino acid
residues except C in gray and Cl in green. Characteristic short distances
between inhibitors and the protein are indicated by dashed lines. Panel
(D) is an enlarged view of the interactions between compound 5 with
the L1 active site.
investigated by determining the MICs (see Supporting
Information for details) of cefazolin in the presence and absence
of 1024 μg/mL 1−16 (Table 3A). E. coli-DH10B cells harboring
pBC SK(+)-L1 were used in these assays. A significant (8-fold)
decrease in MIC for 6 and 8 was observed, restoring the
antibacterial activity of cefazolin. However, the other compounds
did not result in decreased MICs, indicating that there might be
problems with entry into the bacteria. Inhibitors alone (1024 μg/
mL) without antibiotic did not inhibit cell growth. For the active
compounds, 6 and 8, we also established dose-dependency and
found that compound 6 decreased MIC by one serial dilution at
256 μg/mL and compound 8 at 128 μg/mL (Table 3B). No
antibacterial effect of the compounds alone at 1024 μg/mL
against E. coli with and without L1 plasmid was observed.
In summary, given the enormous clinical importance of
metallo-β-lactamases (MβLs), a new scaffold, N-substituted
carbamylmethyl mercaptoacetate thioethers, was constructed
1
and characterized by H and 13C NMR and MS. The obtained
molecules 1−16 inhibited a range of MβLs, but preferentially L1,
with an IC50 value range from 0.4 to >20 μM. Compound 5 was
found to be the most potent inhibitor of L1, with an IC50 value of
0.41 μM using cefazolin as substrate, with 6, 3, and 13 being
almost as potent. The mechanism of inhibition of both 4 and 11
was mixed. ITC assays confirmed the findings from UV−vis
spectrophotometric experiments. SAR analysis reveals that an
D
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ACS Medicinal Chemistry Letters
Letter
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ASSOCIATED CONTENT
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ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00058.
Experimental details; NMR and ESI mass data for target
compounds; stability data; inhibition data with additional
MβLs; ITC data (PDF)
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M.; Baldwin, J. E.; Frere, J.-M.; Gololobov, M.; Schofield, C. J.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +8629-8153-5035. E-mail: kwyang@nwu.edu.cn.
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(17) Simm, A. M.; Loveridge, E. J.; Crosby, J.; Avison, M. B.; Walsh, T.
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ORCID
Ke-Wu Yang: 0000-0003-2560-9659
Author Contributions
K.-W.Y. and P.O. conceived and designed the experiments,
analyzed the data, and wrote the paper. Y.-N.C. performed the
experiments. Y.-J.Z., Y.X., and C.C. purified proteins. Y.-J.Z.
performed cytotoxicity, and W.-M.W. performed ITC assays. All
authors have given approval to the final version of the
manuscript.
Funding
This work was supported by grants 21272186, 21572179, and
81361138018 (to K.W.Y.) from the National Natural Science
Foundation of China.
(18) Walter, M. W.; Valladares, M. H.; Adlington, R. M.; Amicosante,
G.; Baldwin, J. E.; Frere, J.-M.; Galleni, M.; Rossolini, G. M.; Schofield,
C. J. Hydroxamate inhibitors of Aeromonashydrophila AE036 metallo-β-
lactamase. Bioorg. Chem. 1999, 27, 35−40.
Notes
The authors declare no competing financial interest.
(19) Toney, J. H.; Fitzgerald, P. M. D.; Grover-Sharma, N.; Olson, S.
H.; May, W. J.; Sundelof, J. G.; Vanderwall, D. E.; Cleary, K. A.; Grant, S.
K.; Wu, J. K.; Kozarich, J. W.; Pompliano, D. L.; Hammond, G. G.
Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of
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(20) Siemann, S.; Evanoff, D. P.; Marrone, L.; Clarke, A. J.; Viswanatha,
T.; Dmitrienko, G. I. N-arylsulfonyl hydrazones as inhibitors of IMP-1
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(21) Cornaglia, G.; Giamarellou, H.; Rossolini, G. M. Metallo-β-
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ABBREVIATIONS
ITC, isothermal titration calorimetry; MβL, metallo-β-lactamase;
MIC, minimum inhibitory concentration
■
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