Mercaptoacetate thioesters and their hydrolysate mercaptoacetic acids jointly inhibit metallo-β-lactamase L1

Article abstract of DOI:10.1039/c8md00091c

The 'superbug' infection caused by metallo-β-lactamases (MβLs) including L1 has grown into an emerging threat. To probe whether mercaptoacetate thioesters inhibiting L1 is a contribution of the thioester itself or its hydrolysate, ten mercaptoacetate thioesters 1-10 were synthesized, which specifically inhibited L1, exhibiting IC₅₀ values ranging from 0.17 to 1.2 μM, and 8 was found to be the best inhibitor (IC₅₀ = 0.17 μM). These thioesters restored the antimicrobial activity of cefazolin against E. coli expressing L1 by 2-4-fold. UV-vis monitoring showed that 1, 8 and 9 were unhydrolyzed in Tris buffer (pH 6.0-8.5), but hydrolyzed by L1; further HPLC monitoring indicated that 1/3 of the thioester 9 was converted to mercaptoacetic acid. STD-NMR monitoring suggested that both the thioester and its hydrolysate mercaptoacetic acid jointly inhibited L1.

Full text of DOI:10.1039/c8md00091c

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Mercaptoacetate thioesters and their hydrolysate mercaptoacetic
acid jointly inhibit Metallo-β-Lactamase L1
Cheng Chen‡, Yang Xiang‡, Ya Liu, Xiangdong Hu, and Ke-Wu Yang*
Received 00th January 20xx,
Accepted 00th January 20xx
The ‘superbug’ infection caused by metallo-β-lactamases (MβLs) including L1 has grown into an emerging threat. To probe
a truth that mercaptoacetate thioester inhibiting L1 is contribution of the thioester itself or its hydrolysate, ten
mercaptoacetate thioesters 1-10 were synthesized, which specifically inhibited L1, exhibiting an IC50 ranging from 0.17 to
1.2 μM, and 8 was found to be the best inhibitor (IC50 = 0.17 μM). These thioesters restored 2-4-fold antimicrobial activity
of cefazolin against E. coli expressing L1. UV-Vis monitoring shown that 1, 8 and 9 was unhydrolyzed in Tris buffer (pH 6.0-
8.5), but hydrolyzed by L1, further HPLC monitoring indicated that 1/3 of the thioester 9 were converted to
mercaptoacetic acid. STD-NMR monitoring suggested that both thioester and its hydrolysate mercaptoacetic acid jointly
inhibited L1.
DOI: 10.1039/x0xx00000x
www.rsc.org/
ebselen13 and AMA14. The mercaptoacetate thioesters have been
reported to inhibit MβLs through mercaptoacetic acid, the
hydrolysate of thioester, binds to the metal coordinating cysteine of
Introduction
Effectiveness of β-lactam antibiotics has been threatened by the
emergence of pathogenic bacteria producing β-lactamases the in
clinical settings1, 2. These enzymes are divided in four classes (A–D)
based upon DNA sequence similarity3. The class A, C and D enzymes
are called serine β-lactamases (SβLs), which utilize an active-site
serine as a nucleophile to attack the β-lactam carbonyl. The class B
enzymes whose mechanism relies upon the presence of one or two
active site zinc ions to hydrolyze β-lactams thus are called metallo-
β-lactamases (MβLs). MβLs have been further subdivided into B1-
B3 three subclasses, based on their amion acid sequence and
mental occupancies4. The strategy that co-administration of
antibiotic adjuvants capable of maintaining the activity of existing
antibiotics is widely effective in treating infections due to bacteria
expressing SβLs. Clinically relevant SβL 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 inhibitors of
the MβLs available for clinic to date 5.
enzyme. However, the presence of the phenyl substituent
attenuated the cleavage of the thioester bond and inhibition of
MβLs was not accomplished through chelation of the Zn(II) ions15. In
addition, MALDI-TOF showed that two molecules of
mercaptoacetate bind to L1, when the enzyme incubated with
thioester16. Recently, our studies indicated that mercaptoacetate
thioester is a highly promising scaffold for the development of L1
inhibitor, exhibiting IC50 values in the submicromolar range. This
scaffold was further optimized to yield more potent L1 inhibitors17,
18. Also, the carbamylmethyl mercaptoacetate thioether have been
reported to be the potential inhibitors of L119
.
There have been many reports on mercaptoacetate thioesters
as inhibitors of L1 to date. However, almost all these reports
indicated that the essence of thioesters inhibiting L1 is the
contribution of either the thioester itself, or its hydrolysate
mercaptoacetic acid. To probe the truth, in this work, we
constructed
a series of phenyl substituted mercaptoacetate
thioesters and to use them in combination with β-lactams to
combat antibiotic resistant bacteria that produce MβLs. These
thioesters were tested as inhibitors against the purified L1, STD-
NMR was employed to monitor the interaction of L1 with thioester
and its hydrolysate, and their binding affinity was evaluated by
isothermal titration calorimetry (ITC). Also, the capacity of these
inhibitors to restore antibacterial activity of cefazolin against E. coli
expressing L1 was evaluated, and their cytotoxicity against L-929
mouse fibroblastic cells was tested.
Now MβLs are of increasing clinical concern, because they
hydrolyze almost all β-lactam antibiotics6. Ongoing attempts have
been made to prevent the hydrolysis of antibiotics by MβLs, various
small molecule inhibitors have been reported7, including thiols8,
sulfates9, dicarboxylic acids10
,
hydroxamates11
,
tetrazoles12
,
Results and discussion
Towards the above goal, ten mercaptoacetate thioesters were
synthesized through a synthetic pathway shown in Scheme S1. The
This journal is © The Royal Society of Chemistry 20xx
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structures of the synthesized thioesters are shown in Fig. 1. These
O
R'
1
thioesters were all characterized by H and 13C NMR and confirmed
S
COOH
(S)
N
by MS (see ESI).
R
H
O
To confirm the molecular structure of the mercaptoacetate
thioesters, the yellow crystals suitable for X-ray analysis were
obtained through slow evaporation of a solution of 1 in methanol-
ethyl acetate. The crystal structures were given in Fig. 2, and the
resulting structures based on X-ray diffraction confirmed the S-
configuration. Parameters in CIF format were available as Electronic
Supplementary Information from Cambridge Crystallographic Data
Center (CCDC: 1544802).
R' =
N
H
4-Cl
4-Br
3-Br
6
4-CF3
7
R = 4-NO2 3-NO2
4-F
3
1
2
4
5
To test whether these thioesters were MβL inhibitors, MβLs
from subclasses B1 (NDM-1), B2 (ImiS) and B3 (L1) were over-
expressed and purified as previously described methods20. Steady-
state kinetic studies of thioesters as inhibitors against MβL were
conducted on an Agilent UV8453 spectrometer. Hydrolysis of
substrate (cefazolin for NDM-1 and L1; imipenem for ImiS) was
monitored at 262 and300 nm. The initial reaction rates were
determined in the absence and presence of inhibitors in triplicate,
and the average values were recorded. Percent inhibition, defined
as enzyme activity without inhibitor (100%) minus residual activity
with 20 μM inhibitor, is showed for all enzymes tested in Fig. S1.
R' =
S
R = 4-NO2
8
4-Cl
9
3-Br
10
Fig. 1. Structures of the synthesized mercaptoacetate thioesters
The percent inhibition data indicated that all these thioesters
exhibited more than 85% inhibition against L1, but no obvious
inhibition on NDM-1 or ImiS was observed at an inhibitor
concentration up to 20 μM (Fig. S1). Therefore, for the remainder of
this report, we focused on inhibition studies of L1. The inhibitor
concentration causing 50% decrease of enzyme activity (IC50) of
compound 1-10 against L1 were determined in 30 mM Tris, pH 7.0
using cefazolin as substrate. The collected IC50 data (Table 1)
indicated that all inhibitors have excellent inhibitory activity against
L1, exhibiting a IC50 value ranging from 0.17 to 1.2 μM, and 8
showed the lowest IC50 value of 0.17 μM, revealing that
mercaptoacetate thioesters potentially inhibit L1, which is
consistent with their high % inhibition (90−95%) at an inhibitor dose
of 20 μM. These results are comparable to what we have previously
reported. Also, we performed assays of time- and concentration-
dependent inhibition of L1 by thioester 9, the results indicated that
the thioester exhibited maximum inhibition after incubation with
enzyme for 1.5 h and at a concentration around 1.0 μM (Fig. S2).
(
Fig. 2. Crystal structure of compound 1
Table1. Inhibitory activities of mercaptoacetate thioesters 1-10
against MβL L1
inhibitor
IC50 (μM)
0.37±0.04
0.80±0.12
0.75±0.06
0.19±0.02
0.88±0.08
inhibitor
IC50 (μM)
0.62±0.07
1.03±0.14
0.17±0.03
0.94±0.09
1.20±0.15
Isothermal titration calorimetry (ITC) is an essential technique
to probe protein-ligand interactions by directly titrating one
solution into another and the key is to identify the source of the
heat absorbed or released. MicroCal-ITC200 was employed to
investigate the interaction of mercaptoacetate thioesters with L1 in
multiple injection mode at 25 °C21. The calorimetry profiles for
binding of two inhibitors 8 and 9 by L1 are shown in Fig. 3. In the
top panel, each peak represents a single injection of the inhibitor
into protein solution, while the bottom panel shows an integrated
plot of the amount of heat liberated per injection as a function of
the molar ratio of the inhibitor to protein. As shown in Fig 3,
negative heat deflection was observed, indicating that the binding is
an exothermic process. The enthalpy change (∆H), binding affinity
(Kb), entropy changes (ΔS) and binding stoichiometry (N) shown in
Table 2 were given by fitting the data for single sites, using the
sequential binding model, and the dissociation constant (Kd) and
1
2
3
4
5
6
7
8
9
10
Cefazolin was used as substrate; inhibitor concentrations were
varied between 0.01 and 2 μM.
Table 2.ITC derived thermodynamic parameters for the binding of
mercaptoacetate thioesters8 and 9 by L1 at 25 °C, pH 7.0.
Kd
N
ΔH
TΔS
ΔG
Compd
(μM)
(Site)
(kcal/mol)
(kcal/mol/deg)
(kcal/mol)
PAGE 2
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8
9
5.5
0.49
-16.78
-28.24
-9.60
-7.18
-6.43
To probe the mechanism of mercaptoacetate thioester
interaction with L1, we firstly evaluated the effect of pH and L1
enzyme on thioester stability (Fig. S3), and found that the thioesters
1, 8 and 9 was unhydrolyzed when incubated it in Tris buffer with
a pH ranging from 6.0 to 8.5 over 24h. However, it was hydrolyzed
when incubated with L1 as monitored by UV-Vis spectra (Fig. S3).
Further, hydrolysis of the thioester in the presence of enzyme L1
was monitored by HPLC (Fig. 4), and found that 1/3 of the
thioesters were converted to mercaptoacetic acid under the same
conditions as above IC50 assay. This implied that the thioester as
well as its hydrolysate mercaptoacetic acid may jointly inhibited the
L1.
19.7 0.71
-21.81
Time (min)
10 20 30
Time (min)
0
40
0
10
20
30
40
0
-1
-2
0
-2
-4
-6
0
0
-4
-4
-8
-8
-12
-16
-20
-12
To verify this hypothesis, STD-NMR was employed to monitor
the binding of thioester and/or mercaptoacetate to L1. STD
experiments were performed on a Bruker Avance III 400-MHz
spectrometer at 25 °C22. The selective saturation of STD was
achieved by a train of Gauss-shaped pulse with length of 50 ms,
alternating between on and off resonance with water suppression
using excitation sculpting with gradients and spin-lock to suppress
protein signals-STDDIFFESGP.3. Sinc1.1000 shaped pulse was
applied to suppress solvent signal. The subtraction of on- and off-
resonances is performed after every scan using phase cycling which
gives the STD signal. Fig.5A-B presents the reference 1H NMR
spectrum and STD-NMR spectra of L1 mixed with thioester 9. It is
clearly observed that there are three components of thioester 9, its
hydrolysate N-substituted methionine, and mercaptoacetic acid in
the reference 1H NMR spectrum (Fig. 5A). On the other hand, as
shown in Fig 5B, the resonances of proton-7, 9/13, 10/12, 19 of 9
and proton-7’ of the mercaptoacetic acid were enhanced in STD
spectrum with the increase of inhibitor concentration from 2 to 10
mM, indicating that they bind to L1 specifically with defined binding
conformation, whereas another hydrolysate N-substituted
methionine does not present STD-NMR signals, indicating that it
does not bind L1, which is consistent with their inhibitory activities
0.0
0.5
1.0
Molar Ratio
1.5
0
1
2
3
Molar Ratio
Fig. 3. ITC profiles of titration of a solution of inhibitor 8 (left) or 9
(right) into a L1 protein solution at 25 °C. The concentrations of 8 or
9 were 0.5 mM, and concentration of protein was 60 μM.
Fig. 4.The hydrolysis of mercaptoacetatethioester9 in the presence
(Table S1). These results suggested that thioester
9 and its
of enzyme L1 as monitoring by HPLC under the condition of IC50
hydrolysate mercaptoacetate jointly target the active sites of L1.
assay
.
The ability of the mercaptoacetate thioesters to restore the
antibacterial activity of cefazolin against E. coli expressing L1 was
investigated by determining the minimum inhibitory concentrations
(MICs) of the antibiotic in the presence and absence of 1-10. The
bacterial strain of E. coli BL21(DE3) containing plasmids pET26b-L1,
as well as E. coli BL21 control without plasmids, were used to assess
these inhibitors. The concentration of inhibitor was 16 μg/mL. The
collected MIC data are listed in Table 3. It indicate that the
inhibitors 1-10 resulted in a 2-4-fold reduction in MIC of cefazolin
on E. coli expressing L1, indicating successful inhibition of L1 in vivo,
and effectively restoring the antibacterial activity of the antibiotic.
Table 3. MICs of cefazolin (μg/mL) against E. coli cells expressing L1
in the presence and absence of mercaptoacetate thioesters at a
concentration of 16 μg/mL
Inhibitor
E. coli
E. coli-L1
Inhibitor
E. coli
E. coli-L1
Blank
4
4
4
4
4
4
32
8
6
7
4
4
4
4
4
16
16
8
1
2
3
4
5
16
16
8
8
9
16
16
10
To explore potential binding mode of mercaptoacetate
thioesters alone as potent L1 inhibitor, two typical representatives
of the thioesters 4 and 9 were docked into the active sites of L123,
24. The conformations shown in Fig. 6 are the lowest-energy
conformations of those clusters, with binding energy of -12.1 and -
10.8 kcal/mol for the L1/4 and L1/9 complexes, respectively.
16
the Gibbs energy changes (ΔG) were calculated using equation (Kd =
1/Kb, and ∆G = ∆H − T∆S). The Kd values of 8-L1 and 9-L1 binding are
5.5 and 19.7 μM, respectively, which is fully consistent with the IC50
data (0.17 and 0.94 μM) of 8 and 9 against L1.
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Fig. 5. Reference1H NMR (A) and STD-NMR spectrum (B) for the variation of the STD amplification factor with the increase of inhibitor 9
concentration. L1 stock solution was prepared by dissolving 10 mg of protein in 3 mL of PBS buffer in D2O (50 µM) and was stored at 4° C.
Thioester 9 was dissolved in 5 mL of DMSO-d6 (final concentration = 2-10 mM). STD experiments were performed on a 400 MHz
spectrometer in DMSO-d6/D2O PBS, pH 7.5, at 298 K.
B
C
A
9
4
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Fig.6. Lowest-energy docking conformations of compound 4 and 9 (A-B) and the binding mode of mercaptoacetic acid targeting to the
subclass B3 metallo-β-lactamase SMB-1(PDB: 3VQZ) (C). 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 4 and 9 are also shown as sticks with the same color code as amino acid residues
except C in white and Cl in green. Characteristic short distances between inhibitors and the protein are indicated by dashed lines. These
figures were generated with PyMOL.
For the complexes of L1/4 and L1/9 shown in Fig. 6A and 6B, the
carboxylate oxygen of the two compounds coordinated with the
Conflicts of interest
two Zn(II) ions (1.9 and 2.2 Å for L1/4, 1.9 and 2.0 Å for L1/9,
The authors declare no competing interest.
respectively) and forms a H-bond with ASP120 (2.7 in L1/4 and 3.0
Å in L1/9), tightly anchoring these inhibitors in the active site, as
seen previously with amino acid thioesters17, 18. Also sulfur atom
and ketonic oxygen of 4 and 9 interacted with Tyr 32 in L1 (3.3 and
3.7 Å, respectively) via an H-bond. For compound 4, the tyrosine
side chain forms extra H-bond with Pro226 (2.2 Å), compared with
methionine side chain with 9. The hydrolysate mercaptoacetic acid
may coordinated with the Zn(II) ions of L1 similar to the binding
Acknowledgements
This work was supported by grants 81361138018 and 21572179 (to
K.W.Y.) from the National Natural Science Foundation of China
.
mode of it targeting to B3 subclass MβL SMB-1 (Fig. 6C) 25
.
Notes and references
The potential toxicity of enzyme inhibitors is a major concern
biomedically. A selection of mercaptoacetate thioesters 8 and 9 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 Fig. S5, none of them affected viability of the L-
929 mouse fibroblastic cells at a concentration up to 200 μM,
indicating that these compounds have low cytotoxicity.
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thioester inhibiting L1 is the contribution of thioester itself or its
hydrolysate, ten mercaptoacetate thioesters 1-10 were synthesized
and characterized. The obtained molecules specifically inhibited
MβL L1, exhibiting an IC50 value ranging from 0.17 to 1.2 μM, and 8
was found to be the best inhibitor, with an IC50 value of 0.17 μM
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Jointly
IC50 = 0.17 µM
MIC = 4-fold
Metallo-β-
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