Kinetic, thermodynamic, and crystallographic studies of 2-triazolylthioacetamides as verona integron-encoded metallo-β-lactamase 2 (VIM-2) inhibitor

Article abstract of DOI:10.3390/biom10010072

Inhibition of β-lactamases presents a promising strategy to restore the β-lactams antibacterial activity to resistant bacteria. In this work, we found that aromatic carboxyl substituted 2-triazolylthioacetamides 1a–j inhibited VIM-2, exhibiting an IC₅₀ value in the range of 20.6–58.6 μM. The structure-activity relationship study revealed that replacing the aliphatic carboxylic acid with aromatic carboxyl improved the inhibitory activity of 2-triazolylthioacetamides against VIM-2. 1a–j (16 mg/mL) restored the antibacterial activity of cefazolin against E. coli cell expressing VIM-2, resulting in a 4–8-fold reduction in MICs. The isothermal titration calorimetry (ITC) characterization suggested that the primary binding 2-triazolylthioacetamide (1b, 1c, or 1h) to VIM-2 was a combination of entropy and enthalpy contributions. Further, the crystal structure of VIM-2 in complex with 1b was obtained by co-crystallization with a hanging-drop vapour-diffusion method. The crystal structure analysis revealed that 1b bound to two Zn(II) ions of the enzyme active sites, formed H-bound with Asn233 and structure water molecule, and interacted with the hydrophobic pocket of enzyme activity center utilizing hydrophobic moieties; especially for the phenyl of aromatic carboxyl which formed π-π stacking with active residue His263. These studies confirmed that aromatic carboxyl substituted 2-triazolylthioacetamides are the potent VIM-2 inhibitors scaffold and provided help to further optimize 2-triazolylthioacetamides as VIM-2 even or broad-spectrum MβLs inhibitors.

Full text of DOI:10.3390/biom10010072

biomolecules
Article
Kinetic, Thermodynamic, and Crystallographic
Studies of 2-Triazolylthioacetamides as Verona
Integron-Encoded Metallo-β-Lactamase 2
(VIM-2) Inhibitor
Yang Xiang 1,2, Yue-Juan Zhang 1, Ying Ge 1, Yajun Zhou 3, Cheng Chen 1,
Weixiao Yuan Wahlgren 4 , Xiangshi Tan 3, Xi Chen 1 and Ke-Wu Yang 1,
*
1
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, China; xiangyanglovelove@163.com (Y.X.);
phoebe9203@163.com (Y.-J.Z.); geyingxgs@163.com (Y.G.); chencheng_wly@163.com (C.C.);
xchen@nwu.edu.cn (X.C.)
School of Physical Education, Yan’an University, Yan’an 716000, China
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China;
zhouyajun@foxmail.com (Y.Z.); xstan@fudan.edu.cn (X.T.)
2
3
4
Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-40530 Gothenburg,
Sweden; weixiao.yuanwahlgren@chem.gu.se
*
Correspondence: kwyang@nwu.edu.cn; Tel.: +86-29-8153-5035
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 December 2019; Accepted: 24 December 2019; Published: 1 January 2020
Abstract: Inhibition of
antibacterial activity to resistant bacteria. In this work, we found that aromatic carboxyl substituted
2-triazolylthioacetamides 1a inhibited VIM-2, exhibiting an IC50 value in the range of 20.6–58.6 M.
β-lactamases presents a promising strategy to restore the β-lactams
–
j
µ
The structure-activity relationship study revealed that replacing the aliphatic carboxylic acid with
aromatic carboxyl improved the inhibitory activity of 2-triazolylthioacetamides against VIM-2.
1a–j (16 mg/mL) restored the antibacterial activity of cefazolin against E. coli cell expressing VIM-2,
resulting in a 4–8-fold reduction in MICs. The isothermal titration calorimetry (ITC) characterization
suggested that the primary binding 2-triazolylthioacetamide (1b, 1c, or 1h) to VIM-2 was a combination
of entropy and enthalpy contributions. Further, the crystal structure of VIM-2 in complex with 1b was
obtained by co-crystallization with a hanging-drop vapour-diffusion method. The crystal structure
analysis revealed that 1b bound to two Zn(II) ions of the enzyme active sites, formed H-bound
with Asn233 and structure water molecule, and interacted with the hydrophobic pocket of enzyme
activity center utilizing hydrophobic moieties; especially for the phenyl of aromatic carboxyl which
formed π-π stacking with active residue His263. These studies confirmed that aromatic carboxyl
substituted 2-triazolylthioacetamides are the potent VIM-2 inhibitors scaffold and provided help to
further optimize 2-triazolylthioacetamides as VIM-2 even or broad-spectrum MβLs inhibitors.
Keywords: antibiotic resistance; metallo-β-lactamase VIM-2 inhibitor; 2-triazolylthioacetamides;
thermodynamics; crystallographic study
1. Introduction
β
-lactam antibiotics, including penicillins, cephalosporins, and carbapenems, are the clinically
most important and commonly used antimicrobial agents [ ]. However, the overuse of the -lactam
antibiotics has resulted in a rapid appearance of -lactam-resistant bacteria [ ]. A main mechanism
for bacteria to develop resistance to -lactams is the expression of
1
β
β
2
β
β-lactamase, which hydrolyze the
Biomolecules 2020, 10, 72; doi:10.3390/biom10010072
www.mdpi.com/journal/biomolecules

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β
-lactam ring and renders the antibiotics ineffective. According to the action mechanism,
have been categorized as serine- -lactamases (S Ls) and metallo- -lactamases (M Ls) [
amino acid sequence homology and Zn(II) content, M Ls are further divided into subclasses B1,
B2, and B3. M Ls belonging to B1 and B3 subgroups hydrolyze almost all -lactams [ ]. The S Ls
inhibitors, such as clavulanic acid, tazobactam, sulbactam, vaborbactam, and avibactam have been used
clinically [ ]. Although a large number of M L inhibitors have been reported, including thiols [ ],
carboxylic acids [10 11], rhodanine [12 14], cyclic boronates [15 16], and ebselen [17 18], there are no
L inhibitors for available clinical purposes to date. Consequently, it is an urgent need to develop
novel MβL inhibitors and to investigate their action mechanism.
VIM-2, a B1 subclass M L [19], was initially discovered in P. aeruginosa, which showed a high
β
-lactamases
β
β
β
β
3]. Based on
β
β
β
3
β
4,
5
β
6–9
,
–
,
,
M
β
β
prelevance and broad substrate spectrum, including penicillins, cephalosporins, and carbapenems [20],
it therefore reflects a significant drug target for the treatment of antibiotic-resistant bacterial infection.
The crystal structure of VIM-2 has been reported [21,22]. Moreover, several complexes of crystal
structures of VIM-2 with small molecules have been reported [7,11,13].
Recently, we found that azolylthioacetamides are the potent M
these compounds showed broad-spectrum inhibition on CcrA [24], NDM-1 [23
β
L inhibitors [23
,24,26], ImiS [24
–
–
26],
26],
and L1 [24], as the representative of three subclasses enzymes, respectively. Further, we reported
an aromatic carboxyl substituted azolylthioacetamide as an inhibitor of VIM-2, which provided the
binding via the complex crystal structure of enzyme and inhibitor [27].
In this work, we synthesized a series of 2-triazolylthioacetamides as previously reported [25],
tested their inhibitory activity against VIM-2, and evaluated their efficiency to restore antibacterial
activity of cefazolin against bacteria expressing VIM-2. Further, the binding affinities of
2-triazolylthioacetamides to VIM-2 were determined by isothermal titration calorimetry (ITC). Finally,
the crystal structure of VIM-2 in complex with a 2-triazolylthioacetamide molecule was determined
and the interaction between enzyme and inhibitor was explored by crystallographic studies.
2. Materials and Methods
2.1. Synthesis of 2-Triazolylthioacetamides
The 2-triazolylthioacetamides were synthesized as shown in Scheme 1. Briefly, thiosemicarbazide
reacted with corresponding acid anhydride (phthalic anhydride, succinic anhydride, and glutaric
anhydride) to give thiosemicarbazides. The thiosemicarbazides was refluxed in NaOH aqueous to
offer the thiol triazoles. Corresponding anilines were acylated with chloroacetyl chloride to offer
α
-chloroacetamides. Finally, the thiol triazoles cross-linked with α-chloroacetamide to afford the
2-triazolylthioacetamides under alkaline environment, (1a–j, 2a–e, and 3a–e).
Scheme 1. Synthetic route of the 2-trizolylthioacetamides.
2.2. Over-Expression and Purification of VIM-2
The VIM-2 was overexpressed and purified as previously described [21]. In brief, E. coli BL21
(DE3) cells were transelected with plasmid pET24a-VIM-2. A 10 mL overnight culture of these cells in
lysogeny broth (LB) was used to inoculate of M9 media containing kanamycin (25
were allowed to grow at 37 C with shaking until the optical density at 600 nm (OD600) reached
µ
g/mL). The cells
◦

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0.6–0.8. VIM-2 expression was induced with 1 mM IPTG at 25 ◦C overnight. Cells were collected by
centrifugation and lysed by sonication. The supernatant collected by centrifugation was dialyzed three
times at 4 ◦C and loaded on a Q-Sepharose column. Bound protein was eluted with a 0–500 mM NaCl
gradient in 30 mM Tris, pH 7.6. The further purification of crude VIM-2 was run though a G75 column
(GE Healthcare, Boston, MA, USA) and eluted with 20 mM Tris, pH 7.6, containing 150 mM NaCl, and
5% β-mercaptoethanol. Enzyme purity was analyzed by SDS-PAGE, and determined the concentration
by measuring the absorption at 280 nm (extinction co-efficient: 39,000 M−1cm−1) using the Nanodrop
2000 c spectrophotometer (Thermo Scientific, Waltham, MA, USA).
2.3. Inhibition Studies
The percent inhibition (inhibition%, enzyme activity without inhibitor minus residual activity
with 100
activity) values were measured using a previously established enzyme-inhibition assay on an Agilent
UV8453 UV Vis spectrophotometer using 60 M nitrocefin (monitoring at 482 nm) as a substrate.
The 2-triazolylthioacetamides 1a 2a , and 3a were dissolved with DMSO and diluted with ddH2O.
µM inhibitor) and IC50 (the inhibitor concentrations causing 50% decrease of enzyme
−
µ
–
j,
–e
–e
The VIM-2 and nitrocefin were prepared with a Tris buffer (30 mM, pH 7.6). The final concentration of
DMSO inhibition experiments was below 1%. The IC50 values were determined in triplicate against
VIM-2, where the inhibitor concentrations were varied between 0 and 120 µM, and final concentrations
VIM-2 was 0.6 nM. The tested VIM-2 and inhibitors were premixed at room temperature (30 mM
Tris, pH 7.6), and then the substrate in the same buffer was added to the mixture, the initial rates of
nitrocefin hydrolysis were recorded at each inhibitor concentration. The percentage inhibition was
calculated by the equation inhibition% = 1
average inhibition% against inhibitor concentration and fitting of the data.
−
(Vi/V0) and IC50 values were calculated by plotting the
2.4. Antibacterial Activity Assays
The minimum inhibitory concentration (MIC) of antibiotics, both alone and in the presence
of enzyme inhibitors, was determined using the broth micro-dilution method [28]. E. coli DH10B
containing plasmid pBCSK-VIM-2 grown in MH medium to OD600 = 0.45 were used as inocula,
after an 84-fold dilution to 1
medium to prepare 1024, 512, 256, 128, 64, 32, 16, and 8 for E. coli producing VIM-2 stock solutions,
respectively. Compounds 1a–j, 2a–e, and 3a–e were dissolved in DMSO and diluted with MH media
×
105 CFU/mL in MH medium. Cefazolin was dissolved in MH
to prepare 64
µg/mL stock solutions. The 50
µL prepared stock solutions with different cefazolin
concentrations were diluted with a 50
µL inhibitor stock solution, then 100
µL inoculum was added
sequentially. The final concentration of both the inhibitor and cefazolin were a quarter of the stock
solution concentrations.
2.5. Evaluation of Binding Affinity
Binding affinity measurements were performed by ITC on a Malvern MicroCal-ITC200 at 298 K.
The syringe stirring rate was 750 rpm and kept constant. A stock of VIM-2 and inhibitor (1b
1h) were prepared in Tris buffer (30 mM, pH 7.6) with 1% DMSO. The concentration of VIM-2 was
100 M. Before the injections, the solution of enzyme and inhibitor were degassed by centrifugation
(32,583 g, 5 min), respectively. Then, the prepared VIM-2 solution was transferred into the sample
cell (210 L), and the inhibitor (1 mM) in the syringe was titrated into the sample cell by 18 injections
(0.5 L for the first injection and 1.5 L for the other 17 injections) with 120 s intervals per injection.
, 1c, and
µ
×
µ
µ
µ
The heat of dilution for the inhibitor was determined in the control experiment and subtracted from
the integrated data before curve fitting [29]. The data were fitted and analyzed with single binding
sites using Origin 7.0 software, provided with the instrument.

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2.6. Crystallization, X-ray Data Collection, and Structure Determination
The complex crystal of 1b and VIM-2 was obtained with co-crystallization by the hanging-drop
vapor-diffusion method. The 10 mM inhibitor in a 5% DMSO Tris buffer and 14.9 mg/mL VIM-2
solution were pre-incubated overnight with a 1:1 ratio. The reservoir consisting of 20% polyethylene
glycol (PEG) 8000, 0.1 M MES pH 6.5, 0.1 M magnesium acetate, and 3% DMSO solution and crystals
appeared after 2–10 d. Crystals were transferred to 24% PEG 8000, 0.1 M MES pH 6.5, 0.3 M magnesium
acetate, 3% DMSO, and 25% glycerol and flash-cooled in liquid nitrogen. Data were collected at the
Shanghai Synchrotron Radiation Facility (SSRF) and processed using HKL 3000. The phase was solved
by molecular replacement Phenix [30] and a previously published VIM-2 structure (PDB: 1ko3) [22] as
a search model. Structure refinements were actualized by iterative rounds of the model building using
Wincoot [31] and maximum-likelihood restrained refinement with Phenix. The PyMOL molecular
graphics system was used to produce illustrations and to visualize interactions [32]. The numbering of
all residues is used as a class B β-lactamase numbering system [33].
3. Results and Discussion
3.1. Synthesis of 2-Triazolylthioacetamides
Compounds 1a–j, 2a-e, and 3a-e were re-synthesized and characterized as described
previously [25]. The structures of synthesized 2-triazolylthioactamides are shown in Figure 1.
Figure 1. The structure of 2-triazolylthioactamides.
3.2. Inhibition of VIM-2 by 2-Triazolylthioactamides
To evaluate 2-triazolylthioactamides, the VIM-2 was expressed and purified as previously
described [21]. The steady-state kinetic assays were conducted on an Agilent UV8453 spectrometer
using nitrocefin (60 µM) as a substrate and monitoring its hydrolytic product at 482 nm.
Percent inhibition of 1a
are displayed in Figure 2. It is clearly observed that 1a
while 1h exhibited up to 90% inhibition. However, 2a
–
j
,
2a
–
e
, and 3a
–
e
(100 mM) against VIM-2 was determined and the results
showed more than 50% inhibition on VIM-2,
and 3a with an aliphatic carboxyl had very
–
j
–
e
–e
weak inhibitory activity.

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Figure 2. Percent inhibition of 2-triazolylthioactamides at a concentration of 100
integron-encoded metallo-β-lactamase 2 (VIM-2) using nitrocefin as the substrate.
µM against verona
IC50 values of 1a
of inhibitors were varied between 0 and 120
2-triazolylthioactamides 1a inhibited VIM-2 with an IC50 value in the range 20.6–58.6
was the most potent inhibitor (IC50 = 20.6 M). Moreover, clogP values of 1a (Table 1), as predicted by
–j
against VIM-2 were measured in 30 mM Tris, pH 7.6, in which the concentrations
M. The IC50 data listed in Table 1 indicated that the
M, and 1h
µ
–
j
µ
µ
–j
the ALOGPS 2.1 program, are in the range 1.88–3.05, which are similar with many marked antibacterial
agents [34,35].
Analysis of percent inhibition and IC50 data of the 2-triazolylthioactamides against VIM-2 revealed
a structure activity relationship (SAR), which is that the aromatic carboxyl substituted at triazole
−
ring improves inhibitory activity of the thioacetamides against VIM-2. This SAR is similar to that
in our previous reported on ImiS studies [25], hence it was significant to further probe the potential
interaction of the aromatic carboxyl group to enzyme activity.
Table 1. The inhibition of VIM-2 (IC50), and clogP values of aromatic carboxyl substituted
2-triazolylthioactamides 1a–j.
Compds
IC50 (µM)
clogP
Compds
IC50 (µM)
clogP
1a
1b
1c
1d
1e
40.8 ± 0.3
58.6 ± 0.1
49.1 ± 0.1
52.2 ± 0.5
38.2 ± 0.3
2.28
2.18
2.74
3.05
2.23
1f
1g
1h
1i
45.8 ± 0.2
31.4± 0.4
20.6 ± 0.1
45.2 ± 0.1
57.6 ± 0.4
1.88
3.03
3.05
2.14
2.19
1j
The substrate was nitrocefin and inhibitor concentrations were varied between 0 and 120
µM; data were
mean ± standard.
3.3. Antibacterial Activity Assays
The ability of 2-triazolylthioactamides to restore the antimicrobial activity of cefazolin against
the bacteria producing VIM-2 was investigated by determining the MICs of -lactams in the presence
and absence of inhibitors (16 g/mL). E. coli DH10B containing plasmid pBCSK-VIM-2 was used to
study these inhibitors. A significant decrease in MIC of cefazolin (4–8-fold) was observed for 1a
while 2a and 3a did not (Table 2A). The dose-dependent MIC assays for 1b 1c 1g, and 1h against
E. coli-VIM-2 (Table 2B) indicated that the antibacterial effect of cefazolin increased gradually with
the increasing inhibitor dose. In the presence of 1b 1c 1g, or 1h (128 g/mL), the MICs of cefazolin
were decreased 16-, 16-, 32-, and 32-fold, respectively. Although the strains treated with a high
dose of 2-triazolylthioacetamides (128 g/mL) were still resistant, they are promising in the future.
Inhibitors alone did not show antibacterial activity against the E. coli and E. coli producing VIM-2 at
128 g/mL, implying that the capacity of 2-triazolylthioacetamides to restore -lactams activity lies in
their inhibition of the MβLs harbored in bacteria.
β
µ
–j,
–
e
–e
,
,
,
,
µ
µ
µ
β

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Table 2. Minimum inhibitory concentration (MICsµg/mL) of cefazolin against E. coli DH10B-VIM-2 in
the absence and presence of 2-triazolylthioactamides at 16
µg/mL (
A
), and in the presence of 1b, 1c, 1g,
and 1h in a concentration range of 0–128 µg/mL (B).
(A)
Compds
MIC (µg/mL)
Compds
MIC (µg/mL)
Control
1a
128
32
32
32
32
16
1f
1g
1h
1i
32
16
16
32
16
1b
1c
1d
1e
1j
(B)
Compds\conc
0
16
32
64
128
1b
1c
1g
1h
128
128
128
128
32
32
16
16
32
32
16
8
16
16
8
8
8
4
4
8
The MICs of cefazolin against E. coli cells without VIM-2 was 1 µg/mL.
3.4. Binding Affinity Evaluation
To investigate the binding affinity of 2-triazolylthioactamides to VIM-2, 1b
,
1c, and 1h as enzyme
inhibitors were selected based on the inhibitory activity. ITC experiments were performed on
MicroCal-ITC200 with a multiple injections mode at 298 K. Injection of the inhibitor into the protein
solution was shown as the peak of the binding isotherm (upper panels, Figure 3), the bottom of Figure 3
displayed the integrated plots of heat released per injection as a function of protein-inhibitor molar
ratio. After the best fitting by the single binding site model, the affinity constant (Ka), corresponding
dissociation constant (Kd), and thermodynamic parameters (
∆
H,
∆
S, and
∆
G) were determined and
listed in Table 3.
For the binding of 1b
displayed in Table 3 revealed that the binding affinity of 1h is higher than 1c, and then 1b, which is
consistent with the IC50 values of these inhibitors against VIM-2 (20.6, 49.1, and 58.6 M, respectively).
These results implied that the initial binding 1b 1c, or 1h to VIM-2 was a combination of entropy and
enthalpy contributions. Comparison of the structure of 1b and 1c, the NH group of 1b improved the
hydrophilicity and resulted in an increase of H and a decrease of T S. In addition, replacing the
benzimidazole of 1b or benzothiazole of 1c with a chlorophenyl (1h) caused an obvious raising in
,
1c, and 1h to VIM-2, the Ka and corresponding
∆
G ( G = S)
∆
∆
H
−
T∆
µ
,
∆
∆
∆
H
and enhanced the inhibition of VIM-2, suggesting that the favorable enthalpic contributions which are
assigned to van der Waals interaction, hydrogen bonding, or electrostatic interactions are the main
driving force for improving the inhibitory activity of the 2-triazolylthioactamides on VIM-2 [36].

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Figure 3. Isothermal titration calorimetry (ITC) thermograms of titration of 1b
) solution (1 mM) into 100 M VIM-2 in Tris-HCl, pH 7.6, 298 K (upper panels). The lower panels
represent the integrated heat release of the titration after correcting dilution heat against the molar
ration of 1b 1c, or 1h/VIM-2; the solid lines represent the best-fitted isotherm by the nonlinear fitting
(A), 1c (B), or 1h
(
C
µ
,
with the one-site model.
Table 3. Binding abilities and thermodynamic parameters of 1b, 1c, or 1h to VIM-2, as estimated by
ITC at 298 K in Tris-HCl, pH 7.6.
Ka (M−1
)
Compounds
Kd (µM)
∆H (kcal/mol)
T∆S (kcal/mol)
∆G (kcal/mol)
1.16 × 105
1.26 × 105
1.56 × 105
1b
1c
1h
8.62
7.94
6.41
−2.66
−1.61
−5.10
4.26
5.34
1.98
−6.92
−6.95
−7.08
3.5. Crystallographic Analyses
To explore how the 2-triazolylthioactamides bind to VIM-2, we attempted to obtain the X-ray crystal
structure of VIM-2 in complexes with each 1a compound. However, only the crystal structure of
VIM-2 in complex with 1b VIM-2:1b) was obtained successfully through co-crystallization. Maybe the
–
j
(
low solubility was the possible reason which resulted in other inhibitors without providing satisfactory
complexes. VIM-2:1b was solved to 1.78 Å resolution (PDB ID: 6KW1), crystallized in space group C2
with two protein molecules in the asymmetric unit (ASU) in Figure 4, and refined to Rwork/Rfree value
of 17.65%/20.63%. The details for the collection and refinement statistics were listed in Tables 4 and 5.
The crystallographic analyses of the binding mode of 1b to VIM-2 were shown in Figure 5. The OMIT
Fo-Fc map and final 2Fo-Fc density map of ligand were shown at 3.0 and 1.0 sigma, respectively.
As is typical for B1 MβLs, there are two Zn(II) ions in the active sites of the enzyme. Zn1 was
coordinated by His116, His118, while Zn2 was coordinated by Asp120, Cys221, and His263. In Figure 4,
two Mg2+ were located between the two symmetry VIM-2 molecules, which has been observed in the
published crystal structure of VIM-2 and considered to be a crystallographic artefact [8,37]. Moreover,
two Cl− and four Na+ ions have been observed. The protein active site structure, including Zn–ligand
and the distances between the two Zn ions (3.9 Å) agreed with the publications [11,21,22,27].
The active sites structure of VIM-2 in complex with 1b (Figure 5B) showed that the triazole ring of
1b replaced the hydroxide and interacted with both Zn(II) ions (2.1 and 2.2 Å), which identified the
previous SAR that the azolylthioactamides containing the triazole group was better for inhibition of
NDM-1 (belong to the same subclass with VIM-2) than thiazole or oxazole substituted compounds [24];
the carboxyl moiety of 1b interacted with Zn2 (2.4 Å), and formed H-bond with the backbone N atom

Biomolecules 2020, 10, 72
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of Asn233 (3.2 Å) on L10 loop; the O atoms of carboxyl interacted with the W1 (2.6 Å) and the structural
water molecule W2 (2.7 Å) through hydrogen-binding, respectively, which was similar to the interactions
for debris fastening with the active site of VIM-2 [11]. Importantly, we found that the phenyl at R1
position formed
reasonably explained the SAR that the aromatic carboxyl substituted 2-triazolylthioactamides (1a
exhibited a better inhibitory activity against VIM-2 than the aliphatic carboxyl substituted compounds
2a and 3a ). Moreover, Tony et al. reported that the binding of compound 1c) caused a flip of
π-π stacking with the imidazole ring of His263, the favorable hydrophobic interaction
–j)
(
–e
–
e
1 (
the backbone and side-chain of Trp87 and believed that the conformational changes of Trp87 were
important to further optimize triazolylthioacetamide inhibitors [27]. In our crystal structure, residues
Trp87 kept a native pose, yet the binding of 1b to the active sites of VIM-2 resulted in a conformational
change of the residue Phe61 on the L3 loop and formed a potential clash with Phe61 utilizing the
benzimidazole group (Figure 5C).
Figure 4. Two VIM-2 molecules in the asymmetric unit with a ribbon diagram. Helices were shown in
cyan (chain A) or red (chain B) and β-strands are shown in red (chain A) or yellow (chain B). 1b binding
to the active site was shown in stick and colored by atom (H, white; C, cyan; N, blue; O, red; S, yellow).
Zn2+, Cl−, Mg2+, and Na+ ions are shown as gray, green, yellow, and purple spheres, respectively.
Table 4. Data collection.
Structure (PDB ID)
6A61
Data Collection
Space group
a, b, c (Å)
SSRF BL18u
C 1 2 1
103.759, 79.421, 67,981
α, β, γ (◦)
90, 130.938, 90
a
Mol/ASU
2
39.19–1.78 (1.839–1.78)
39948
Resolution range (Å)
Number of unique reflections
Completeness (%)
I/σ(I)
99.13
6.3 (3.8)
16.10
Wilson B Factor (Å2)
a
Mol/ASU: Molecules per asymmetric unit.

Biomolecules 2020, 10, 72
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Figure 5. Crystallographic analyses of the binding mode of 1b to VIM-2. (
Fo-Fc map was shown in green at 3.0 and final 2Fo-Fc density map in blue at 1.0
) crystal structure of compound 1b bind to the activity site of VIM-2; ( ) the structure alignment of
A
) The ligand structure,
σ
σ
, respectively;
(
B
C
native structure VIM-2 (pink, 1 ko3) and the complex structure of VIM-2 and 1b (cyan, PDB: 6 KW1).
Table 5. Refinement statistics.
Overall B factor (Å2)
Protein B factor (Å2)
24.15
22.80
50.62
31.79
0.006
0.83
Ligand B factor (Å2)
Water B factor (Å2)
b
RMSD from Ideal Bond Length (Å)
RMSD from Ideal Angles (◦)
b
Final Rwork (%)
Final Rfree (%)
17.65%
20.63%
b
RMSD: Root mean square deviation.
4. Conclusions
The 2-triazolylthioacetamides 1a
that compounds 1a were potent inhibitors of VIM-2 with an IC50 value in the range 20.6–58.6
and 1h was found to be the best inhibitor (IC50 = 20.6 M). SAR studies revealed that the substituted
aromatic carboxyl at triazole ring improved inhibition of the molecules on VIM-2. MIC tests indicated
that 1a partly restored the antimicrobial effect of cefazolin against E. coli expressing VIM-2, although
the strains treated with a high dose of 2-triazolylthioacetamides (128 g/mL) were still resistant.
–j, 2a
–
e, and 3a
–e
were synthesized, and kinetic assays showed
M,
–
j
µ
µ
–
j
µ

Biomolecules 2020, 10, 72
10 of 12
Thermodynamic characterization suggested that the binding of 1b
,
1c, or 1h to VIM-2 was a combination
of entropy and enthalpy contributions, and replacing the bulky hydrophobic group with a small
hydrophobic group at R2 improved inhibition of 2-triazolylthioactamide on VIM-2. The crystallographic
analysis revealed that 1b replaced the hydroxide which bridges the two Zn(II) ions and bound to
the Zn(II) ions via N atoms on the triazole ring; the carboxyl moiety of 1b interacted with Zn2 and
formed an H-bond with the backbone N atom of Asn233 and the water molecule W2. Although the
crystal structure in the complex with 2-triazolylthioacetamide has been reported [27], we proved
the importance of phenyl at R1 to the inhibitory activity of 2-triazolylthioactamide against VIM-2,
which formed a hydrophobic interaction with active residue His263 and confirmed the above SAR;
further, a new hydrophobic interaction has been found where the benzimidazole formed a potential
disturbed with Phe61 and resulted in a conformational change of the residue Phe61 on the L3 loop.
This work revealed that the aromatic carboxyl at R1 was essential for the activity of inhibitors on VIM-2,
further, the m-chlorophenyl at R2 promoted activity of the compounds on VIM-2. The information
gained in this work is valuable for further optimizing 2-triazolylthioacetamides as VIM-2, even as
broad-spectrum MβLs inhibitors.
Author Contributions: All authors contributed to the writing of the manuscript. K.-W.Y. and Y.X. designed the
experiment, prepared, and revised the draft; Y.X., Y.-J.Z., Y.G., and Y.Z. conducted the kinetic, thermodynamic,
and crystallographic analysis; C.C., W.Y.W., X.T., and X.C. participated in the data analysis. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by grants (21572179 and 2019KW-068 to K.-W.Y) from the National Natural
Science Foundation of China and Shaanxi Province International Cooperation Fund and the grants (205040223
and 2019053 to Y.X.) from Natural Science Foundation of Yan’an University and Shaanxi Provincial Sports Bureau.
Acknowledgments: X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF,
BL18U), China.
Conflicts of Interest: The authors declare no conflict of interest.
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