Rhodanine as a Potent Scaffold for the Development of Broad-Spectrum Metallo-β-lactamase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00548

A series of rhodanines was constructed, their Z-configuration was confirmed by small molecule X-ray crystal structures, and their activity against metallo-β-lactamases (MβLs) was measured. The obtained 26 molecules and a thioenolate specifically inhibited the MβL L1 with an IC₅₀ range of 0.02-1.7 μM, and compounds 2h-m exhibited broad-spectrum inhibition of the MβLs NDM-1, VIM-2, ImiS, and L1 with IC₅₀ values <16 μM. All inhibitors increased the antimicrobial effect of cefazolin against E. coli cells expressing L1, resulting in a 2-8-fold reduction in MIC. Docking studies suggested that the nitro (NDM-1, CphA, and L1) or carboxyl group (VIM-2) of 2l coordinates one or two Zn(II) ions, while the N-phenyl group of the inhibitor enhances its hydrophobic interaction with MβLs. These studies demonstrate that the diaryl-substituted rhodanines are good scaffolds for the design of future broad-spectrum inhibitors of MβLs.

Full text of DOI:10.1021/acsmedchemlett.7b00548

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Rhodanine as a potent scaffold for the development
of broad-spectrum metallo-#-lactamase inhibitors
Yang Xiang, Cheng Chen, Wen-Ming Wang, Li-Wei Xu, Kewu Yang, Peter Oelschlaeger, and Yuan He
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00548 • Publication Date (Web): 22 Mar 2018
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Rhodanine as a potent scaffold for the development of broadꢀ
spectrum metalloꢀβꢀlactamase inhibitors
Yang Xiang,1‡ Cheng Chen,1‡ Wen-Ming Wang,1 Li-Wei Xu,1 Ke-Wu Yang,1* Peter Oelschlaeger,2
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and Yuan He1*
1Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biolo-
gy Innovation Laboratory, College of Chemistry and Materials Science, Northwest University, Xi’an 710127, P. R.
China
2Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, 309 East
Second Street, Pomona, California 91766, United States
KEYWORDS: antibiotic resistance, metallo-β-lactamases, broad-spectrum inhibitor, rhodanine
ABSTRACT: A series of rhodanines was constructed, their Z-conformation confirmed by small molecule X-ray crystal
structures, and their activity against metallo-β-lactamases (MβLs) measured. The obtained twenty-six molecules and a
thioenolate specifically inhibited the MβL L1 with an IC50 range of 0.02-1.7 μM, 2h-m exhibited broad-spectrum inhibition
of the MβLs NDM-1, VIM-2, ImiS, and L1 with IC50 values < 16 μM. All inhibitors increased the antimicrobial effect of
cefazolin against E. coli cells expressing L1, resulting in a 2-8-fold reduction in MIC. Docking studies suggested that the
nitro (NDM-1, CphA, and L1) or carboxyl group (VIM-2) of 2l coordinates one or two Zn(II) ions, while the N-phenyl
group of the inhibitor enhances its hydrophobic interaction with MβLs. These studies demonstrate that the diaryl-
substituted rhodanines are good scaffolds for the design of future broad-spectrum inhibitors of MβLs.
The development of β-lactam antibiotics over the past
70 years has led to the availability of drugs to treat a wide
range of bacterial infections. However, the wide-spread
use of β-lactam containing antibiotics has resulted in a
large number of bacteria that are resistant to almost all
antibiotics. Most commonly, bacteria become resistant to
β-lactam antibiotics by producing β-lactamases, which
hydrolyze the C–N bond in the four-membered ring of β-
lactam antibiotics. The β-lactamases have been catego-
rized as serine β-lactamases (SβLs) and metallo-β-
lactamases (MβLs), according to their mechanism of ac-
tion.1 MβLs are further divided into subclasses B1, B2, and
B3, based on amino acid sequence homology and Zn(II)
content.1 The B1 and B3 subclasses hydrolyze almost all β-
lactam antibiotics, including penicillins, cephalosporins,
and carbapenems. In contrast, the B2 subclass enzymes
preferentially hydrolyze carbapenems, which have been
called ‘last resort’ antibiotics.2
MβLs. Spicer and co-workers reported that a rhodanine
with a trichlorobenzylidene substituent (see Scheme S1)
showed an inhibitory effect on the Verona Integron-borne
Metallo-β-lactamase 2 (VIM-2) and imipenemase 1 (IMP-
1).11 Further mechanistic studies by Brem et al. indicated
that the rhodanine hydrolysis product thioenolate
(Scheme S1) also inhibited B1 MβLs.12
Our goal is to develop specific or broad-spectrum in-
hibitors of MβLs and to use them in combination with β-
lactams to combat bacterial infections, in which the bac-
teria produce MβLs. Based on the above information and
to investigate whether the rhodanine alone is a potent
inhibitor of MβLs, we constructed a series of novel
rhodanines with benzyl, heterocyclic, naphthyl, aliphatic
and aromatic carboxyl substituents (Fig. 1). These com-
pounds were tested as inhibitors against the purified
MβLs VIM-2, New Delhi Metallo-β-lactamase 1 (NDM-1),
Imipenemase-1 from Aeromonas veronii bv. sobria (ImiS),
and β-lactamase 1 from Stenotrophomonas maltophilia
(L1), which are representative enzymes belonging to the
B1, B2, and B3 subclasses of MβLs, respectively.13 Further-
more, the ability of these inhibitors to restore the antimi-
crobial activity of existing antibiotics against antibiotic-
resistant strains was evaluated.
Facing the emergence of drug resistance mediated by
MβLs, a large number of MβL inhibitors have been re-
ported, such as β-lactam analogues,3 hydroxamic acid,4
azolylthioacetamides,5 and cyclic boronates.6 Ebselen7 and
aspergillomarasmine A (AMA)8 have also been described
to be inhibitors of MβLs.
Twenty-six rhodanines 1a-m and 2a-m (Fig. 1) were syn-
thesized by a previously reported method. The synthetic
route is shown in Fig. 1. Briefly, amines reacted with car-
The rhodanines, unique non-transitional state analogs
that inhibit penicillin-binding proteins (PBPs)9 and
SβLs,10 have recently been described to be inhibitors of
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ACS Medicinal Chemistry Letters
bon disulphide in NaOH aqueous solution at room tem-
perature for 16 h, sodium chloroacetate was added and
stirred for 3 h, and the resulting mixture was acidified
with HCl and refluxed for 16 h to give N-substituted
rhodanines 1 and 2.14 Knoevenagel condensation of aryl
aldehyde and 1 or 2 in acetic acid offered the desired
rhodanines.15, 16
Page 2 of 7
and B3 (L1) were overexpressed and purified as previously
described.17-20 The inhibition experiments with these
compounds under steady-state conditions were conduct-
ed on an Agilent UV8453 spectrometer using cefazolin (50
μM, monitoring at 262 nm) as substrate for NDM-1, VIM-
2, and L1, and imipenem (60 μM, monitoring at 300 nm)
for ImiS. The concentrations of inhibitors were varied
between 0 and 50 μM.
1
2
3
4
5
6
7
8
To confirm the molecular structures of the rhodanines,
crystals suitable for X-ray analysis were obtained by slow
evaporation of a solution of 1l and 2m in methanol-
acetone. The crystal structures are given in Fig. S1, and
the resulting structures based on X-ray diffraction con-
firmed the expected structures. Coordinates of 1l and 2m
in CIF format are available as Electronic Supplementary
Information from the Cambridge Crystallographic Data
Center (CCDC: 1480089 and 1479978, respectively). All
The concentrations of compounds 1a-m and 2a-m caus-
ing 50% decrease in enzyme activity (IC50) were deter-
mined in 50 mM Tris, pH 7.0 (at this pH, the rhodanine
was not hydrolyzed, see the following discussion). The
IC50 data (Table 1) indicate that all of these rhodanines
exhibited excellent inhibition of L1 with IC50 values rang-
ing from 0.02 to 1.7 μM, and 2m was found to be the most
potent inhibitor (IC50 =0.02 μM). For NDM-1, 1b, 1g, 1i-l,
2a-d and 2g-m showed potency with IC50 values ranging
from 0.69 to 47 μM, and 2b had the lowest IC50 (0.69 μM).
1f, 1h-l, 2d-e and 2h-m inhibited VIM-2 with IC50 values
ranging from 0.19 to 27.9 μM, and 2l was the most potent
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1
13
synthesized rhodanines were characterized by H and C
NMR and HRMS (see Supporting Information).
inhibitors (IC50=0.19 μM). ImiS was inhibited by 1d, 2a and
2e-m with IC50 values ranging from 3.0 to 19.1
μM with 2l
being the most potent inhibitor (3.0 μM).
It should be noted that 2h-m were potent broad-
spectrum inhibitors of all four tested MβLs, exhibiting
IC50 values < 16 μM, and 2l was found to be the most po-
tent broad-spectrum inhibitor with IC50 values ≤ 3 μM.
Given the broad-spectrum potency of 2l, the inhibition
curves of 2l against the four MβLs tested were analyzed in
more detail (Fig. 2). 2l exhibited more than 95% inhibi-
tion (< 5% residual activity) against NDM-1, VIM-2, ImiS,
and L1 at concentrations of 20, 4, 40 and 0.8 μM, respec-
tively.
Brem et al. reported that the rhodanine hydrolysis
product thioenolate with a trichlorobenzylidene substitu-
ent (see Scheme S1) exhibited inhibition of MβLs.12 To
investigate whether rhodanine alone inhibits MβLs in
case it is not hydrolyzed, we assayed the stability of 2g (50
μM) in MES (pH 5.5, 6.0, and 6.5), Tris (pH 7.0, 7.5, 8.0,
and 8.5), and Tris containing L1 (pH 7.0) through moni-
toring its absorbance change at 401 nm for 24 h. The re-
sults (Fig. S3) show that 2g was hardly hydrolyzed at pH
values ranging from 6.0 to 7.0 (Fig. S3B-D), even in the
presence of L1 enzyme (Fig. S3H), perhaps because our
rhodanines are not “activated” by multiple chlorines on the
phenyl ring as in Brem et al.’s report. These results indicate
that rhodanine 2g is not hydrolyzed by the MβL and that
the rhodanine itself and not its hydrolysis product is re-
sponsible for the MβL inhibition observed.
To check whether the rhodanine hydrolysis product in-
hibits MβLs, 3a was prepared by hydrolysis of 2g with
NaOH and acidification with HCl (Fig. 1) and confirmed
1
by H and 13C NMR and HRMS (see Supporting Infor-
Fig. 1. Synthetic route and the structures of rhodanine deriva-
tives and thioenolate 3a.
mation). Inhibition assays (in 50 mM Tris, pH 7.0) indi-
cated that 3a inhibited L1 and VIM-2 with IC50 values of
0.32 and 4.7
μ
M, respectively, but not NDM-1 or ImiS at
M (Table 1), indicating that
To test whether these rhodanines were MβL inhibitors,
MβLs from subclasses B1 (VIM-2 and NDM-1), B2 (ImiS),
concentrations of up to 50
μ
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Table 1. Inhibitory Activity (IC50,
ꢁ
M) of Rhodanines against Four MβLs in 50 mM TrisꢀHCl, pH 7.0
1
2
3
4
5
6
7
8
9
B3
B1
B2
B3
L1a
B1
B2
Compds
Compds
NDMꢀ1a
VIMꢀ2a
ImiSb
NDMꢀ1a
0.69±0.02
14.3±0.4
1.13±0.06
ꢀ
VIMꢀ2a
ꢀ
ImiSb
ꢀ
L1a
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.32±0.02
1.7±0.1
2b
2c
2d
2e
2f
0.18±0.02
0.59±0.02
0.21±0.02
0.12±0.01
0.10±0.01
0.31±0.02
0.070±0.002
0.030±0.008
0.080±0.003
0.070±0.008
0.050±0.003
0.020±0.004
0.32±0.01
1a
1b
1c
1d
1e
1f
47±1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.77±0.09
0.16±0.03
0.37±0.01
0.28±0.01
0.6±0.1
0.92±0.02
1.9±0.3
ꢀ
ꢀ
19.1±0.6
16.5±0.3
6.9±0.2
15.0±0.3
7.7±0.9
7.6±0.2
3.6±0.1
7.8±0.5
3.0±0.2
15.9±0.8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
27.9±0.3
ꢀ
2g
2h
2i
12±1
ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25.1±0.8
ꢀ
12.2±0.9
10.6±0.8
8.4±0.4
10.6±0.5
1.31±0.05
6.7±0.2
ꢀ
3.6±0.1
3.5±0.2
6.9±0.2
2.36±0.09
0.19±0.03
3.9±0.3
4.7±0.4
1g
1h
1i
15.3±0.8
25.1±0.4
14.4±0.6
12.7±0.9
16.8±0.4
ꢀ
0.22±0.06
0.080±0.002
0.20±0.02
0.11±0.02
0.22±0.01
0.10±0.01
0.21±0.01
21.5±0.8
15.4±0.9
14.3±0.9
27.4±0.4
ꢀ
ꢀ
2j
ꢀ
2k
2l
1j
ꢀ
1k
1l
ꢀ
2m
3a
ꢀ
1m
2a
8.7±0.9
ꢀ
18.3±0.2
ꢀ: Percent inhibition was under 50% at a concentration of 50 ꢁM; the antibiotics used were cefazolin (a) and imipenem (b).
the rhodanine hydrolysis product thioenolate (3a) inhib-
its only specific MβLs, including VIM-212 and L1.
The IC50 data listed in Table 1 reveal a structure-activity
relationship (SAR), which is that the diaryl-substituted
rhodanines with electron-accepting atoms or groups (2h-
m), such as chlorine, fluorine, nitro and trifluoromethyl,
exhibit broad-spectrum inhibition of MβLs, and the N-
aromatic carboxyl (R1 in the 2 series) makes the inhibitors
more potent than the aliphatic carboxyl (R1 in the 1 series)
implying that the phenyl may interact with the hydro-
Fig. 2. Inhibition curves of 2l against NDM-1, VIM-2, ImiS, and
L1, where RA means residual activity.
phobic pocket of MβLs.
Table 2. Antibacterial Activities (MICs, ꢁg/mL) of Cefazolin or Imipenem against E. coli DH10B Expressing MβLs in the
Absence and Presence of Rhodanines at a Concentration of 32 ꢁg/mL (A), and Cefazolin MICs against E. coli Expressing L1
at a 2l and 2m Concentration Range of 16ꢀ256 ꢁg/mL (B)a
A
b
b
c
b
b
b
b
Inhibitors
Blank
Inhibitors
E. coliꢀImiSc
E. coliꢀNDMꢀ1
E. coliꢀVIMꢀ2
E. coliꢀImiS
E. coliꢀL1
E. coliꢀNDMꢀ1
E. coliꢀVIMꢀ2
E. coliꢀL1
8
512
2
32
2a
1
512
1
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
8
8
8
8
16
8
4
8
4
4
256
512
512
512
512
512
512
512
256
256
256
256
512
2
2
2
2
2
1
2
2
4
2
1
2
1
8
8
8
8
8
8
8
8
16
8
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
3a
4
4
2
>64
8
2
1
1
2
4
512
512
128
128
512
>512
128
128
256
128
128
128
256
1
1
2
1
1
0.5
1
1
1
1
8
8
8
8
8
4
8
8
8
8
4
4
16
1j
1k
1l
4
4
8
8
4
4
4
1
16
1
1
2
1m
B
Compd\Conc
0
16
32
64
4
128
2
256
2l
32
16
4
2
2m
32
8
4
2
2
1
a The MICs of cefazolin and imipenem alone against E. coli cells not expressing MβL were 1 and 0.125 ꢁg/mL, respectively. The antibiotꢀ
ics used were cefazolin (b) and imipenem (c).
The ability of the rhodanines to inhibit MβLs and to re-
store the antimicrobial activity of antibiotics was investi-
gated by determining the minimum inhibitory concentra-
tions (MICs) of existing antibiotics in the presence and
absence of 1a-m, 2a-m and 3a.21 E. coli DH10B cells ex-
pressing VIM-2, NDM-1, ImiS, or L1 from pBCSK plasmids
were used (Table 2A). The concentration of inhibitors
used was kept constant at 32 μg/mL.
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The MIC data indicate that all tested rhodanines 1a-m,
demonstrated previously in a crystal structure of Zn-
dependent carboxypetidase A in complex with 2-benzyl-3-
nitropropanoic acid.23 Also in the NDM-1/2l and CphA/2l
1
2
3
4
5
6
7
8
2a-m and the thioenolate 3a increased the antimicrobial
effect of cefazolin against E. coli expressing L1, and the
largest effect was observed to be from 1l, 1m, 2g, 2l and
2m, resulting in an 8-fold reduction in MIC. 2a, 2d, 2i-g
and 2m increased the antimicrobial effect of cefazolin
against E. coli producing NDM-1, resulting in a 4-8-fold
reduction in MIC. 2d-e, 2h-i and 2k-m also increased the
antimicrobial effect of cefazolin against E. coli harboring
VIM-2, resulting in a 4-fold reduction in MIC. Against E.
coli expressing ImiS only 2g had an effect larger than one
dilution factor.
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Given the excellent broad-spectrum inhibitory effect of
2l and 2m, dose-dependent MIC assays were performed
for these compounds against cells expressing L1 (Table
2B). The MIC data indicate that the antimicrobial effect of
cefazolin increased gradually with an increasing inhibitor
dose. At the highest dose of 2l and 2m tested (256 μg/mL),
the MICs of the antibiotic were decreased 16- and 32-fold,
nearly and completely, respectively, restoring the antibac-
terial effect of cefazolin in the absence of MβLs. No anti-
bacterial effect of the rhodanines alone against the E. coli
with and without MβLs was observed at the same inhibi-
tor doses, indicating that the rhodanines’ ability to restore
antibiotic activity is due to their inhibitory effect on the
MβLs. We further monitored the pH of the culture medi-
um during MIC assays (Fig. S4) and found that during the
first 20 hours it ranged from 6.6 to 7.1, a pH range at
which the rhodamine was shown to be stable in the sta-
bility assays (Fig. S3).
Also, the representative inhibitors 1l, 1m, 2l, 2m and 3a
were subjected to a cytotoxicity assay using mouse fibro-
blast cells (L929) with different working concentrations
(12.5, 25, 50, 100, 200 and 400 μM). As shown in Fig. S5,
more than 80% of the cells tested maintained viability in
the presence of the inhibitors at concentrations up to 200
μM (except the thioenolate 3a), indicating that these
rhodanines have low cytotoxicity and are not (or only to a
small degree) converted to thioenolates.
To explore potential binding modes, compound 2l
was docked into the active sites of NDM-1, VIM-2, CphA
(in lieu of ImiS, which has not been crystallized, yet, and
with which it shares 96% sequence identity), and L1. The
conformations shown in Fig. 3 are the lowest-energy con-
formations of those clusters, with binding energies of -
13.6, -13.1, -11.2 and -15.3 kcal/mol for the NDM-1/2l, VIM-
2/2l, CphA/2l and L1/2l complexes, respectively. Views of
2l in complex with the four enzymes represented as a sur-
face are shown in Fig. S7. They indicate that 2l fits very
tightly into the substrate binding sites of the four en-
zymes.
Fig. 3. Lowest-energy conformations of 2l docked into the
active sites of different enzymes. Graphs A-D show key elec-
trostatic interactions of 2l with Zn(II) ions and residues of
the MβLs NDM-1, VIM-2, CphA (as a proxy of ImiS) and L1,
respectively, indicated by dashed lines, while hydrophobic
interactions are shown in green. The capital letters A and B
following the amino acid residue numbers show the protein
chain A and chain B in the crystal structure. All 2D images
As shown in Fig. 3A and 3C (as well as Fig. S6A and
S6C), 2l adopted similar binding modes to NDM-1 and
CphA. The nitro group acted as a bidentate ligand of a
Zn(II) ion, and one oxygen of the nitro group formed hy-
drogen bonds with Asp124 in NDM-1 and Asp120 and
His196 in CphA. These residues are Zn(II) ligands in the
two enzymes.22 The nitro group as a zinc ligand has been
were
generated
with
PoseView
(www.biosolveit.de/PoseView/), and redrawn with ChemBi-
oDraw 14.0.
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Corresponding Author
complexes, the benzene ring at the R1 position formed a
1
2
3
4
5
6
7
8
π-π stacking with Phe70 in NDM-1 and a hydrophobic
interaction with Gly160 in CphA. In the complex VIM-2/2l
(Fig. 3B and S6B), the carboxyl group coordinated Zn2 (1.8
Å) and formed an H-bond with Asp118 (2.9 Å). Carbox-
ylate as a Zn(II) ion ligand has been reported24 and is also
seen in aspartate as a Zn(II) ligand in MβLs. In addition,
the nitro oxygen interacted with Gln60 in VIM-2 (2.9 Å)
via an H-bond, and the two benzene rings interacted with
Trp87 and Gln60 via hydrophobic interactions. In the
complex L1/2l shown in Fig. 3D and S6D, the nitro group
bridged the two Zn(II) ions (1.8 Å), which is reminiscent
of the binding mode of a micromolar inhibitor of the IMP-
*Tel/Fax: +8629-8153-5035. E-mail: kwyang@nwu.edu.cn;
Yuanhe@nwu.edu.cn.
Author Contributions
The manuscript was written through contributions of all
authors.
‡These authors contributed equally to this work.
Funding Sources
9
This work was supported by grants 81361138018 and 21572179
(to K. W. Y.) and 31400663 (to Y. H.) from the National Natu-
ral Science Foundation of China and by grant 2014JQ3090
from Natural Science Foundation of Shaanxi Science and
Technology Department.
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enzyme.25 Also, the carboxyl oxygen formed two hydro-
gen bonds with the backbone amide and side chain hy-
droxyl of Thr33 (2.5 Å), and the R1 benzene ring formed a
hydrophobic interaction with Tyr32.
Notes
The authors declare no competing financial interest.
In summary, twenty-six rhodanines and one thioenolate
were synthesized and characterized. Z-Configurations of
rhodanine were confirmed by X-ray crystal structure reso-
lution of 1l and 2m. Biochemical evaluation revealed that
all rhodanines tested strongly inhibited L1, exhibiting IC50
values ranging from 0.02 to 1.7 μM. Specifically 2h-m
showed broad-spectrum inhibition of all MβLs tested
(NDM-1, VIM-2, ImiS, and L1), with IC50 values < 16 μM.
SAR studies revealed that the diaryl-substituted
rhodanines with electron-accepting atoms or groups ex-
hibited broad-spectrum MβL inhibition, and an N-
aromatic carboxyl made the inhibitors more potent than
an aliphatic carboxyl. MIC tests indicated that all
rhodanines tested and a thioenolate enhanced the anti-
microbial effect of cefazolin againts E. coli expressing L1,
and the largest effect was observed to be from 1l, 1m, 2g,
2l and 2m, resulting in 8-fold reduction in MIC. Dose-
dependency assays showed that the antimicrobial effect of
cefazolin increased with increasing dose of inhibitors 2l
or 2m. Docking studies suggest that the nitro group
(NDM-1, CphA, and L1) or the carboxyl group (VIM-2) of
2l coordinates one or two Zn(II) ion(s), while the N-
phenyl of the inhibitor enhances its hydrophobic interac-
tion with the MβLs.
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Rhodanine as a potent scaffold for the development of broadꢀ
spectrum metalloꢀβꢀlactamase inhibitors
Yang Xiang,1‡ Cheng Chen,1‡ Wen-Ming Wang,1 Li-Wei Xu,1 Ke-Wu Yang,1* Peter Oelschlaeger,2 and Yuan He1*
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