Biochemistry Advanced

Read the Wang et al. PLOS article assigned. Briefly (5-9 sentences) answer the below questions.  Please type your answers.
Name __________________________________
Read the Wang et al. PLOS article assigned in class and available through the class blackboard page.  Briefly (4-9 sentences) answer the below questions.  Please type your answers.

  1. What was the main purpose of and justification for the study?
  2. What computational and experimental methods were used in the study?
  3. What are the main results of the study?
  4. What is main conclusion of the study?
  5. Is the conclusion justified? Specifically, do the methods and results support the conclusion?
  6. Is the conclusion important?
  7. Identify and comment on a strength of the article.
  8. Identify and comment on a weakness of the article.

Discovery of Novel New Delhi Metallo-β-
Lactamases-1 Inhibitors by Multistep Virtual
Xuequan Wang1‡
, Meiling Lu1‡
, Yang Shi1
, Yu Ou1
*, Xiaodong Cheng2
1 School of Life Science and Technology, China Pharmaceutical University, Nanjing, People’s Republic of
China, 2 Department of Integrative Biology & Pharmacology, The University of Texas Health Science Center,
Houston, United States of America
‡ These authors contributed equally to this work.
* (YO); (XC)
The emergence of NDM-1 containing multi-antibiotic resistant “Superbugs” necessitates
the needs of developing of novel NDM-1inhibitors. In this study, we report the discovery of
novel NDM-1 inhibitors by multi-step virtual screening. From a 2,800,000 virtual drug-like
compound library selected from the ZINC database, we generated a focused NDM-1 inhibitor
library containing 298 compounds of which 44 chemical compounds were purchased
and evaluated experimentally for their ability to inhibit NDM-1 in vitro. Three novel NDM-1
inhibitors with micromolar IC50 values were validated. The most potent inhibitor, VNI-41, inhibited
NDM-1 with an IC50 of 29.6 ± 1.3 μM. Molecular dynamic simulation revealed that
VNI-41 interacted extensively with the active site. In particular, the sulfonamide group of
VNI-41 interacts directly with the metal ion Zn1 that is critical for the catalysis. These results
demonstrate the feasibility of applying virtual screening methodologies in identifying novel
inhibitors for NDM-1, a metallo-β-lactamase with a malleable active site and provide a
mechanism base for rational design of NDM-1 inhibitors using sulfonamide as a
functional scaffold.
Antibiotics being used to treat or prevent infectious disease have revolutionized the practice of
medicine. Without them numerous modern therapies such as organ transplantation and cancer
chemotherapy would simply not be possible [1]. Unfortunately, overuse and/or misuse of
antibiotics in farming and clinical practices has resulted in rising multidrug-resistance bacterial
strains, among which gram-negative bacteria producing β-lactamases become the most prevalent
[2–4]. According to the U.S. Centers for Disease Control and Prevention, more than two
million people are affected by infectious diseases with antibiotic resistance and at least 23,000
people died each year in the United States [5].
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 1 / 17
Citation: Wang X, Lu M, Shi Y, Ou Y, Cheng X
(2015) Discovery of Novel New Delhi Metallo-β-
Lactamases-1 Inhibitors by Multistep Virtual
Screening. PLoS ONE 10(3): e0118290. doi:10.1371/
Academic Editor: Horacio Bach, University of British
Columbia, CANADA
Received: July 20, 2014
Accepted: January 12, 2015
Published: March 3, 2015
Copyright: © 2015 Wang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the National
Natural Science Foundation of China (No: 81302795
and No: 81328023). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: Xiaodong Cheng is an AE.
This does not alter the authors’ adherence to PLOS
ONE Editorial policies and criteria.
β-lactamases have been classified into four classes (A-D) based on their structures (Ambler
classification) [6–8], among which the class B enzymes also known as metallo-β-lactamases
(MBLs) requiring bivalent metal cation, normally Zn2+, as cofactors are further classified into
B1, B2, and B3 subclasses [9]. B1 and B3 MBLs containing two zinc binding sites exhibit a
broad substrate spectrum profile including the last antibiotic defense lines carbapenems, therefore
pose a looming pandemic threat [10]. One typical example is the global dissemination of
bacteria harboring B1 subgroup member New Delhi metallo-β-lactamase (NDM-1). These bacteria
often carry several different resistance genes in addition to NDM-1 gene, blaNDM-1, and
are resistant to almost all antibiotics and only partially susceptible to colistin, tigecycline, and
fosfomycin, creating enormous challenges in managing these multi-resistance “Superbugs”
[11–13]. In addition, colistin and tigecycline resistant NDM-1 harboring bacteria have been reported
[14–16] and NDM-1 gene has been isolated in more than 11 bacterial species from natural
environment [17–19]. Given the biomedical importance role of MBLs, development of
MBLs inhibitors become an urgent need. High throughput screening (HTS) and virtual screening
(VS) are the two main methods to identify novel scaffolds for drug discovery. Indeed, HTS
has successfully identified a number of MBLs inhibitors [20–22], yet structure-based drug design
and virtual screening have not been widely applied in MBLs inhibitors development [23].
Since the force field and zinc parameter has been optimized for metalloenzymes, molecular
docking has proved to be a feasible way to found inhibitors or predict actual substrates of
metalloenzymes structures [24–28]. Five inhibitors of CcrA, a B1 subclass of MBLs, with apparent
Ki values less than 120 μM have been screened based on virtual screen method [29]. Recent
high-resolution x-ray crystallographic analyses of multiple three-dimensional structures
of NDM-1 reveal that it shares a common structural-fold with other B1 MBLs [30–34]. In addition,
all three subclasses of MBLs share a common substrate hydrolysis mechanism [31]. These
findings suggest that discovery of NDM-1 inhibitors via structure-based design and in silica
screening may be productive. The aim of this study is to identify novel inhibitors of NDM-1
using virtual screening methods.
Materials and Methods
Bacterial strains and plasmids
MBL DNA sequences of NDM-1, VIM-2 and SIM-1 lacking the signal sequences were codonoptimized
for expression in E. coli, chemically synthesized and inserted into pUC-19. Sequencing-validated
MBL genes were further cloned into the pET28a expression vector using the
NcoI/XhoI sites. E. coli DH5α (ATCC 53868) was used routinely as host for molecular cloning
and plasmids amplifying while E. coli BL21 (DE3) was used for MBLs expression. Bacteria were
grown in Luria–Bertani (LB) medium supplemented with appropriate antibiotics.
Protein expression and purification
Recombinant NDM-1, VIM-2 and SIM-1 proteins were induced to express in E. coli BL21 (DE3)
cells by 0.1 mM IPTG for 10 h at 2°C when the optical density (OD600 nm) reached 0.7–0.8.
Cells were harvested and cell lysate was prepared by sonication at 4°C. The protein expression
levels in soluble and insoluble fractions were analyzed by 12% SDS-PAGE after ultracentrifugation.
Individual MBL was purified from the lysate supernatant using Ni2+-affinity column (Bio
Basic Inc, Markham, Canada). All three recombinant proteins showed an abundant expression
after induction for 10 h and could be purified with an estimated purity around 95% (Figure A in
S1 File). MBL activity analysis was carried out using the nitrocefin assay at 30°C in 300 μL
HEPES buffer (30 mM HEPES, 10 μM ZnCl2, 100 mM NaCl, 20 μg/mL BSA, pH 6.8) at 482 nm
with a UV-2400PC spectrophotometer (Shimadzu, Tokyo, Japan). The Michaelis constants,
Discovery of Novel NDM-1 Inhibitors
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determined under initial velocity conditions by Lineweaver-Burk plot, for NDM-1, Vim-2 and
SIM-1 were 9.54 ± 0.43 μM, 14.48 ± 0.68 μM and 31.3 ± 0.24 μM, respectively. These values are
consistent with those previously reported [33, 35].
Selection and preparation of structure models
22 reported NDM-1 X-ray crystallographic structures were analyzed [30–34,36] (Table A in
S1 File) using protein alignment and superpose biopolymer module in Molecular Operating
Environment suit (MOE, version 2009.10; Chemical Computing Group Inc; Montreal, QC,
Canada) or the Protein Model Portal (PMP) [37] to facilitate the structure-based virtual screening.
Structure 3Q6X (Figure B.A in S1 File) with a resolution value of 1.30 Å was selected for
the screening process. The structural file contains two almost identical NDM-1 molecules with
an RMSD value of 0.21 Å for Cα atoms [31]. The second structure after removing ligands and
non-conserved water molecules in the active site, was processed for Protonate 3D and Energy
Minimize using MOE. All hydrogen atomic coordinates were refined by the conjugate gradient
method using the MMFF94x (Merck Molecular Force Field 94x) force field [38]. Other 21
NDM-1 structures were also processed with ligand and solvent deletion, protonate 3D and energy
minimization using the same parameters and superposed together.
Initial virtual screening
Hydrolyzed ampicillin, L-captopri, ampicillin and other 9 β-lactams (cefepime, cefotaxime, ceftazidime,
cefuroxime, faropenem, imipenem, meropenem, penicillin G, piperacillin) structures
downloaded from ZINC database were docked into the NDM-1 active site using different
docking simulations in MOE and docking protocols in Discovery Studio (ADS, version 2.5;
Accelrys Inc, San Diego, USA) according to the following procedure: the docking box was generated
around the active site using the site finder module in MOE (Figure B.B in S1 File). The
dimensions of the docking box were manipulated to accommodate all the amino acid residues
present in the active site. Default parameters were used for all computational procedures unless
otherwise stated.
A virtual collection drug-like compounds subset taken from ZINC database containing
2,800,000 compounds was served as the screening library [39]. The hits with firm binding conformations
were collected and redocked into the active site using the libdock protocol in ADS.
Those compounds with high libdock scores were selected as a focused library used for the
further analysis.
Docking results analysis
Energy calculations and analysis of docking poses were performed on MOE. The resulting protein-inhibitor
or protein-β-lactam complexes were analyzed using the protein–ligand interaction
fingerprint (PLIF) implemented in MOE [40]. The hydrolyzed ampicillin and NDM-1
residue interaction energies were calculated for the docked pose with the least RMSD value, assigning
energy terms in kcal mol-1 for each residue. LigX-interaction application was used to
provide ligand-interaction diagram to understand the binding type of those docked hits [41].
A 96-well assay for NDM-1 inhibitor screening
Preliminary screening of the selected compounds was performed in 96-well plates using
nitrocefin as a substrate. Final assay conditions include compounds (30 μM), NDM-1 (1
nM), HEPES (30 mM), ZnCl2 (10 μM), NaCl (100 mM), BSA (20 μg/mL) at pH 6.8. EDTA
(30 μM) was used as a positive control. After incubation at 30°C for 20 min, nitrocefin
Discovery of Novel NDM-1 Inhibitors
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hydrolysis (100 μM) was monitored by following absorbance readings at 490 nm using a PR
4100 Microplate Reader (BIO-RAD; USA). The assay was performed in quadruplicate for all
compounds and controls.
IC50 Determination
Ten different concentrations of compounds VNI-24, VNI-34 and VNI-41 ranging from 0 μM
to 45.0 μM were used to determine the half-maximal inhibitory concentration (IC50) against
NDM-1 (1 nM) using nitrocefin (20 μM) as substrate. The assay was performed in the buffer
for inhibitor screening in the presence or absence of 0.01% Triton X-100 [42]. Each data point
was performed in quadruplicate and the inhibition data were analyzed by a standard dose response
curve fitting in the Origin 8.0 software.
Analysis of NDM-1/VNI-41 complexes by molecular dynamics study
NDM-1 and VNI-41 after optimized with partial charge were then subjected to molecular dynamics
simulation (MD) employing the NVT (N = constant number, V = volume, and T =
temperature) statistical ensemble and Nosé-Poincaré-Andersen (NPA) algorithm with the periodic
boundary conditions applied to analysis stability of binding model of the compound.
The complex was solvated in water molecules in a sphere mode with a 10 Å width layer. The
molecular dynamics simulations were performed at a temperature of 310 K for 2000 ps. The
data of position, velocity and acceleration were saved every 0.5 ps.
Results and Discussion
NDM-1 structural superposition and optimization
Structural superposition of the 22 reported NDM-1 structures using force realignment and refined
with gaussian distance weight showed that most of the independently solved structures
(except 3S0Z) shared a high degree of structural similarity with each other (Figure 1A, 1B,
Figure C in S1 File). The average pair-wised RSMD for all atoms in these structures is 0.801 Å
(Fig. 1A) and 3Q6X showed a high similarity with 4EYF, 4HOD, 4HL1, 4HL2, 4EYB and 4EY2
with RMSD values below 0.3 Å (Fig. 1A, Table A in S1 File). A notable variation is the distance
between zinc ions ranging on average from 3.48 to 4.6 Å (Table A in S1 File, Figure B.C
in S1 File). This indicates that the metal ions are relative flexible to move within the active site.
While NDM-1 structures complexed with hydrolyzed antibiotics share a greater metal-ion separation
(4.53 ± 0.11Å, 3Q6X 4.59 Å) with a slight outward flexing of His120 and His122, the
binuclear Zn distance appears to be significantly less (3.72 ± 0.17Å, p < 0.001) in most apoNDM-1
structures (Table A in S1 File). Since the binuclear Zn distance is compatible with μ-
η1:η1carboxylate coordination, such distance changes also prevail in all other binuclear Zn
MBLs [43], and this inherent flexibility of metal ions in the active site is likely important for
substrate binding and turnover [36,44]. 4H0D and 4HL1, where the Zn ions replaced by Mn2+
or Cd2+ showed a similar hydrolyzed ampicillin binding framework as 3Q6X [45]. A detailed
RMSD analysis for 3Q6X reveals that residues in loop L3 (Leu65-Gly73) adjacent to the active
site possess greatest variation (Fig. 1C). Moreover, L3 displayed the greatest deviations with
the exception of the N terminal signal peptide when structures with hydrolyzed antibiotics
(4HKY, 4EY2, 4EYF, 4HL1, 4HL2, 4H0D. 4EYB and 3Q6X) were superposed with apo structures
while L3 showed less difference among 3Q6X, 4HL2 (Hydrolyzed Ampicillin), 4EYB (Hydrolyzed
Oxacillin), 4EYF (Hydrolyzed Benzylpenicillin) and 4EY2 (Hydrolyzed Methicillin)
(Figure C.C & C.D in S1 File). These results suggest that L3 is involved in substrate binding in
NDM-1. Loop L10 (Gly205-His228) showed the subordinate deviation after L3 among NDM-1
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structures (Fig. 1C, Figure C.C in S1 File). Asn220 in L10 interacts with Zn1 to provide an
oxyanion hole in polarizing the lactam carbonyl upon binding, and facilitates nucleophilic attack
by the adjacent hydroxide [32]. Regions of Ala121–Met129 flanking NDM-1 active site
Fig 1. Comparative analyses of 21 published NDM-1 X-ray crystal structures. (A) Pairwise RMSD matrix table of NDM-1 structures superimposed with
force realignment method and refine with Gaussian Weights in MOE. PDB codes for structures with hydrolyzed substrate in the active site are highlighted in
red. (B) Superposition of the 22 NDM-1 structures. 3S0Z, 4GYU, 4GYQ, 3SPU, and 3Q6X2 are highlighted in thick line and colored as shown in the index
panel. (C) The RMSD-residue index 3D waterfall plots of NDM-1 structures compared with 3Q6X structure. (D) Superimposition of the active site among the
reported NDM-1 structures (without 3S0Z and NDM-1 mutants 4GYQ and 4GYU) showing the metal chelating residues (Oliver) and conserved water
molecules (Red) in the active site of NDM-1 structures. Residues from 3Q6X are highlighted in green.
Discovery of Novel NDM-1 Inhibitors
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also showed a slightly difference among NDM-1 structures especially when NDM-1 mutants
4GYQ (D223A) and 4GYU (A121F) superposed with 3Q6X (Figure C.D in S1 File). A detailed
analysis showed that the loop spanning residues Phe163–Asn176 in 3S0Z adopt a very different
conformation from those observed in the other structures, and the so called “ceiling” region in
3S0Z is a loop while in most other reported structures it is an α-helix (Figure C.F in S1 File).
Quantitative stability-flexibility relationship analysis revealed that NDM-1 had several regions
with significantly increased rigidity when compared with other four B1 MBLs [46]. In most
NDM-1 structures (except mutants and 3S0Z), RMSD of these regions were blow 0.5 Å
(Figure C.A in S1 File). These evolutionary traits of NDM-1, with more rigid regions out of
the active site together with the more plastic and more hydrophobic L3 loop [31] as compared
to other MBLs, may provide more flexibility to accommodate a broader spectrum of substrates.
Based on our detailed analysis, many NDM-1 structures shares some identical waters in the
active site (Fig. 1D), which may play a role in the overall structure stability or in substrate
binding and product turnover. The bridging water in the active site showed different distances
among these structures, a likely consequence of change in distance between the metal ions
(Figure B.D in S1 File) or the pH conditions that the protein crystallization were used. Unlike
most other MBLs, NDM-1 functions well at high pH conditions [47]. Our analyses suggest that
3Q6X is of high resolution and possesses a high degree of the structural similarities with other
NDM-1 structures, therefore is suitable for docking and screening studies.
Molecular docking
Hydrolyzed ampicillin, L-captoril, ampicillin and other 9 β-lactams (cefepime, cefotaxime, ceftazidime,
cefuroxime, faropenem, imipenem, meropenem, penicillin G, piperacillin) were
docked into NDM-1 active site using different docking simulations in MOE and docking protocols
in ADS to evaluate the ability of these programs to reproduce the experimental binding
modes. For all programs the binding modes of the docked hydrolyzed ampicillin structures
were found in a narrow range of RMSDs (Fig. 2A). The RMSDs of hydrolyzed ampicillin were
1.53–2.07 Å, 1.86–2.62 Å, 1.98–2.78 Å and 1.79–2.31 for Triangle Matcher, Alpha PMI, Alpha
triangle, Proxy triangle placement in MOE respectively, while 1.46–2.65 Å for libdock in ADS
receptor-ligand interactions protocols. In general, poses with an RMSD < 2 Å are considered a
success, and dockings with RMSDs between 2 and 3 Å are considered a partial success [48].
For L-captopril, the RMSD values between poses docked into the active sites and the determined
ligand structure in 4EXS arranged from 0.72 to 2.03 Å and the 2D interaction map of
the docked L-captopril was similar to that in 4EXS (Fig. 3). Hydrolyzed ampicillin-residue interaction
energies for the best docked pose and the structure reference were calculated [49].
The interaction of best docked hydrolyzed ampicillin and ampicillin showed similar interactions
as revealed by the X-ray structure (Fig. 2B, 2C, 2D). Among the conserved residues in
the active site, Leu65, Gln123, Asp124, His189, Cys208, Lys211, Asn220 interact with the hydrolyzed
ampicillin in both the structure complex and the docked pose. PLIF analysis showed
that Gln123, His189 and Asn220 interacted with the docked hydrolyzed ampicillin at a high
frequency (Fig. 4A). The residue interaction energies between NDM-1 and hydrolyzed ampicillin,
and the 2D interaction map (Fig. 2E) well defined the dock results.
After ampicillin and other β-lactams were docked into the active site, the 2D binding pattern
of these binding pose were analyzed by the ligX-interactions and RLIF. 404 docked poses of 10
different β-lactams showed that those substrates interacted with His120, His122, Asp124,
His189, Lys211, Ser249, Asn220, Zn1 and Zn2 at a high frequency compared with other residues
around the docking site. On the other hand, other residues Ile35, Phe70, Asp212 and
Ser217 interacted less frequently (Fig. 4B). Docking poses also formed the inhibited
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Fig 2. Docking of the hydrolyzed ampicillin in the active site of NDM-1. (A) Molecular surface of NDM-1
(PDB 3Q6X) active site with docked hydrolyzed ampicillin. The structurally determined hydrolyzed ampicillin
is shown in gray stick representation while docked poses are shown in colored stick. 2D ligand-protein
interaction maps showing the detailed binding pattern of structurally determined hydrolyzed ampicillin (B),
docked hydrolyzed ampicillin (C) and docked ampicillin (D) in the active site of 3Q6X. (E) Residue-ligand
interaction energies between NDM-1 (3Q6X) and hydolyzed ampicillin (vdw_ref) or docked hydolyzed
ampicillin (vdw_pose). The hydrolyzed ampicillin and NDM-1 residue interaction energies were calculated for
the best pose (RMSD = 1.53 Å).
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conformers at a low frequency (< 10%), in which the carboxylic group of β-lactams coordinated
with two zinc ions and kept the amide group away from the metal ions as described before
[50]. These findings, along with the high flexibility of NDM-1 active site, is consistent with
NDM-1’s broad substrate spectrum, as well as the fact that most reported MBL inhibitors interact
with Zn2+ or Zn2+ chelating residues [51].
Structure-based screening and analysis
Triangle Matcher placement method, followed by molecular mechanics refinement and scoring,
was used for the first round docking based screening process. The placement stage was
scored by E_place. Binding free energy values (Gbinding) was quantified using London dG [52]
and Affinity dG [53]. After 1000 binding orientations for each compounds were refined, 30
Fig 3. L-captopril docked in the active site of NDM-1. 2D ligand-protein interaction maps showing the detailed binding pattern analysis of structurally
determined (A) and docked (B) L-captopril. (C) Molecular surface of NDM-1 active site with docked hydrolyzed ampicillin.
Discovery of Novel NDM-1 Inhibitors
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conformations with lowest binding free energy, lowest affinity dG and London dG values was
produced. In the screening process, we adopted the strategy that the most anticipated hits
would exhibit the desirable scores in all the evaluation algorithms and be in conformity with
screening threshold of different screening methods.
Docking poses without major clashes were scored for receptor complementarity and were
further screened using the criteria that affinity dG value was less than -10 kcal/mol and that
london dG was less than -20 kcal/mol. E_refine for refinements using GridMIn was limited to
190 kca1/mol. E_conf, the energy of the conformer calculated at the end of the refinement was
Fig 4. PLIF analysis of the docking process. The interaction frequency of individual residue with the
docking poses of (A) hydolyzed ampicillin; (B) 10 beta-lactams (ampicillin, cefepime, cefotaxime, ceftazidime,
cefuroxime, faropenem, imipenem, meropenem, penicillin G, piperacillin); (C) 298 virtual hit compounds.
Each columns of every residue are denoted by some of the following characters to indicate the interaction
role of each residue: side chain hydrogen bond acceptor, backbone hydrogen bond donor, backbone
hydrogen bond acceptor, solvent hydrogen bond, ionic attraction or surface contact to the atom of the
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confined to less than 9 kcal/mol (Figure C.D in S1 File). 2218 compounds satisfied these filter
settings were selected from the database.
In the second round of screening, the receptor-ligand interactions protocols of ADS 2.5
were used to dock these 2218 compounds into the docking box of NDM-1. For each ligand, another
30 different conformations for each compound were generated by the libdock process.
On the basis of the docking scores, the compounds were ranked, and 1388 conformations of
298 compounds with libdock score above 150, Absolute Energy under 200 kcal/mol; Relative
Energy under 25 kcal/mol were selected.
The 1388 screened conformations displaying in a camel-like appearance (Figure D.A, D.B &
D.C in S1 File) were further analyzed by PLIF. During the PLIF screening, we focused on interactions
with His122, His189, Asn220, His250, Zn1, and Zn2 because all these elements showed
high interaction frequency in the β-lactam based docking (Fig. 4B). 1,388 conformations (poses)
of 298 compounds satisfied with above specific binding requirement were selected as a focused
library, and most of which also interact with Ile35, Gln123, Asp124, Lys211, Ser217, Gly219 and
Ser251 at a high frequency (Fig. 4C). In addition, these molecules were inspected visually for features
not captured in the docking calculation.
Biological activity analysis of the screened compounds in vitro
Based on the docking scores, chemical diversity, 2D ligX-interactions map, commercial availability,
and an overall balance between polar and nonpolar complementarity to the binding site, 44
molecules (Table B in S1 File) were ultimately selected and purchased from the ChemDiv (San
Diego, CA) for experimental validation using in vitro assays. When the selected 44 chemicals
were tested for their ability to inhibit NDM-1 activity, eleven compounds showed more than
25% inhibition at 30 μM concentration. Among these eleven compounds VNI-24, VNI-34, and
VNI-41 inhibited NDM-1 by more than 50% at 53.2% ± 2.2%, 56.5% ± 2.6%, and 56.8% ± 3.0%,
respectively (Fig. 5A). Dose-dependent analyses further revealed that compounds VNI-41, VNI34
and VNI-24 inhibited NDM-1 with an apparent IC50 value of 29.6 ± 1.3 μM, 31.4 ± 1.2 μM,
or 37.6 ± 0.9 μM, respectively (Fig. 5B, 5C). Similar results were obtained using buffer containing
0.01% Triton X100 (Figure E in S1 File).
Activity of the three compounds against VIM-2 and SIM-1 was also tested. Within the aqueous
solubility limit of these compounds (Table C in S1 File), none of the three compounds
showed significant inhibition for SIM-1. While 45 μM VNI-24 and VNI-41 inhibited VIM-2
activity by 19.6% ± 3.1% and 34.2% ± 5.2%, respectively, VNI-34 was ineffective in blocking
VIM-2 activity. These results suggest that VNI-24, VNI-34 and VNI-41 are selective NDM-1
inhibitors capable of discriminating among various MBLs. Taken together, our study shows
that it is feasible to develop novel NDM-1 specific inhibitors via in silico screening.
Molecular dynamic study of the NDM-1/VNI-41 complex
To investigate stability of the active site cavity in response to the binding of VNI-41, the most
potent NDM-1 inhibitor validated in our study, MD simulations were performed. RMSD for
zinc ions, VNI-41 and the active site atoms (atoms in Fig. 1D) of NDM-1 from their initial positions
(t = 0) was calculated. Overall, the RMSD values of NDM-1 active site fluctuated from
0.5 to 2.5 Å and reached a steady state (Fig. 6A) that the systems were equilibrated and the predicted
pose of each inhibitor was compatible with the pocket in the catalytic cavity of NDM-1
structure. Close examination of MD simulation snapshots (N = 10, with different time intervals)
of the VNI-41/NDM-1 complex relative to the original pose revealed a coordinated movement
of L3, L10 and L12 around the active site (Fig. 7A, 7B). The distance of the zinc ions
maintained a steady state during the dynamic simulation and the RMSD of the zinc was less
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Fig 5. Experimental validation of selected virtual screening hits. (A) Percent inhibition of NDM-1 activity in the presence of 30 uM individual compounds.
Data are presented as mean ± standard deviation (n = 4). (B) Dose-dependent inhibitions of NDM-1 by VNI-24, VNI-34 and VNI-41 against NDM-1. Each
data point indicated the remaining activity of NDM-1 after incubated with inhibitors, and were presented as mean ± standard deviation (n = 4). (C) Structures
of three active compounds.
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Fig 6. Molecular dynamic profile of NDM-1 and VNI-41 complex. (A) RMSD of two Zn2+, conserved H2O,
VNI-41, and active site (atoms shown in Fig. 1D) spanning the 2000 ps molecular dynamic simulation
process; (B) The overall molecular surface of NDM-1 colored by activeLP (white, hydrophobic; blue, polar;
red, H-Bonding); (C) The 2D interaction map VNI-41 in the active site of NDM-1 at 1500 ps after the MS
reached equilibrium; (D) Molecular surface of NDM-1 around the VNI-41 binding site with a cutoff limit of 4.5 Å
(black, hydrophilic; purple, neutral; green, lipophilic). VNI-41 and adjacent NDM-1 residues shown in stick
representation with carbon atoms colored by green and dark yellow respectively.
Discovery of Novel NDM-1 Inhibitors
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 12 / 17
Fig 7. Structure movements during molecular dynamic simulation process. Overlaid snapshots of the
ribbon diagrams of NDM-1 Cα atoms and VNI-41 compound around the active site before (A) (snapshots
interval 10 ps, N = 10) and after (B) (snapshots interval 150 ps, N = 10) the system reached equilibrium.
Surface analyse of the active site cavity was performed on apo NDM-1(C), NDM-1/hydrolyzed ampicillin (D)
and NDM-1/NVI-41(E). Atoms in the active site active site cavity are highlighted in colored balls. (F) The
ribbon diagrams showing the active site associated loops (L3, L6, L10) moving toward the ligand and
contraction of the active site. The structure of apo NDM-1, NDM-1/hydrolyzed ampicillin and NDM-1/NVI-41 is
colored in blue, green and black respectively.
Discovery of Novel NDM-1 Inhibitors
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 13 / 17
than 0.5 Å. While the ligand underwent a maximal change with a RMSD value of 1.6 Å, the active
site showed a change with a RMSD value about 2.0 Å. The conservative water in the active
site fluctuated and this may be caused by the solvent used to dissolve NDM-1 in the MD simulation
competing with the water kept before MD simulation (Fig. 6A).
It is reported that the expanded cavity volume of the active site in the surrounding loops
(Loop L1, L3, L10 and L12 in Figure B.A in S1 File) is important for the broad substrate spectrum
of NDM-1 [33]. To investigate residue movement in the active site cavity in response to
VNI-41 binding, a surface analysis was performed on apo NDM-1 and NDM-1/NVI-41 complex
after MD simulation[54]. Apo NDM-1 has the largest cavity (surface area = 392.4 Å2
; volume
= 693.2 Å3
) while NDM-1-hydrolyzed ampicillin complex has a smaller cavity after
removing the ligand (surface area = 376.2 Å2
; volume = 633.5 Å3
). Active site cavity of NDM1-VNI-41
complex is the smallest after removing the docked ligand (surface area = 345.3 Å2
volume = 596.3 Å3
) (Fig. 7C, 7D, 7E). Our study shows that VNI-41 clamped into the groove
surrounded by active site, induced L3 and L10 movement and narrowed the active cavity
(Fig. 7F).
Interactions between NDM-1 and compound VNI-41 among the MD generated steady conformations
during MD simulation were analyzed. The benzoxadiazole moiety binding to
NDM-1 hydrophilic site adopted an appropriate conformation with the double π-π stacking interactions
with His122, and the ring-to-ring distances were 3.03 and 2.79 Å for the five member
ring and the six number ring interacted with His122, respectively (Fig. 6B, Fig. 6C). Moreover,
one oxygen atom from the sulfonamide group interacted with Zn2 via a metal contact (score
100%, distance 2.5 Å), forming a solvent contact with the bridge water (H2O in the 2D interaction
map) (score 33%, distance 3.0 Å), and ionic contacted with His122, His120 and His189
(score 61%, distance 1.9 Å; score 42%, distance 1.8 Å; score 25%, distance 1.8 Å). The other oxygen
atom interacted with Asn220 (score 69%, distance 2.06 Å). The naphthalene group
clamped into hydrophobic groove around the active site and contacted with His250 and Ile30
(Fig. 6D). Mechanisms study has showed that the bridging hydroxide-zinc serving as the general
base while a surrounding water molecule serving as the nucleophile responsible for the nucleophilic
attack which results in a negatively charged intermediate stabilized by oxyanion hole
of NDM-1 [45]. In the conformation of VNI-41 interacting with NDM-1, the bridge water
formed solvent contacting with oxygen atom from sulfonamide group may prevent the proton
transfer from the surrounding water to the bridging water in the active site.
To date, various sulfamide/sulfonamide/sulfamate containing metalloenzyme inhibitors,
such as diuretic and antiglaucoma agents (acetazolamide, methazolamide, dichlorophenamide,
and brinzolamide), have been clinically used to inhibit carbonic anhydrases [55]. Sulfamide/
sulfonamide/sulfamate containing MBL inhibitors have also been reported. The crystal structure
of 4-nitrobenzenesulfonamide interacting with BJP-1, a B3 subclass MBLs, reveals that
binding of sulfonamide changes coordination number and geometry for Zn1 by adding one oxygen
atom of sulfonamides to the Zn2 and the nitrobenzene moiety form a hydrophobic pocket
in the active site [56]. MBL inhibitor DansylCnSH can be docked and shown to interact with
the core region of the active site of IMP-1 via sulfamide [57]. Since the zinc ions of NDM-1 are
essential for catalytic activity and participate directly in the catalysis, the ability of the sulfonamide
group of VNI-41 to interact with the Zn ion in the active site of NDM-1 suggests that
sulfamide/sulfonamide/sulfamate containing compounds may represent promising leads for
developing clinically effective NDM-1 inhibitors.
In summary, we have identified novel inhibitors of NDM-1 using a multistep docking methodology.
Dynamic and ligX-interaction analyses have revealed that VNI-41 interacts with Zn1.
This study has demonstrated the feasibility of identifying inhibitors of NDM-1 with a plastic
active site by virtual screening. Further investigations and future modifications studies for
Discovery of Novel NDM-1 Inhibitors
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 14 / 17
rational design of NDM-1 inhibitors using sulfonamides as a functional scaffold will lead to a
better understanding of their exact mechanism of action, laying a solid foundation for further
structure-based hit-to-lead optimization.
Supporting Information
S1 File. Supporting Information.
Author Contributions
Conceived and designed the experiments: XW ML YO XC. Performed the experiments: XW
ML YS. Analyzed the data: XW ML YS YO XC. Wrote the paper: XW ML YS YO XC.
1. Morar M, Wright GD. The genomic enzymology of antibiotic resistance. Annu Rev Genet. 2010; 44:
25–51. doi: 10.1146/annurev-genet-102209-163517 PMID: 20822442
2. Fisher JF, Meroueh SO, Mobashery S. Bacterial resistance to beta-lactam antibiotics: compelling opportunism,
compelling opportunity. Chem Rev. 2005; 105: 395–424. PMID: 15700950
3. Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology,
and detection of this important resistance threat. Clin Microbiol Rev. 2001 14: 933–951. PMID:
4. Laxminarayan R, Duse A, Wattal C, Zaidi AKM, Wertheim HFL, Sumpradit N, et al. Antibiotic resistance—the
need for global solutions. The Lancet Infectious Diseases 2013; 13: 1057–1098. doi:
10.1016/S1473-3099(13)70318-9 PMID: 24252483
5. U.S. Centers for Disease Control and Prevention. Antibiotic resistance threats in the united states.
Accessed 2013 Apr 23.
6. Ambler RP. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci. 1980; 289: 321–
331. PMID: 6109327
7. Jaurin B, Grundstrom T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary
origin from that of beta-lactamases of the penicillinase type. Proc Natl Acad Sci U S A. 1981; 78: 4897–
4901. PMID: 6795623
8. Dale JW, Godwin D, Mossakowska D, Stephenson P, Wall S. Sequence of the OXA2 beta-lactamase:
comparison with other penicillin-reactive enzymes. FEBS Lett. 1985; 191: 39–44. PMID: 3876949
9. Bush K. Metallo-beta-lactamases: a class apart. Clin Infect Dis. 1998; 27 Suppl 1: S48–53. PMID:
10. Galleni M, Lamotte-Brasseur J, Rossolini GM, Spencer J, Dideberg O, Frere JM, et al. Standard numbering
scheme for class B beta-lactamases. Antimicrob Agents Chemother. 2001; 45: 660–663. PMID:
11. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al. Characterization of a new metallobeta-lactamase
gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic
structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother. 2009;
53: 5046–5054. doi: 10.1128/AAC.00774-09 PMID: 19770275
12. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a
new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological
study. Lancet Infect Dis. 2010; 10: 597–602. doi: 10.1016/S1473-3099(10)70143-2 PMID:
13. Karthikeyan K, Thirunarayan MA, Krishnan P. Coexistence of blaOXA-23 with blaNDM-1 and armA in
clinical isolates of Acinetobacter baumannii from India. J Antimicrob Chemother. 2010; 65: 2253–2254.
doi: 10.1093/jac/dkq273 PMID: 20650909
14. Arpin C, Noury P, Boraud D, Coulange L, Manetti A, Andre C, et al. NDM-1-producing Klebsiella pneumoniae
resistant to colistin in a French community patient without history of foreign travel. Antimicrob
Agents Chemother. 2012; 56: 3432–3434. doi: 10.1128/AAC.00230-12 PMID: 22450982
15. Stone NR, Woodford N, Livermore DM, Howard J, Pike R, Mushtag S, et al. Breakthrough bacteraemia
due to tigecycline-resistant Escherichia coli with New Delhi metallo-beta-lactamase (NDM)-1
Discovery of Novel NDM-1 Inhibitors
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 15 / 17
successfully treated with colistin in a patient with calciphylaxis. J Antimicrob Chemother. 2011; 66:
2677–2678. doi: 10.1093/jac/dkr337 PMID: 21846669
16. Wailan AM, Paterson DL. The spread and acquisition of NDM-1: a multifactorial problem. Expert Rev
Anti Infect Ther. 2014; 12: 91–115. doi: 10.1586/14787210.2014.856756 PMID: 24308710
17. Isozumi R, Yoshimatsu K, Yamashiro T, Hasebe F, Nguyen BM, Ngo TC, et al. bla(NDM-1)-positive
Klebsiella pneumoniae from environment, Vietnam. Emerg Infect Dis. 2012; 18: 1383–1385. doi: 10.
3201/eid1808.111816 PMID: 22840532
18. Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New
Delhi environment and its implications for human health: an environmental point prevalence study. Lancet
Infect Dis. 2011; 11: 355–362. doi: 10.1016/S1473-3099(11)70059-7 PMID: 21478057
19. Shahid M. Environmental dissemination of NDM-1: time to act sensibly. Lancet Infect Dis. 2011; 11:
334–335. doi: 10.1016/S1473-3099(11)70074-3 PMID: 21478055
20. Schneider G. Virtual screening: an endless staircase? Nat Rev Drug Discov. 2010; 9: 273–276. doi:
10.1038/nrd3139 PMID: 20357802
21. Rollinger JM, Stuppner H, Langer T. Virtual screening for the discovery of bioactive natural products.
Prog Drug Res. 2008; 65: 213–249.
22. Attene-Ramos MS, Austin CP, Xia M. High Throughput Screening. In: Wexler P, editor. Encyclopedia
of Toxicology ( Third Edition). Oxford: Academic Press. 2014; pp. 916–917.
23. Thomas PW, Spicer T, Cammarata M, Brodbelt JS, Hodder P, Fast W. An altered zinc-binding site confers
resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by
high-throughput screening. Bioorg Med Chem. 2013; 21: 3138–3146. doi: 10.1016/j.bmc.2013.03.031
PMID: 23591260
24. Hu X, Shelver WH. Docking studies of matrix metalloproteinase inhibitors: zinc parameter optimization
to improve the binding free energy prediction. J Mol Graph Model 2003; 22: 115–126. PMID: 12932782
25. Hu X, Balaz S, Shelver WH. A practical approach to docking of zinc metalloproteinase inhibitors. J Mol
Graph Model 2004; 22: 293–307. PMID: 15177081
26. Olsen L, Pettersson I, Hemmingsen L, Adolph HW, Jorgensen FS. Docking and scoring of metallobeta-lactamases
inhibitors. J Comput Aided Mol Des. 2004; 18: 287–302. PMID: 15562992
27. Mohamed MS, Hussein WM, McGeary RP, Vella P, Schenk G, Abd ERH, et al. Synthesis and kinetic
testing of new inhibitors for a metallo-β-lactamase from Klebsiella pneumonia and Pseudomonas aeruginosa.
European Journal of Medicinal Chemistry 2011; 46: 6075–6082. doi: 10.1016/j.ejmech.2011.
10.030 PMID: 22051063
28. Shen B, Yu Y, Chen H, Cao X, Lao X, Fang Y, et al. Inhibitor Discovery of Full-Length New Delhi
Metallo-beta-Lactamase-1 (NDM-1). PLoS One 2013; 8: e62955. doi: 10.1371/journal.pone.0062955
PMID: 23675445
29. Irwin JJ, Raushel FM, Shoichet BK. Virtual screening against metalloenzymes for inhibitors and substrates.
Biochemistry. 2005; 44: 12316–12328. PMID: 16156645
30. King DT, Worrall LJ, Gruninger R, Strynadka NC. New Delhi metallo-beta-lactamase: structural insights
into beta-lactam recognition and inhibition. J Am Chem Soc. 2012; 134: 11362–11365. doi: 10.1021/
ja303579d PMID: 22713171
31. Zhang H, Hao Q. Crystal structure of NDM-1 reveals a common beta-lactam hydrolysis mechanism.
FASEB J. 2011; 25: 2574–2582. doi: 10.1096/fj.11-184036 PMID: 21507902
32. King D, Strynadka N. Crystal structure of New Delhi metallo-beta-lactamase reveals molecular basis
for antibiotic resistance. Protein Sci. 2011; 20: 1484–1491. doi: 10.1002/pro.697 PMID: 21774017
33. Kim Y, Tesar C, Mire J, Jedrzejczak R, Binkowski A, Babnigg G, et al. Structure of apo- and monometalated
forms of NDM-1—a highly potent carbapenem-hydrolyzing metallo-beta-lactamase. PLoS One 6:
2011; e24621. doi: 10.1371/journal.pone.0024621 PMID: 21931780
34. Guo Y, Wang J, Niu G, Shui W, Sun Y, Zhou H, et al. A structural view of the antibiotic degradation enzyme
NDM-1 from a superbug. Protein Cell. 2011; 2: 384–394. doi: 10.1007/s13238-011-1055-9
PMID: 21637961
35. Docquier JD. On functional and structural heterogeneity of VIM-type metallo-beta-lactamases. Journal
of Antimicrobial Chemotherapy. 2003; 51: 257–266. PMID: 12562689
36. Green VL, Verma A, Owens RJ, Phillips SE, Carr SB. Structure of New Delhi metallo-beta-lactamase 1
(NDM-1). Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011; 67: 1160–1164. doi: 10.1107/
S1744309111029654 PMID: 22102018
37. Haas J, Roth S, Arnold K, Kiefer F, Schmidt T, Bordoli L, et al. The Protein Model Portal—a comprehensive
resource for protein structure and model information. Database (Oxford) 2013; bat031.
Discovery of Novel NDM-1 Inhibitors
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 16 / 17
38. Halgren TA. MMFF VI. MMFF94s option for energy minimization studies. Journal of Computational
Chemistry. 1999; 20: 720–729.
39. Irwin JJ, Shoichet BK. ZINC—a free database of commercially available compounds for virtual screening.
J Chem Inf Model. 2005; 45: 177–182. PMID: 15667143
40. Marcou G, Rognan D. Optimizing fragment and scaffold docking by use of molecular interaction fingerprints.
J Chem Inf Model. 2007; 47: 195–207. PMID: 17238265
41. Labute P. On the perception of molecules from 3D atomic coordinates. J Chem Inf Model. 2005; 45:
215–221. PMID: 15807481
42. McGovern SL, Helfand BT, Feng B, Shoichet BK. A specific mechanism of nonspecific inhibition. J Med
Chem. 2003; 46: 4265–4272. PMID: 13678405
43. Tamilselvi A, Mugesh G. Zinc and antibiotic resistance: metallo-beta-lactamases and their synthetic analogues.
J Biol Inorg Chem. 2008; 13: 1039–1053. doi: 10.1007/s00775-008-0407-2 PMID: 18648861
44. Selevsek N, Rival S, Tholey A, Heinzle E, Heinz U, Hemmingsen L, et al. Zinc ion-induced domain organization
in metallo-beta-lactamases: a flexible “zinc arm” for rapid metal ion transfer? J Biol Chem.
2009; 284: 16419–16431. doi: 10.1074/jbc.M109.001305 PMID: 19395380
45. Kim Y, Cunningham MA, Mire J, Tesar C, Sacchettini J, Joachimiak A. NDM-1, the ultimate promiscuous
enzyme: substrate recognition and catalytic mechanism. FASEB J. 2013; 27:1917–1925. doi: 10.
1096/fj.12-224014 PMID: 23363572
46. Brown MC, Verma D, Russell C, Jacobs DJ, Livesay DR. A case study comparing quantitative stabilityflexibility
relationships across five metallo-beta-lactamases highlighting differences within NDM-1.
Methods Mol Biol. 2014; 1084: 227–238. doi: 10.1007/978-1-62703-658-0_12 PMID: 24061924
47. Li T, Wang Q, Chen F, Li X, Luo S, Fang H, et al. Biochemical characteristics of New Delhi metallobeta-lactamase-1
show unexpected difference to other MBLs. PLoS One 2013; 8: e61914. doi: 10.
1371/journal.pone.0061914 PMID: 23593503
48. Cole JC, Murray CW, Nissink JW, Taylor RD, Taylor R. Comparing protein-ligand docking programs is
difficult. Proteins. 2005; 60: 325–332. PMID: 15937897
49. Goto J, Kataoka R, Hirayama N. Ph4Dock: pharmacophore-based protein-ligand docking. Journal of
Medicinal Chemistry. 2004; 47: 6804–6811. PMID: 15615529
50. Yuan Q, He L, Ke H. A potential substrate binding conformation of beta-lactams and insight into the
broad spectrum of NDM-1 activity. Antimicrob Agents Chemother. 2012; 56: 5157–5163. doi: 10.1128/
AAC.05896-11 PMID: 22825119
51. Fast W, Sutton LD. Metallo-beta-lactamase: Inhibitors and reporter substrates. Biochim Biophys Acta.
2013; 1834: 1648–1659. doi: 10.1016/j.bbapap.2013.04.024 PMID: 23632317
52. Wildman SA, Crippen GM. Evaluation of ligand overlap by atomic parameters. J Chem Inf Comput Sci.
2001; 41: 446–450. PMID: 11277735
53. Feher M, Schmidt JM. Multiple flexible alignment with SEAL: a study of molecules acting on the colchicine
binding site. J Chem Inf Comput Sci. 2000; 40: 495–502. PMID: 10761156
54. Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J. CASTp: computed atlas of surface topography
of proteins with structural and topographical mapping of functionally annotated residues. Nucleic
Acids Res. 2006; 34: W116–118. PMID: 16844972
55. Mincione F, Scozzafava A, Supuran CT. The development of topically acting carbonic anhydrase inhibitors
as anti-glaucoma agents. Curr Top Med Chem. 2007; 7: 849–854. PMID: 17504129
56. Docquier JD, Benvenuti M, Calderone V, Stoczko M, Menciassi N, Rossolini GM, et al. High-resolution
crystal structure of the subclass B3 metallo-beta-lactamase BJP-1: rational basis for substrate specificity
and interaction with sulfonamides. Antimicrob Agents Chemother. 2010; 54: 4343–4351. doi: 10.
1128/AAC.00409-10 PMID: 20696874
57. Chen J, Liu Y, Fang M, Chen H, Lao X, Gao X, et al. Combined Support-Vector-Machine-Based Virtual
Screening and Docking Method for the Discovery of IMP-1 Metallo-β-Lactamase Inhibitors Supplementary
Data. American Journal of Biomedical Research. 2013; 1: 120–131.
Discovery of Novel NDM-1 Inhibitors
PLOS ONE | DOI:10.1371/journal.pone.0118290 March 3, 2015 17 / 17

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