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In Silico Evaluation of Bioactive Compounds from Cucumis anguria L. as

Potential Inhibitors of Antibiotic-Resistant New Delhi Metallo-β-Lactamase
(NDM-1)

Article in ACS Omega · May 2025

DOI: 10.1021/acsomega.5c02627

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http://pubs.acs.org/journal/acsodf Article

In Silico Evaluation of Bioactive Compounds from Cucumis anguria L.

as Potential Inhibitors of Antibiotic-Resistant New Delhi Metallo-β-
Lactamase (NDM-1)
Soundar Rajan Kulandhaivel, Pandiyan Muthuramalingam, Balasubramanian Sivaprakasam,
Manikandan Ramesh, Arun Muthukrishnan, Kapildev Gnanajothi, Vidhyavathi Ramasamy,
Thamaraiselvi Chandran, Hyunsuk Shin,* and Jesudass Joseph Sahayarayan*

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ABSTRACT: Traveler’s diarrhea (TD), manifested by loose

stools, is a critical health issue affecting the digestive system. It
poses a significant health risk with documented mortality and
morbidity. Escherichia coli and Klebsiella are primary causative
organisms. The emergence of New Delhi metallo-β-lactamases
(NDM-1), initially reported in E. coli and Klebsiella pneumoniae, has
driven the rapid dissemination of antibiotic-resistant strains. The
bla NDM-1 gene encodes NDM-1, an enzyme that confers
resistance to β-lactam antibiotics. Traditionally, Cucumis anguria
L., which is native to Africa and is widely distributed in East and
Southern Africa, has been used to alleviate various stomach
disorders. The NDM-1 metallo-β-lactamase protein is involved in
the production of β-lactams. In this study, we have conducted in
silico screening of natural bioactive compounds, including ethylenediaminetetraacetic acid (EDTA), to identify potential inhibitors of
metallo-β-lactamase protein. Molecular docking is performed to evaluate the binding interactions between the compounds and
NDM-1. Subsequently, the ADME/Tox properties of the lead compounds and EDTA are predicted. The phytocompounds
homogentisic acid, caffeic acid, and protocatechuic acid have Glide g-scores of −8.818, −8.663, and −8.121 kcal/mol, respectively.
They form hydrogen bonds with GLN 123 and ASN 220, along with metal coordination involving Zn2+ ions. Molecular dynamics
(MD) simulations, including RMSD, RMSF, Rg, and hydrogen bond analyses, are conducted on the top-ranked protein−ligand
complexes and EDTA. The results indicate that the protein−ligand complexes remained stable throughout the 100 ns simulation
period. This assessment provides the first evidence supporting the specificity and compatibility of potential phytochemicals in C.
anguria against TD by inhibiting β-lactam proteins.

1. INTRODUCTION Sometimes, β-lactamase resists all of the antibiotics.4 Thus,

Traveler’s diarrhea (TD) is an intestinal infection characterized multidrug resistance mechanisms emancipated by TD account
by loose stools and abdominal cramps, commonly resulting for critical health hazards. Every year, around 80 million
from the consumption of contaminated food or water. tourists traveling abroad experience diarrhea. As a result, TD
Travelers visiting regions with climates and sanitation will remain a global concern.5
standards that differ from those of their hometowns face a TD is mainly caused by the New Delhi β-lactamase (NDM-
higher risk of illness. This risk often arises from inadequate 1) protein, which inactivates all β-lactams, except aztreonam.6,7
sanitation practices among food handlers or the consumption The gene renders bacteria resistant to antibiotics of the
of contaminated food.1,2 The most frequent cause of TD is carbapenem family. NDM-1 was also reported in a Swedish
believed to be bacterial infection, accounting for 80−90% of
cases. According to the area, studies suggest that between 9 Received: March 21, 2025
and 79% of people traveling to Eastern and Southeastern Asia Revised: April 30, 2025
suffered from TD during or after their trip.3 These incidences Accepted: May 7, 2025
are high in warmer climates, where sanitation and hygiene Published: May 30, 2025
practices are deplorable. Various antibiotics such as loper-
amide, rifaximin, and fluoroquinolones are used to treat TD.
© 2025 The Authors. Published by
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Figure 1. Ligand-based virtual screening workflow implemented in this study. The workflow includes the identification of active phytocompounds,
molecular docking, ADMET prediction, and molecular dynamics simulations.

patient of Indian origin in 2008, incited by Escherichia coli, activity, leading to treatment failures and high mortality rates.16
although initial isolation correlates to Klebsiella pneumoniae.8 Targeting NDM-1 with novel zinc-chelating agents from
Subsequently, the prevalence was confirmed in the UK, the US, natural resources offers a promising strategy to restore the
Canada, Japan, India, and Pakistan. The bla NDM-1 gene effectiveness of β-lactam antibiotics without side effects and
encodes NDM-1 protein, a carbapenemase β-lactamase combat the growing challenge of antimicrobial resistance.
enzyme that hydrolyzes and inactivates carbapenem anti- Structural conformations and changes are deciphered as
biotics.9 The first documented fatality caused by bacteria serine or zinc ions that are present at the dynamic centers of β-
expressing the NDM-1 enzyme occurred in August 2010. A lactamases and have electrostatic interactions with carbapene-
significant quantity of the bla NDM-1 gene was isolated from mase enzymes, hence called metallo-β-lactamases (MBLs).
microbes found in tap water and sewage water collected from Most of the MBLs have two zinc metal ions, in which Zn1 can
hospitals and urban areas.10,11 bind to the molecule through hydroxyl groups and Zn2 with
NDM-1 has recently been reported to be resistant to the substrate and is mediated by a metal coordination bond
carbapenemases, spreads rapidly, and has major consequences and Zn2.17 The MBLs can be categorized into three subclasses,
in the field of clinical microbiology.12,13 Different types of such as B1, B2, and B3, according to sequence similarity.18 The
NDM-1 inhibitors have been tested against microbes. Thus, metal chelators, like ethylenediaminetetraacetic acid (EDTA),
the presence of an infection carrying the NDM-1 gene poses a are used to inhibit NDM-1 by sequestering essential zinc ions
serious threat and consequently may lead to mortality and fatal from its active site.19,20
sequel.14 Creating novel β-lactam molecules that are resistant Mutations in NDM-1 can significantly influence protein
to hydrolysis by these enzymes and the development of β- stability, catalytic efficiency, and substrate specificity, impacting
lactamase inhibitors, which can be combined with antibiotics their role in antibiotic resistance.21 Some mutations can
to create a single product because β-lactamase inhibitors enhance the thermodynamic or structural stability of NDM-1,
extend the shelf life of outdated β-lactam antibiotics, are the allowing it to function more effectively in diverse bacterial
two major strategies used to neutralize β-lactamases. Clinically hosts or under varying environmental conditions. Mutations
approved metallo-β-lactamase inhibitors are still unknown.15 near the active site, especially those involving residues that
Based on the CDC 2024 report, there are no available vaccines coordinate with zinc ions or interact directly with β-lactam
or medicines to cure TD. However, antibiotics such as antibiotics, can alter the enzyme’s hydrolytic activity. Natural
Azithromycin, Ciprofloxacin, Levofloxacin, and Rifamycin have mutations in NDM-1 variants (like NDM-4, NDM-5, etc.)
been used to reduce the risk of illness. NDM-1 poses a have shown increased resistance to the antibiotics.20
significant public health threat by hydrolyzing a broad range of Cucumis anguria L., commonly known as bur gherkin, is an
β-lactam antibiotics because of the absence of clinically underutilized traditional medicinal plant that belongs to the
approved inhibitors available to neutralize its zinc-dependent Cucurbitaceae family and genus Cucumis. This type of plant
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produces bitter fruits and nonbitter fruits.22 The leaves and process ensures the accuracy and reliability of the protein
fruits of this bur gherkin are consumed as pickles and used as structure for further study.
fresh fruits in salads. Every part of this plant belongs to the 2.3. Drug Likeness and ADME/Tox Prediction:
realm of traditional medicine, combating stomach-related Computational Approach. Compounds with unconditional
problems and kidney stones.23,24 The fruit from this plant pharmacokinetic properties like absorption, distribution,
contains large amounts of protein, iron, potassium, phospho- metabolism, and excretion (ADME) have been considered
rus, calcium, and vitamin C. Further, the plant is mainly unsuitable for drug development.32 The selected compounds
consumed and cultivated in many countries like the United from the plant C. anguria root were subjected to ADME
States, Brazil, India, Cuba, Africa, and Zimbabwe.25 Phyto- properties by using the Qikprop version to decipher the drug
chemicals from C. anguria are used to treat jaundice, likeness (QPlog Po/w, QPlogS, QPlog HERG, QPlog BB
hemorrhoids, stomach aches, and kidney stones. The reported levels) and human oral absorption percentage and rule of 5
phytochemicals include Cucurbitacin B, Cucurbitacin D, and (RO5) properties aiding the drug discovery process. ProTox-
Cucurbitacin G, where Cucurbitacin B is used to prevent III (https://tox.charite.de/protox3/index.php?site=
cancer. Compounds from C. anguria L. have high levels of compound_input) is an online web server that predicts the
antioxidant activity.26 Japanese gherkins displayed a low toxicity of small molecules.33
concentration of carotenoids, nondetectable amounts of 2.4. Molecular Docking: Ligand-Based Screening. The
anthocyanins, and vitamin C, and a moderate source of sitemap module was used to identify the substrate-binding
phenolic compounds. The antibacterial properties of C. anguria pocket of the bla NDM-1 receptor. A comprehensive structural
leaf extract were studied and found to act as a potent analysis revealed the remarkable conservation of the binding
antibacterial agent, notably against K. pneumoniae, S. aureus, pocket, suggesting a promising possibility for developing
and E. coli.27 potential inhibitors targeting bla NDM-1. Compounds derived
Here, we chose the N-terminally truncated NDM-1 metallo- from medicinal plants that inhibit NDM-1 activity are
beta lactamase [PDB ID: 6TWT] crystal structure for docking considered promising candidates for traditional drug design.
studies. In this structure, two positively charged zinc metal ions In this screening, the selected compounds underwent a
are present at the active site and are responsible for the sequential docking process, including ligand docking and
hydrolysis of the β-lactam ring in NDM-1, a major factor extra precision (XP) modes with default parameters to retain
contributing to antibiotic resistance in certain bacteria.28 This the best-scoring conformations. Docking calculations were
protein corresponds to the B1 subclass (broad-spectrum conducted by using the OPLS-2005 force field. Following a
enzyme), and coordination spheres of zinc ions are present successful screening analysis, the top compounds were selected
at the active center of B1 with water molecules.29 Therefore, based on predefined inclusion criteria.34
developing selective metal chelators from the phytocompounds 2.5. Free Binding Energy Analysis: Prime MM-GBSA.
from C. anguria that specifically target the zinc ions in NDM-1 To evaluate the relative free energy of ligand binding at the
without affecting other metalloproteins is a promising strategy protein’s active site, the top molecules identified through Glide
to combat antibiotic resistance mediated by this enzyme. docking were analyzed. The prime MM-GBSA module was
used for this analysis. This approach utilizes the OPLS-2005
force field to compute the energies of the complex. The
2. METHODOLOGY equation used to calculate the binding energy is as follows
The workflow combines computational methodologies to G binding = Gcomplex [ Gprotein + G ligand ]
systematically identify and characterize potential drug-like
compounds, as depicted in Figure 1. By integrating ligand- where ΔGbinding is the free energy of the protein−ligand
based screening, docking, molecular dynamics, and quantum complex, and ΔGprotein and ΔGligand are minimized energy
chemical calculations, this approach facilitates the identifica- values of the protein and ligand, respectively.35
tion of promising phytocompounds from C. anguria for NDM- 2.6. Density Functional Theory (DFT) Analysis. DFT
1 inhibition. calculations were applied to the top-scoring compounds, and
2.1. Data Set. Phenolic compounds from the roots of C. EDTA was obtained from molecular docking and ADMET
anguria were collected through a literature survey,30 and studies. The Jaguar module, Maestro, Schrodinger 2023-2, was
compound structures were retrieved from the PubChem used to calculate various electrostatic properties such as
database. The retrieved compounds were optimized with the HOMO, LUMO, dipole moment, electron density, and
LigPrep module from Schrodinger and subjected to further electrostatic potential of the lead molecules. DFT is also
analysis. The crystal structure of bla NDM-1 (PDB ID: 6TWT) useful to get detailed geometric features of the molecule.36
with reasonable resolution was retrieved from the PDB The lead compounds were optimized using the Lee−Yang−
database for screening studies with the GLIDE (GLIDE, Parr correlation functional (B3LYP) theory with the 6-31G**+
Maestro, Schrodinger 2023-2) docking protocol. Water + basis set to determine the compound-binding capabilities
molecules were removed from the crystal structure and more effectively. The water solvation system, Poisson−
preprepared by the Protein Preparation Wizard in Schrodinger. Boltzmann finite, dispenses the molecules. The electron
The OPLS-2005 force field was employed to minimize energy. donor and acceptor region of the molecules were identified
This study mainly focused on identifying potent compounds by HOMO and LUMO, and their energy gaps well defined the
from the roots of C. anguria that can inhibit the bla NDM-1 stability and bioactive properties of the lead molecules.37 The
receptor.31 charge distribution of the molecule, indicated by its van der
2.2. PROCHECK Analysis. The geometrical parameters of Waals contact area, was measured by electrostatic potential
the protein were evaluated using PROCHECK, which can energy represented by the colored surface of the molecule. The
validate the quality of protein models by assessing the dihedral positive potential regions of the molecule were colored deep
angles (phi and psi), bond lengths, and bond angles. This blue, while the negative regions were deep red. The
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Table 1. In Silico Pharmacokinetic and ADME Analyses of Selected Compounds Using the QikProp Toola
Pubchem ID compound name % HOA QPlog Po/w QPlogS QPlog HERG QPlog BB RO5
1183 vanillin 82.06 1.004 −1.134 −3.376 −0.653 0
8468 vanillic acid 66.92 1.045 −1.395 −1.606 −0.881 0
444539 trans-cinnamic acid 79.52 1.910 −1.645 −2.393 −0.555 0
10742 syringic acid 66.05 0.971 −1.621 −1.612 −0.978 0
338 salicylic acid 76.70 2.259 −1.616 −1.615 −0.651 0
5280343 quercetin 52.19 0.385 −2.884 −5.075 −2.380 0
72 protocatechuic acid 52.90 0.031 −0.799 −1.515 −1.223 0
135 p-hydroxybenzoic acid 64.10 0.585 −1.537 −1.632 −0.789 0
637542 p-coumaric acid 67.47 1.439 −1.676 −2.280 −1.081 0
637540 o-coumaric acid 69.34 1.487 −1.654 −2.280 −0.995 0
5280863 kaempferol 64.12 1.061 −3.142 −5.178 −1.864 0
780 homogentisic acid 58.09 0.444 −0.971 −1.549 −1.164 0
5280373 biochanin A 90.84 2.562 −3.601 −5.119 −0.873 0
5281672 myricetin 27.40 −0.281 −2.645 −4.973 −2.903 1
1794427 chlorogenic acid 16.49 −0.250 −2.520 −3.311 −3.317 1
689043 caffeic Acid 54.36 0.557 −1.308 −2.181 −1.547 0
445858 ferulic acid 67.23 1.376 −1.871 −2.242 −1.177 0
439246 naringenin 74.458 1.653 −3.423 −4.964 −1.396 0
10621 hesperidin 0 −1.349 −3.739 −6.398 −4.685 3
9064 cianidanol 60.577 0.479 −2.614 −4.744 −1.881 0
7121 3,4-dimethoxybenzoic acid 82.587 2.155 −2.007 −1.552 −0.455 0
3469 2,5-dihydroxybenzoic acid 58.853 0.792 −1.060 −1.513 −1.147 0
Chelating Agent
6049 EDTA 0 −3.374 0.920 2.311 −2.554 0
a
The key parameters are summarized here, including lipophilicity (logP), aqueous solubility (logS), drug likeness, and absorption metrics.

electronegativity, hardness, and softness of the compounds (Rg), and hydrogen bonding were employed for system
were determined as follows38 analysis. The graph was plotted using Xmgrace tool.40
(a) The energy gap is the difference between the energies of
the LUMO and the HOMO: 3. RESULTS
energy gap = E LUMO E HOMO The potential of natural compounds, particularly antimicrobial
agents, remains largely unexploited. Screening phytocom-
pounds against specific biological targets combined with
(b) Hardness (η) is a measure of the resistance to changes in structural dynamics studies is a highly recommended strategy
electron density: in traditional drug design for the development of novel and
effective drug molecules.
= E LUMO E HOMO/2
3.1. Structural Validation of the Target Protein.
NDM-1 remains the major target hindering the outcomes of
(c) Softness (s) is the reciprocal of hardness: TD and is a critical therapeutic target. The 3-dimensional
structure of the β-lactamase receptor was acquired from the
s = 1/ Protein Data Bank (PDB ID: 6TWT). It was determined by X-
ray diffraction at a resolution of 0.95 Å and was visualized by
2.7. Molecular Dynamics Simulations. The structural
stability of the lead phytocompounds complexed with protein the Schrodinger tool, and no mutations were observed in the
and EDTA was determined by using GROMACS-2019 and the structure. The sequence length of the protein was 243 amino
Charmm36 all-atom force field to generate ligand topology. acids. The Ramachandran plot from PROCHECK indicates
The systems were equilibrated under periodic boundary that 91.6% of the residues fall within the most favored region,
conditions with a cubic box of 1.0 nm and solvated with the 7.9% in the additionally allowed region, and 0.5% in the
TIP3P water model. Next, chlorine and sodium ions were disallowed region.
added to neutralize the system, followed by energy 3.2. In Silico Pharmacokinetic and ADMET Analysis.
minimization using the steepest descent and conjugate gradient The pharmacokinetic properties are among the most
algorithms for 50,000 steps. To equilibrate the system, NPT challenging aspects of designing a drug. These properties are
and NVT ensembles were utilized for maintaining a constant crucial for determining whether a molecule is a viable drug
temperature of 300 K and constant pressure of 1 bar. Finally, candidate. They depend on several factors, including the
the MDS was performed for a 100 ns time period for each Lipinski rule of five, as well as the ADME characteristics
system.39 depicted in Table 1.
The results were analyzed using tools integrated into the Based on the protocol of the Qikprop module, molecules
GROMACS package. The root mean square deviation with human oral absorption ≥30% are considered drug
(RMSD) shows structural stability, root mean square candidates. The selected phytocompounds show human oral
fluctuations (RMSF) show fluctuation, radius of gyration absorption >30%, except hesperidin, which violates the three
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Table 2. Docking Scores, Binding Energies, and Glide Energy for Lead Phytocompounds from C. anguria against NDM-1
(PDB ID: 6TWT)
protein Pubchem ID com. name docking score (kcal/mol) XPG score glide score glide energy
(NDM-1) PDB ID: 6TWT 780 homogentisic acid −8.818 −8.818 −8.818 −46.498
689043 caffeic acid −8.663 −8.663 −8.663 −44.076
72 protocatechuic acid −8.121 −8.121 −8.121 −32.843
5281672 myricetin −8.020 −8.066 −8.066 −42.318
637540 o-coumaric acid −7.842 −7.844 −7.844 −24.610
637542 p-coumaric acid −7.839 −7.839 −7.839 −23.703
3469 2,5-dihydroxybenzoic acid −7.750 −7.750 −7.750 −24.732
Chelating Agent
6049 EDTA −5.178 −7.258 −7.258 −32.3

Figure 2. Molecular docking interactions of compounds from C. anguria and chelating compound EDTA with the active site of the NDM-1 protein.
The interactions with 6TWT protein residues are shown for (A) homogentisic acid, (B) caffeic acid, (C) protocatechuic acid, and (D) EDTA.

rule of five, chlorogenic acid, myricetin, and chelating agent 124, 154, 189, 207, 208, 210, 211, 212, 214, 215, 216, 217,
(EDTA), which cannot be considered a drug molecule. 219, 220, 248, 249, 250, 251, 301, 302) were defined in the
3.3. Molecular Docking Studies. Toward the compre- grid generation. The Glide gscore ranges from −9.107 to
hensive search of phytocompounds, a total of 22 compounds −5.076 kcal/mol. The molecular docking results of the
were docked against the NDM-1 receptor through the Glide- compounds against the NDM-1 receptor are shown in Table 2.
XP mode in Schrodinger to analyze the binding affinity. Before The three hit lead molecules are homogentisic acid, caffeic
docking the binding site was predicted by the Sitemap module acid, and protocatechuic acid with Glide g-scores of −8.818,
in Schrodinger, and the binding site residues (93, 122, 123, −8.663, and −8.121 kcal/mol, respectively. It formed hydro-
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gen bonds with GLN 123 and ASN 220, and metal 3.5. DFT Calculation. The top three scoring molecule
coordination with Zn2+ ions, as depicted in Figure 2. conformations were analyzed by using frontier molecular
Toxicity assessments for the lead compounds and the orbital analysis to predict the electrostatic properties, chemical
chelating agent were conducted using the ProTox-III web stability, and reactivity. Electrostatic potential maps were
server, as shown in Table 3. The compounds protocatechuic generated to identify regions of high and low electron density
acid (72) and EDTA demonstrated activity in carcinogenicity, within the molecule. The HOMO, LUMO, and MESP regions
which may lead to cancer. of the compounds are depicted in Figure 3, in which the red
color shows the negative phase and the blue color shows the
Table 3. Predicted Toxicity Profiles of the Top Five Lead positive phase in the HOMO and LUMO plots. The energy
Compounds from C. anguria Using the ProTox-II Web gap of lead molecules was in the range of 0.1480 to 0.4148 eV,
Server, Including Various Toxicity End Points as shown in Table 4 with its electronegativity, hardness, and
softness.
chelating
compounds agent Hardness (η) and softness (s) are key concepts in DFT that
homogentisic caffeic protocatechuic
describe a molecule’s resistance or susceptibility to changes in
toxicity acid acid acid EDTA its electron density. Molecules with low hardness and high
hepatotoxicity inactive inactive inactive inactive softness are highly reactive in nature.
neurotoxicity inactive inactive inactive inactive 3.6. Molecular Dynamics (MD) Simulation. MD
cardiotoxicity inactive inactive inactive inactive simulation was performed to study the conformational stability
carcinogenicity inactive inactive active active of the protein and ligands. Variation at the atomistic level of
immunotoxicity inactive inactive inactive inactive the protein−ligand and its complexes in the dynamic
mutagenicity inactive inactive inactive inactive environment was determined by using MD simulation.41 In
cytotoxicity inactive inactive inactive inactive the present study, the parameters such as RMSD, RMSF, Rg,
and hydrogen bond formation for the apo and complexes were
evaluated over a 100 ns simulation.
3.4. Prime: MM-GBSA Analysis. The binding free 3.6.1. RMSD. RMSD values were calculated for the
energies of the selected hit molecules within the active site backbone atoms of the protein and each complex relative to
of NDM-1 were estimated by using MM/GBSA calculations in their respective initial structures. A low RMSD value indicates
the protein−ligand complexes. This analysis reveals the that the protein maintains a similar structure, suggesting
binding energy of the top three hit molecules complexed stability throughout the simulation, and vice versa. The
with the NDM-1 receptor, i.e., homogentisic acid (−46.498 dynamic variation in the backbone of the three lead complexes
kcal/mol), caffeic acid (−48.076 kcal/mol), protocatechuic was determined by monitoring the RMSD during the
acid (−35.843 kcal/mol), and EDTA (−24.09 kcal/mol). This molecular simulation. The plot of the complex reveals that
results in the strong binding affinity of the lead three molecules RMSD ranges between 0.1 and 0.3 nm, and the average is
and EDTA, which supports the glide docking results. noticed at 0.15 nm. It was found to be very consistent, and no

Figure 3. HOMO, LUMO and MESP regions of the phytocompounds from C. anguria. (A) homogentisic acid, (B) caffeic acid, (C) protocatechuic
acid, and (D) chelating agent EDTA.

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Table 4. HOMO and LUMO Energies of the Phytocompounds Homogentisic Acid, Caffeic Acid, Protocatechuic Acid, and
Chelating Agent EDTA
s. no. compound HOMO (eV) LUMO (eV) energy gap (eV) hardness (eV) softness (eV)
1 homogentisic acid −0.0201 0.1280 0.1481 0.07 13.51
2 caffeic acid −0.2314 0.0936 0.3247 0.16 6.17
3 protocatechuic acid −0.0273 0.1418 0.4148 0.20 4.83
4 EDTA 0.1433 0.380 0.5233 0.26 3.84

significant deviations are observed in Figure 4. RMSD plots

indicate that the binding of inhibitors does not induce any
greater conformational changes in the backbone of the NDM-1
receptor.

Figure 5. RMS fluctuation analysis of protein−ligand complexes over

a 100 ns MD simulation. The fluctuation of the apo 6TWT_Protein-
(Apo) (magenta) and complexes 6TWT_72 (protocatechuic acid),
6TWT_780 (homogentisic acid), 6TWT_689043 (caffeic acid), and
6TWT_EDTA is represented by the black, red, green, and blue lines,
respectively.
Figure 4. RMSD of the protein backbone atoms during the 100 ns
MD simulation. The deviations in apo 6TWT_Protein(Apo)
(magenta) and complexes 6TWT_72 (protocatechuic acid),
6TWT_780 (homogentisic acid), 6TWT_689043 (caffeic acid),
and 6TWT_EDTA are represented by the black, red, green, and
blue lines, respectively.

3.6.2. RMSF. The RMSF graph illustrates the fluctuation of

residues within the protein throughout the molecular dynamics
simulation. In this graph, the x-axis represents the residue
index, while the y-axis indicates the RMSF value in nanometers
(nm). Regions exhibiting higher RMSF values correspond to
residues with greater flexibility, often indicating the protein’s
loop regions or terminal ends. Fluctuations occur in the
residues between 63 and 75, 120−125, 144−156, 169−177,
and 213−229 in the loop regions, as shown in Figure 5.
3.6.3. Radius of Gyration. The graph representing Rg
provides insights into a molecular system’s overall compactness
or expansion under varying conditions. It measures the mass
distribution around the center of mass over a 100 ns time Figure 6. The plot shows the variation in the radius of gyration,
interval of the molecules, indicating their structural con- highlights the structural transitions, and shows the overall
formation. The low Rg values indicate that the structure of conformation of the complexes. 6TWT_Protein(Apo), 6TWT_72
proteins is more compact and atoms are closer to the center of (protocatechuic acid), 6TWT_780 (homogentisic acid),
6TWT_689043 (caffeic acid), and 6TWT_EDTA are represented
mass, while higher values indicate that the atoms are spread
by the magenta, black, red, green, and blue lines, respectively.
further from the center. The gyration values range between
1.65 nm and 1.7 nm, suggesting that the structure of the
molecule is more compact, as shown in Figure 6. throughout the trajectory suggested that the native protein
3.6.4. Hydrogen Bond Analysis. Hydrogen bond analysis maintained its native secondary and tertiary structures under
provides insight into the structural stability and compactness of simulated conditions. In the present study, the number of
the protein and complex during the simulation period, as intramolecular hydrogen bonds in both the apo form and
depicted in Figure 7. In the intramolecular hydrogen bond ligand-bound complexes remained relatively stable during the
analysis, a consistent number of hydrogen bonds formed 100 ns simulation (Figure 7A), indicating no major structural
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Figure 7. Hydrogen bond formation throughout the MD simulation. (A) Intramolecular hydrogen bonds. (B) Hydrogen bonds between the
complexes 6TWT_72 (protocatechuic acid), 6TWT_780 (homogentisic acid), 6TWT_689043 (caffeic acid), and 6TWT_EDTA are represented
by the black, red, green, and blue lines, respectively.

distraction. This suggests that ligand binding did not adversely efficacy of β-lactam antibiotics by developing novel inhibitors,
affect the protein’s internal hydrogen bonding network, such as zinc-chelating agents or structure-based small
supporting the structural integrity and reliability of the molecules designed to block its active site.20 Therefore,
protein−ligand complexes throughout the simulation period. addressing NDM-1 through innovative drug discovery
The phytocompounds complexed with the receptor revealed approaches is vital for managing and mitigating the growing
that 2−4 hydrogen bonds and EDTA revealed that 1−2 bonds threat of antimicrobial resistance.
were formed throughout the simulation (Figure 7B). These The molecules that chelate the characteristics of Zn+ ions in
results showed that the screened phytocompounds maintained the NDM-1 gene act as an inhibitor48 and inhibit the enzyme’s
a good interaction with the active site and suggested that the ability to hydrolyze β-lactam antibiotics.49 Few compounds
docking complexes were stable throughout the 100 ns may act as viable inhibitors that bind to the active site of
simulation. NDM-1 and prevent the substrate from binding to antibiotics,
leading to resistance.14 Bioactive compounds from medicinal
4. DISCUSSION plants may lead to synergistic effects, enhancing the overall
The emergence of NDM-1 poses a significant global health inhibitory activity against NDM-1.50,51 Many compounds in C.
threat by conferring resistance to β-lactam antibiotics, anguria have metal-binding properties that chelate zinc ions in
including carbapenems, which are often considered the last the active site of NDM-1, essential for its enzymatic activity.
line of defense against multidrug-resistant bacterial infec- Molecular docking results revealed that several phytocom-
tions.42 Standard antibiotic treatments for TD are still pounds, chlorogenic acid, hesperidin, homogentisic acid,
ineffective, necessitating the use of more potent and expensive caffeic acid, and protocatechuic acid from C. anguria exhibited
antibiotics.43 strong binding affinities toward the active site of NDM-1,
E. coli and K. pneumoniae are responsible for many comparable to the metal chelator EDTA. Among them,
nosocomial infections in immunocompromised patients, and chlorogenic acid and hesperidin did not meet ADME
these organisms carry the NDM-1 gene responsible for TD.44 properties, and thus cannot be considered drug molecules. In
The infections caused by bacteria that produce NDM-1 are addition, EDTA showed 0% drug-likeness and is immunotoxic.
associated with longer duration of illness, increased risk of Notably, compounds such as homogentisic acid, caffeic acid,
medical complications, and higher healthcare costs due to the and protocatechuic acid demonstrated significant interactions
need for more intensive treatment and prolonged hospital with key catalytic residues GLN 123, ASN 220, and Zn2+ ions,
stays; in some cases, it leads to death.45 These bacteria, which are essential for enzymatic activity. These interactions
equipped with NDM-1, are a critical target in the treatment of suggest that the identified compounds can effectively chelate
antibiotic-resistant infections due to their ability to hydrolyze zinc cofactors, which are crucial for the hydrolysis of β-lactam
and inactivate a broad spectrum of β-lactam antibiotics, antibiotics. The compounds taniborbactam and xeruborbactam
including penicillins, cephalosporins, and carbapenems, which are currently in clinical trials targeting both MBLs and SBLs,
are often used as last-resort treatments.46 and further clinical research is essential to confirm their
NDM-1 is particularly challenging because of the lack of efficacy. Additionally, ANT2681 is a promising molecule
clinically approved inhibitors capable of neutralizing its zinc- specifically designed to inhibit MBLs and is advancing toward
dependent enzymatic activity, as traditional β-lactamase clinical development.52 4-Amino-1,2,4-triazole-3-thione deriv-
inhibitors are ineffective against metallo-β-lactamases.47 atives, 3-aryl-6,7-dihydro-[1,2,4]triazolo[3,4-b][1,3]thiazines,
Targeting NDM-1 offers a promising way to restore the 3-phenyl-5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3] thiazoles, 1-
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substituted-1H-imidazole-2-carboxylic acid derivatives,53 In- 5. CONCLUSIONS

dole-2-carboxylic acid derivatives, 3,4-dihydroxyphenylacetic NDM-1 is critical for developing effective inhibitors to combat
acid derivatives are the zinc inhibitors, evaluated for inhibitory antibiotic-resistant bacterial infections. The selective com-
activity against five representative MBLs (L1, VIM-4, VIM-2, pounds from C. anguria are a natural source for developing
NDM-1, and IMP-1), all compounds exhibited activity against NDM-1 inhibitors for treating TD by chelating the zinc ions in
at least one enzyme, with the most promising inhibitor the NDM-1 enzyme. By targeting the metalloenzyme’s active
demonstrating significant potential against MBLs.54 β-Lactam site through metal chelation, which acts as a competitive
conjugates with pyridyl-containing chelators, tormentic acid, inhibition mechanism, these compounds could potentially
and combined pyridyl- and carboxylate-containing chelators, β- restore the effectiveness of β-lactam antibiotics against resistant
lactam conjugates with carboxylate-containing chelators, bacterial strains. A promising hypothesis arising from this study
carboxylate-containing chelators, and 2,6-dipicolinic acid55 is that phytocompounds from C. anguria can restore the
are the metal chelators and its toxicity experiments effectiveness of β-lactam antibiotics by competitively binding
demonstrated that a carboxylic derivative exhibited low in to the zinc-dependent active site of NDM-1, thereby disrupting
vitro cytotoxicity and minimal in vivo toxicity. Additionally, in its hydrolytic activity. This study suggests that phytocom-
vivo studies revealed that the combined treatment of this pounds derived from C. anguria exhibit potential therapeutic
derivative with meropenem significantly improved the survival effects against TD, particularly by targeting the NDM-1
probability of mice infected with MBL-producing K. pneumo- enzyme associated with antibiotic resistance. Comprehensive
in vitro and in vivo experiments are essential to evaluate the
niae.55 Prime MM-GBSA is a widely used postdocking method
efficacy of these compounds in inhibiting NDM-1 activity.
for calculating the binding free energies. The phytocompounds Such validation will help researchers to develop effective
show better binding free energies when compared to EDTA. treatments for TD and other infections caused by NDM-1-
The stability of lead phytocompounds and NDM-1 producing pathogens. This holistic approach validates tradi-
complexes was assessed further through MD simulations. tional remedies and fosters the modernization and global-
Deviation, fluctuation, and radius of gyration analyses ization of plant-based bioactive molecules in contemporary
indicated that the protein−ligand and EDTA complexes medicine.
remained stable during the simulation, with only minor
fluctuations. Furthermore, hydrogen bond analysis confirmed
the persistence of interactions over time, reinforcing the
potential inhibitory activity of the selected phytocompounds.
■ AUTHOR INFORMATION
Corresponding Authors
There is some disruption in the hydrogen bonding interactions Hyunsuk Shin − Division of Horticultural Science, College of
between EDTA and the receptor during the simulation, Agriculture and Life Sciences, Gyeongsang National
indicating that the hydrogen bonds do not remain consistently University, Jinju 52725, South Korea; orcid.org/0000-
stable throughout the trajectory. This fluctuation suggests that 0002-2167-4843; Email: [email protected]
dynamic interactions are potentially influenced by conforma- Jesudass Joseph Sahayarayan − Department of
Bioinformatics, Science Campus, Alagappa University,
tional changes in the receptor−ligand complex or solvent
Karaikudi 630003 Tamil Nadu, India; orcid.org/0009-
effects. 0006-3184-4347; Email: [email protected]
Natural inhibitors offer several potential advantages over
synthetic compounds in combating NDM-1-mediated anti- Authors
biotic resistance.56 The compounds derived from various plant Soundar Rajan Kulandhaivel − Department of
sources possess complex and unique chemical scaffolds that Bioinformatics, Science Campus, Alagappa University,
may enhance their binding specificity and affinity for biological Karaikudi 630003 Tamil Nadu, India; orcid.org/0000-
targets like NDM-1 enzyme with lower toxicity and better 0003-3452-7161
biocompatibility, reducing the risk of adverse effects that are Pandiyan Muthuramalingam − Division of Horticultural
commonly seen with synthetic drugs.57 Naturally derived Science, College of Agriculture and Life Sciences, Gyeongsang
inhibitors may exhibit multitarget activity, helping to suppress National University, Jinju 52725, South Korea; orcid.org/
multiple resistance mechanisms simultaneously, which can 0000-0001-7294-1625
reduce the chances of bacteria developing resistance. Their Balasubramanian Sivaprakasam − Department of
abundance and ecofriendly nature also make them attractive Bioinformatics, Science Campus, Alagappa University,
compounds for sustainable drug discovery. These character- Karaikudi 630003 Tamil Nadu, India
istics support the potential use of natural compounds as Manikandan Ramesh − Department of Biotechnology, Science
Campus, Alagappa University, Karaikudi 630003 Tamil
promising leads for the development of safer and more
Nadu, India
effective NDM-1 inhibitors.
Arun Muthukrishnan − Department of Biotechnology,
This study highlights the potential of C. anguria bioactive Bharathiar University, Coimbatore 641 046 Tamil Nadu,
compounds as natural inhibitors of NDM-1. However, India
although computational predictions provide valuable insights, Kapildev Gnanajothi − Translational Plant Research
experimental validation through enzyme inhibition assays, Laboratory, Department of Microbial Biotechnology,
bacterial susceptibility tests, and toxicity profiling of Bharathiar University, 641 046 Coimbatore, Tamil Nadu,
phytocompounds is essential to confirm their efficacy as a India; orcid.org/0000-0001-5448-4838
potential drug candidates. Future studies should also explore Vidhyavathi Ramasamy − Department of Bioinformatics,
structural modifications to enhance potency and pharmacoki- Science Campus, Alagappa University, Karaikudi 630003
netic properties for potential drug development. Tamil Nadu, India
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Thamaraiselvi Chandran − Department of Biotechnology, Sequence Type 14 from India. Antimicrob. Agents Chemother. 2009, 53
Mother Teresa Women’s University, Kodaikanal 624101 (12), 5046−5054.
Tamil Nadu, India (9) Sahayarayan, J. J.; Thiyagarajan, R.; Prathiviraj, R.; Tn, K.; Rajan,
K. S.; Manivannan, P.; Balasubramanian, S.; Mohd Zainudin, M. H.;
Complete contact information is available at: Alodaini, H. A.; Moubayed, N. M. S.; Hatamleh, A. A.; Ravindran, B.;
https://pubs.acs.org/10.1021/acsomega.5c02627 Mani, R. R. Comparative Genome Analysis Reveals Putative and
Novel Antimicrobial Resistance Genes Common to the Nosocomial
Author Contributions Infection Pathogens. Microb. Pathog. 2024, 197, No. 107028.
Conceptualization: S.R.K. and J.J.S.; methodology: S.R.K. and (10) Villa, L.; Poirel, L.; Nordmann, P.; Carta, C.; Carattoli, A.
J.J.S.; investigation: S.R.K., P.M., M.R., A.M., K.G., V.R., T.C. Complete Sequencing of an IncH Plasmid Carrying the blaNDM-1,
and H.S.; writing-original draft, S.R.K. and B.S.; formal blaCTX-M-15 and qnrB1 Genes. J. Antimicrob. Chemother. 2012, 67
analysis: P.M. and J.J.S.; validation: S.R.K. and P.M.; writing- (7), 1645−1650.
review and editing: P.M., M.R., A.M., K.G., V.R., T.C., H.S. (11) Puljko, A.; Rozman, S. D.; Barišić, I.; Maravić, A.; Jelić, M.;
and J.J.S.; funding acquisition: H.S.; project administration: Babić, I.; Milaković, M.; Petrić, I.; Udiković-Kolić, N. Resistance to
Critically Important Antibiotics in Hospital Wastewater from the
J.J.S.; supervision: J.J.S.
Largest Croatian City. Sci. Total Environ. 2023, 870, No. 161805.
Notes (12) Lascols, C.; Hackel, M.; Marshall, S. H.; Hujer, A. M.;
The authors declare no competing financial interest. Bouchillon, S.; Badal, R.; Hoban, D.; Bonomo, R. A. Increasing
Prevalence and Dissemination of NDM-1 Metallo-β-Lactamase in

■ ACKNOWLEDGMENTS
J.J.S. thanks AURF Seed Money, Dt.10.12.2024, DST- Fund
India: Data from the SMART Study (2009). J. Antimicrob. Chemother.
2011, 66 (9), 1992−1997.
(13) Zhao, Q.; Sha, L.; Wu, Z.; Meng, L.; Yang, F.; Wu, L.; Yu, C.;
for Improvement of S&T Infrastructure in Universities and Zhang, H.; Yu, J.; Jin, Z. Evolution of Carbapenem Resistance in
Higher Educational Institutions (FIST) (SR/FST/LSI-667/ Klebsiella pneumoniae and Escherichia coli Carrying blaNDM-1 Gene:
2016)(C), MHRD-RUSA 2.0, New Delhi (F.24-51/2014-U), Imipenem Exposure Results in Sustained Resistance Memory of
Policy (TNMulti-Gen), Dept. of Edn. Govt. of India, Dt. Strains in Vitro. Ann. Clin. Microbiol. Antimicrob. 2023, 22 (1), No. 46.
09.10.2018, and Tamil Nadu State Council for Higher (14) Nagulapalli Venkata, K. C.; Ellebrecht, M.; Tripathi, S. K.
Education (TANSCHE-RGP) (RGP/2019-20/BU/HECP- Efforts towards the Inhibitor Design for New Delhi Metallo-Beta-
0018; Dt. 27.04.2021). Lactamase (NDM-1. Eur. J. Med. Chem. 2021, 225, No. 113747.
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