REVIEW JURNAL
1. JUDUL PENELITIAN
Ginger (Zingiber officinale) phytochemicals-gingerenone-A and shogaol inhibit
saHPPK : moleculer docking, molecular dynamics simulations and in vitro
approaches.
2. NAMA JURNAL
Annals of Clinical Microbiology and Antimicrobials.
3. VOLUME DAN HALAMAN
Volume 17 dan halaman 1-16
4. TAHUN 2018
5. PENULIS
Shailima Rampogu, dkk.
6. TANGGAL TERBIT
-
7. PENDAHULUAN
Staphylococcus aureus telah berkembang sebagai salah sau pantogen yangpaling menghancurkan, dimana menunjukkan berbagai resistensi terhadap antibiotik.Staphylococcus aureus adalah bakteri gram positif yang tidak bergerak. Resisensiantibiotik adalah mekanisme pertahana yang dilindungi oleh patogen untuk bertahanhidup dalam kondisi yang tidak menguntungkan. Diantara beberapa korsosiummikroba yang kebal antibiotik, Staphylococcus aureus adalah salah satumikroorganisme yang paling buruk. Staphylococcus aureus mengkodekan enzim unik6-hydroxymethyl-7,8-dihidropterin pyrophosphokinase (SaHPPK), dimana belum adaantibioik yang mampu merusaknya.Staphylococcus aureus ini biasanya terdapat pada membran mukosa dan kulitmanusia, dan biasanya dapat menyebar melalui udara, debu, dan tutup yang menutupiwadah minuman. Staphylococcus aureus Staphylococcus aureus ini dapatmenyebabkan keracunan yang ditandai dengan munta, diare dan sebagainya.
8. METODOLOGI
a. Pemilihan protein dan persiapanya.b. Seleksi dan persiapan ligan.c. Mekanisme docking molekuler.d. Analisis antimikroba in vitro fitokimia.e. Persiapan kultur media.f. Pembuaan inokulum.g. Persiapan disk.h. Analisis statistik.
9. HASIL DAN PEMBAHASAN
Berdasarkan skor dock molekuler, interaksi molekuler dengan residu aktif katalik danstudi simulasi MD, dua phyochemical gingerenone-A dan shogaol telah diusulkansebagai kandidat inhibitor terhadap Staphylococcus aureus. Mereka telahmenunjukkan skor yang lebih tinggi daripada antibiotik yang dikenal dan telahmewakili interaksi dengan residu utama dalam situs aktif. Lebih lanjut, senyawasenyawa ini telah menghasilkan aktivitas penghambatan yang cukup besar ketika diujiin vitro. Selain itu, keunggulan mereka dikuatkan oleh hasil MD stabil yang dilakukanselama 100 ns dengan menggunakan paket GROMACS.
10. KESIMPULAN
Dari penelitian ini dapat disimpulkan bahwa gingerenone-A dan shogaol dapatmenjadi inhibitor SaHPPK potensial atau dapat digunakan sebagai platform dasaruntuk pengembangan inhibitor SaHPPK baru.
11. KELEBIHAN DAN KEKURANGAN
A. KELEBIHANKelebihan dari penelitian ini adalah sudah dijelaskan secara rinci bagaimana caramelakukan penelitian ini dan peneliti sudah meyakinkan pembaca bahwagingerone-A dan Shogaol ini dapat digunakan dan dikembangkan dalam skalabesar.B. KEKURANGANKelemahan dari jurnal ini adalah tidak dicanumkan anggal penerbitan dari jurnalini.
12. SARAN DA REKOMENDASI
Dengan adanya jurnal ini menambah pengetahuan baru mengenai kegunaan dari jahe,sehingga dapat direkomendasikan untuk dimanfaakan oleh masyarakat umum.Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16https://doi.org/10.1186/s12941-018-0266-9
RESEARCH
Ginger (Zingiber ofcinale)
phytochemicals—gingerenone-A and shogaol
inhibit SaHPPK: molecular docking, molecular
dynamics simulations and in vitro approaches
Shailima Rampogu1
, Ayoung Baek1
, Rajesh Goud Gajula2
, Amir Zeb1
, Rohit S. Bavi1
, Raj Kumar1
,
Yongseong Kim3
, Yong Jung Kwon4
and Keun Woo Lee1*
Abstract
Background: Antibiotic resistance is a defense mechanism, harbored by pathogens to survive under unfavorable
conditions. Among several antibiotic resistant microbial consortium, Staphylococcus aureus is one of the most havoc
microorganisms. Staphylococcus aureus encodes a unique enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophospho
kinase (SaHPPK), against which, none of existing antibiotics have been reported.
Methods: Computational approaches have been instrumental in designing and discovering new drugs for several
diseases. The present study highlights the impact of ginger phytochemicals on Staphylococcus aureus SaHPPK. Herein,
we have retrieved eight ginger phytochemicals from published literature and investigated their inhibitory interac
tions with SaHPPK. To authenticate our work, the investigation proceeds considering the known antibiotics alongside
the phytochemicals. Molecular docking was performed employing GOLD and CDOCKER. The compounds with the
highest dock score from both the docking programmes were tested for their inhibitory capability in vitro. The binding
conformations that were seated within the binding pocket showing strong interactions with the active sites residues
rendered by highest dock score were forwarded towards the molecular dynamic (MD) simulation analysis.
Results: Based on molecular dock scores, molecular interaction with catalytic active residues and MD simulations
studies, two ginger phytochemicals, gingerenone-A and shogaol have been proposed as candidate inhibitors against
Staphylococcus aureus. They have demonstrated higher dock scores than the known antibiotics and have represented
interactions with the key residues within the active site. Furthermore, these compounds have rendered considerable
inhibitory activity when tested in vitro. Additionally, their superiority was corroborated by stable MD results con
ducted for 100 ns employing GROMACS package.
Conclusions: Finally, we suggest that gingerenone-A and shogaol may either be potential SaHPPK inhibitors or can
be used as fundamental platforms for novel SaHPPK inhibitor development.
Keywords: Ginger phytochemicals, 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase, GOLD, Shogaol,
Gingerenone-A, MD simulations
© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Open Access
Annals of Clinical Microbiology
and Antimicrobials
*Correspondence: kwlee@gnu.ac.kr
1
Division of Applied Life Science (BK21 Plus Program), Systems
and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology
and Biotechnology Research Center (PMBBRC), Research Institute
of Natural Science (RINS), Gyeongsang National University, Jinju 52828,
Republic of Korea
Full list of author information is available at the end of the articlePage 2 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
Background
Staphylococcus aureus has evolved as one of the most
devastating pathogens, demonstrating a wide range of
antibiotic resistance [1]. Staphylococcus aureus is a gram
positive, non-motile bacterium. Tis facultative anaer
obe is a gram positive, non-motile bacterium hailing
from Staphylococcaceae family, powered to infect every
known mammalian species causing food poisoning [2, 3].
Tis is an ectopic commensal and is niched on mucosal
membranes and skin of humans [4]. It is transmitted to
foods via air, dust, and the lids covering the food con
tainers [5, 6] and the food handlers carry the bacteria
on their heads and noses, hence, has an ability to colo
nize on the normal humans and transmit through direct
contact with the bacteria-colonized person. Staphylo
coccus intoxication occurs due to toxin-contaminated
food consumption. Such condition is symptomized very
quickly (2–8 h) and is associated with vomiting, abdomi
nal cramps, nausea and/or diarrhea [7, 8]. Even though,
staphylococcus intoxication subsides within 48 h, never
theless, it becomes severe in children and elders [9] and
causes several life threatening infections like, impetigo,
ritter disease, osteomyelitis, septic arthritis, endocardi
tis, toxic shock syndrome, pneumonia, thrombophlebitis
and deep skin abscess and infection [10, 11]. Several anti
biotics have been used to combat the bacteria [12–20]
such as, penicillin, methicillin, oxacillin, various vanco
mycins and glycopeptides, daptomycin, tetracyclines,
aminoglycosides, linezolid, chloramphenicol, forfenciol,
macrolides, and streptogramins [21]. However, Staphy
lococcus aureus exerts resistance by several mechanisms
that could be broadly categorized into mutations that
occur at the chromosomal genes and by horizontally
acquired resistance [21]. Specifcally, gaining resistance
through mutations can happen when the inhibitor is una
ble to bind to the accurate drug target, derepressing the
drug resistance efux pumps and by mutations that can
amend the structure and composition of the drug targets
[21]. On the other hand, the horizontally acquired resist
ance may occur by alteration and inactivation of enzy
matic drug, change in the drug binding site, dislocating
the drug from its appropriate position and by drug efux
[21]. Adapting either of the mechanisms, the organism
endeavours to survive avoiding the encounter with the
drug/antibiotic or neutralizes them [22]. Besides these,
antibiotic abuse can also add to the raise the resistance
[23]. Consequently, the efective treatment is hampered
and promotes the infection and enhances the economic
burden [23, 24].
Nevertheless, concerns for this bacterium rise due to its
resistance against methicillin, often called the Methicillin
resistance S. aureus (MRSA) that is prevalent currently
by exhibiting diverse phenotypes [25]. Tis ‘superbug’
was responsible for 19,000 deaths in USA in 1 year [26]
and can be classifed as hospital MRSA (haMRSA), refer
ring to those originating from the hospitals and the com
munity MRSA (caMRSA), indicating to those prevalent
in the community [27]. Besides these there is another
MRSA called as livestock-associated methicillin resist
ant Staphylococcus aureus [28]. However, caMRSAs are
increasingly virulent because of the presence of elevated
levels of alpha toxins and phenol-soluble modulin [29].
Not only to methicillin, this bacterium has also been
shown resistance against several antibiotics including
wonder drug penicillin [1, 22, 30, 31]. Pathogenicity and
antibiotic resistance potential of S. aureus drives our
interest to develop new drugs that can efectively chal
lenge this bacterium.
Choosing an appropriate target for the discovery of
novel antimicrobial drugs is a very important aspect [32].
It will be ideal to identify a target that is confned to path
ogen and is not present in host such that the drugs can
efectively render its efcacy on the target alone, doing
no harm to the host [33]. Accordingly, 6-hydroxyme
thyl-7,8-dihydropterin pyrophosphokinase (HPPK, EC
2.7.6.3) has been chosen as the drug target for the present
investigation [33] as it is not present in humans [34]. Tis
monomeric protein comprises of 158 residues, which cat
alyzes the transfer of pyrophosphoryl group from ATP to
6-hydroxymethyl-7, 8-dihydropterin (HMDP), the sub
strate. It has a molecular weight of 18 kDa pictured by
three-layered α-β-α fold. Typically, the HPPK has three
loops comprising of loop 1 with residues from 12 to 14,
loop 2 consisting of residues from 45 to 51 and loop 3
consisting of residues from 82 to 94, which demonstrate
a signifcant change in the conformation as compared
with the protein structure during catalysis. However,
loop 3 is known to display major conformational changes
[35]. Te rigidity of the ternary structure is attributed
when the pterin substrate binds and thus loop 3 closes
over the binding site. SaHPPK is an ideal target for novel
drug designing due to its expression in pathogen only,
and is not targeted by existing antibiotics. Alternatively,
this enzyme has also been found in other bacteria such
as Yersinia pestis, Haemophilus infuenzae, Streptococcus
pneumonia, Francisella tularensis, Saccharomyces cerevi
siae and Escherichia coli, demonstrating conserved active
sites [36, 37]. Consequently, it can be suggested that the
discovery of drugs against SaHPPK might have inhibitory
efects on its homologues on other pathogens [36].
An overwhelming signifcance of phytochemicals to be
employed as drug molecules is their capability to induce
nutritional values besides acting as a medicine and thus,
redirecting towards the formulation of nutraceuticals.
Nutraceuticals are the food that have both the nutri
tional and the pharmaceutical value [38] and refers to Page 3 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
as a food or part of the food that induces health benefts
while ofering medicinal values and thus participates in
improving the health of an individual. Additionally, they
are cost efective in their formulation and produce no
side efects and serious toxicities upon prolonged usage
[38]. Due to these properties, the phytochemicals have
high advantage to be labelled as drugs [39, 40]. Ginger
(Zingiber ofcinale) is an aromatic, pungent and spicy
herb, enriched with the natural phytochemicals. For a
time, this herbal species has been used as a favoring
agent and has been placed on the top list of folk medi
cines against common cold [41], sore throat [42] etc. It
is therefore understood that ginger is regarded to be safe
[39], however, little is known regarding its mechanism of
action and hence, careful assessment is required before
considering ginger phytochemicals for any therapy [43].
Moreover, ginger extracts were known to showcase its
inhibitory efect on Staphylococcus aureus [44, 45]. All
these intriguing factors focus our research to investigate
ginger phytochemicals against SaHPPK and elaborate
their mechanism of interaction.
In order to accomplish this goal, we have retrieved
eight selected ginger phytochemicals from published
literature based upon their therapeutic ability and anti
microbial activity [44, 46–52]. Te predictive inhibitory
efects of these selected phytochemicals have been evalu
ated against SaHPPK by molecular docking. To further
infer on the mode of binding and interaction with cata
lytic active residues, successful candidates have been
subjected to MD simulations. For the accomplishment of
this objective, docking of seven antibiotics, such as strep
tomycin, ampicillin, amoxicillin, methicillin, penicillin,
trimethoprim, and sulfamethoxazole were also consid
ered for comparative analysis. Finally, gingerenone-A and
shogaol propounded as potential ginger-phytochemicals
against SaHPPK.
Methods
Selection of the protein and its preparation
SaHPPK structure used for current investigation, has
been obtained from RCSB Protein Data Bank (PDB)
with PDB code 3QBC [26], which is in complex with
2-amino-8-sulfanyl-1,9-dihydro-6H-purin-6-one (8MG).
Te protein was prepared employing Discovery Studio
v4.5 (hereinafter D.S v4.5) by removing all heteroatoms
and the addition of hydrogen atoms [53]. Te structure
was energy minimized, until the convergence gradi
ent satisfed was obtained. Te active site was evaluated
10.0 Å around 8MG and the key residues were identifed
as Ala44, Tr43, Val46, and Asn56 [26]. Interrogating
the active site revealed the presence of two Phe residues;
Phe54 and Phe123, that are located on either side of the
8MG [26]. Moreover, the histidine residues of protein
were oriented in accordance with crystal structure to
ND1H protonation state.
Selection and preparation of the ligands
Ginger has several active compounds [54] and to the
best of our knowledge, they have not been tested against
SaHPPK as no reports were retrieved upon performing a
systematic search. Tis triggered our interest to under
stand how these phytochemicals efect the SaHPPK and
therefore, for the current study, phytochemicals namely
6-dehydrogingerdion, gingerenone-A, gingerol, paradol,
shogaol, zingerone, trans-1,8-cineole-3,6-dihydroxy-3-O-
β-d-glucopyranoside and trans-3-hydroxy-1,8-cineole
O-β-d-glucopyranoside were selected that have not been
assessed against SaHPPK [54, 55]. More specifcally the
selection of these phytochemicals was done based upon
their therapeutic ability as reported earlier [46–50]. Te
2D structures of the selected phytochemicals were rep
resented in (Fig. 1). Te corresponding 2D structures of
the eight phytochemicals were sketched on ChemSketch
(http://www.acdlabs.com/resources/freeware/Chem
Sketch/) and were subsequently, imported onto D.S v4.5
to generate their 3D structures.
Molecular docking mechanism
Molecular docking is a promising strategy to mimic
intermolecular binding modes and interactions. Particu
larly, molecular docking relies on binding site topology,
intermolecular afnity and interaction of key residues
with the ligand. For the current study, Genetic Optimi
zation for Ligand Docking (GOLD) v5.2.2 was employed
to perform the docking studies. Goldscore was recruited
to compute the binding afnities between the protein and
ligands, whereas the Chemscore was used for the rescor
ing purpose. Goldscore comprises of external H-bond,
external vdW, internal vdW and internal torsion. Moreo
ver, to obtain an appropriate binding pattern of ligands,
30 docking poses were allowed to generate. Additionally,
for identifying the best pose, the Goldscore, interactions
between the protein’s active site residues and ligand and
the binding modes were considered.
To further validate the obtained results, a second dock
ing programme, CDOCKER implemented on D.S v4.5
has been employed. Te results were evaluated based
upon CDOCKER interaction energy; higher CDOCKER
interaction energy implies greater favourable binding
[56]. Tis is a grid based docking operates by employing
CHARMm and facilities the generation of random ligand
conformations retrieved form the initial structure.
In‑vitro antimicrobial analysis of phytochemicals
In-vitro evaluation of the phytochemicals was performed
to infer the results obtained from the computational Page 4 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
approach. Te phytochemicals and the Staphylococ
cus aeureus were the generous gifts from the Osmania
University, Department of Botany, Hyderabad. To main
tain the prepared medium with no contamination, it
was autoclaved for 15 min at 15 lb pressure along with
petri dishes, spreader, 4–25 ml conical fasks, forceps,
inoculation loops and cotton balls. Te agar media was
then transferred into the petri dishes and was allowed to
solidify.
Preparation of the culture media
Te nutrient agar media was prepared by suspending
28 g of nutrient agar in 1000 ml distilled water according
to Mueller and Hinton [57], and was maintained at pH
7.0 and at room temperature.
Preparation of the inoculum
20 ml of the above-prepared media was transferred onto
the petri-dishes and was allowed to solidify. A loopful of
bacteria was transferred to 10 ml of distilled water in a
test tube and the addition is continued until the turbidity
is equal to standard 0.5 McFarland. Employing the cotton
swabs the inoculum was gently swabbed on the surface of
the media and were then allowed to dry.
Preparation of the disks
Te disks (Whatman 1 flter paper) were prepared with
the help of the punch machine of 6 mm in diameter. For
the current experiment, the two phytochemicals that
have produced the highest dock score along with the
antibiotic amoxicillin were considered for current in vitro
test. Te samples were prepared in the concentration of
1 mg/1 ml.
Te bacterial strain, overnight culture was grown in
broth was adjusted to an inoculum size of 106 CFU/ml
for inoculation of the agar plates. Te sterilized disks
were carefully impregnated with the sample, allowed
to dry for 1 min, and then transferred onto the petri
dishes containing 20 ml on nutrient agar. Te plates were
allowed to incubate for 24 h at 35
±2 °C and was followed
by measuring the zone of inhibition expressed in mm and
was performed in triplicates [58].
6 dehydrogingerdion Gingerenone – A Gingerol Paradol
Zingerone
Shogaol
trans-1,8-cineole-3,
6-dihydroxy-3-O-β-D
glucopyranoside
trans-3-hydroxy-
1,8-cineole -O-β-D
glucopyranoside
Fig. 1 2D structures of the selected phytochemicalsPage 5 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
Minimum inhibitory concentration
To quantify the minimum inhibitory concentration (MIC),
diferent concentration of the phytochemicals were tested
against the Staphylococcus aureus (MTCCB 737) ranging
between 0.05 and 2 mg/ml. Te MIC is defned as the low
est concentration of the phytochemical (highest dilution)
that can inhibit the growth of the bacteria.
Statistical analysis
In vitro results were analyzed employing the GraphPad
Prism v7.02 and were expressed as mean
±standard devi
ation recruiting the Turkey’s method and the correlation
analysis was executed by two-way ANOVA, P value less
than 0.005 was considered as signifcant.
Molecular dynamics simulations to assess the binding
modes of the hits against the reference
To gain further insight into the protein–ligand interac
tions, the selected ginger phytochemicals were subjected
to MD simulations along with reference compound
(8MG) and amoxicillin. Parameters for protein’s topol
ogy and coordinates were developed by CHARMm27 f
[59–62] in GROMACS 5.0.7 [63]. Te topologies of the
ligands and the cofactor were extracted from the Swiss
Param [64]. Te parameters for topology and coordinates
of protein and for corresponding ligand were merged,
and ten independent systems (one for each phytochemi
cal, one for amoxicillin, and one for 8MG) were designed.
Each system was solvated in a dodecahedron box, using
TIP3P water model, and neutralized with counter ions.
Each solvated system was energy minimized by employ
ing steepest descent algorithm for 10,000 steps and an
upper limit of force being lower than 1000 kJ/mol was
employed to remove any bad contacts and steric clashes of
protein–ligand complexes. Every minimized system was
grouped into protein–ligand and solvent-ions to escape
collapse, and subsequently, subjected to equilibration. Te
equilibration of each system was comprised of two com
ponents. First, equilibration was conducted at constant
volume (NVT) for 1 ns at constant temperature of 300 K
using Berendsen thermostat algorithm [65]. Following
this, second equilibration was executed for 1 ns at con
stant pressure (NPT) of 1 bar maintained by Parrinello–
Rahman barostat [66] and LINCS [67] was employed to
constrain all bonds. Particle Mesh Ewald (PME) [68] was
used to calculate long-range electrostatic interactions
with a cut-of of 1.2 nm. All short-range non-bonded
interactions were calculated within a cut-of of 1.2 nm. A
cut-of distance of 12 Å was attributed for Coulombic and
van der Waals interactions. All simulations were executed
using the NPT ensemble for 100 ns, and coordinates were
saved after each 2 fs intervals. Te results were examined
recruiting visual molecular dynamics [69] and D.S v4.5.
Results
Molecular docking
Molecular docking results showed that the eight ginger
phytochemicals have strong interactions with SaHPPK
protein, however, gingerenone-A and shogaol displayed
the highest Goldscore of 63.62 and 55.48 respectively
(Additional fle 1: Table S1). On the other hand, it was
noted that the antibiotics showed lower Goldscore than
the phytochemicals. Among them, amoxicillin has gen
erated the highest dock score of 41.98 (Additional fle 1:
Table S2), and therefore, this antibiotic was considered
for further studies. Hereinafter, the co-crystal, 8MG in
SaHPPK crystal structure, was designated as the refer
ence compound. Characteristically, this is a co-crystal
located at the active site of the protein and imparts
knowledge on the location where the chosen ligands
should be anchored at the proteins binding groove. Addi
tionally, this guides the key residues that are involved in
the inhibition and marked at 10.0 Å around its location.
Moreover, an interaction with these residues labels pro
spective drug candidates as efective. Furthermore, the
reference molecule should logically determine an appro
priate binding mode of the candidate molecules. Tere
fore, the 8MG has been represented as the reference
compound. To further evaluate the best conformation,
the dock scores rendered by GOLD and CDOCKER,
catalytic active residue(s) interactions and the binding
modes were opted as the determinant factors. Te study
was proceeded in the presence of Mg2+-AMPCC.
In‑vitro antimicrobial analysis of phytochemicals
Te ginger phytochemicals have rendered remarkable
results and were comparable with the standard refer
ence antibiotic and a control into which no inoculum was
added. Te mean zone of inhibition of the phytochemi
cal shogaol has recorded to be between 6 and 12 mm
and gingerenone-A was found to be between 2 and
8 mm, respectively. Tese reading are in harmony with
that of the reference antibiotic and were observed to be
4–16 mm. Te minimum zone of inhibition was observed
at 25 µg/ml for the phytochemicals and the reference
antibiotic. Te in vitro results further state that the phy
tochemicals could induce the inhibitory efect in par with
amoxicillin (Fig. 2).
Molecular dynamics simulations
To gain insight into the interaction mechanism of ligands
(selected phytochemicals, amoxicillin, and reference
compound), MD simulations were carried out [70–72]
for 100 ns and the results were analyzed. Trough
out the MD run, dynamic behavior and conformational
changes of all the ligands were monitored. Te root
mean square deviation (RMSD) of backbone atoms Page 6 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
was evaluated to estimate the stability of each protein–
ligand complex. Te reference molecule displayed an
average RMSD of~0.18 nm, while gingerenone-A and
shogaol projected~0.15 and~0.16 nm respectively
(Fig. 3), while amoxicillin has demonstrated a RMSD
of~0.17 nm. Additionally, all RMSD plots were noticed
to be in 0.1–0.27 nm range (Additional fle 1: Figure S1)
suggesting their stability during the simulation [73].
Furthermore, the potential energy analysis notifed that
all the compounds have ranged between −390,000 and
−395,000 kJ/mol, (Fig. 4). Te root mean square fuc
tuation (RMSF) profles of backbone atoms of the cor
responding systems shed light on their fuctuations.
Accordingly, for all the systems, backbone fuctuation
was observed within 0.49 nm and was found to be in sim
ilar manner. However, moderate deviations were noticed
for amoxicillin between 0.1 and 0.4 nm as depicted in
blue box with reference to the residues that lie between
90 and 110 (Fig. 5a) and their corresponding atoms
(Fig. 5b). Tis deviation might be because of the non
bonded water molecule that is present in the active site.
Te binding mode assessment was executed utilizing
the last 20 ns trajectories. Upon superimposition of the
Fig. 2 Antimicrobial activity of shogaol, gingerenone-A and amoxicil
lin expressed by zone of inhibition in mm
Fig. 3 RMSD profles of ten systems during 100 ns. The plots show variations during initial simulations and are stable towards last 20 ns. a Refer
ence, b amoxicillin, c gingerenone-A, d gingerol, e shogaol, f zingerone, g 6dehydrogingerdion, h paradol, i trans-1,8-cineole-3,6-dihydroxy-3-O-β-
d-glucopyranoside, j trans-3-hydroxy-1,8-cineole-O-β-d-glucopyranosidePage 7 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
representative structure of each system, it was observed
that all the ligands have occupied the binding pocket in a
similar manner as was observed for reference compound
(Fig. 6) and displayed similar binding pattern. Te bind
ing pocket is present towards the loop 2 and the cofactor
site is located near to loop 3. Since the target protein is
devoid of cofactor AMPCC and two Mg2+ ions, we have
imported their coordinates from SaHPPK protein with
the PDB code 5ETR. Additionally, the key residues that
shaped the active site were found to interact with the ref
erence compound as well as for the selected phytochemi
cals. Inspecting the molecular interactions revealed that
the reference compound has generated four hydrogen
bonds with key residues of SaHPPK (Fig. 7a). Te car
boxylic oxygen (hereinafter O) of Ala44 has interacted
with N3 of reference compound, whereas, O of Val46 has
H-bond interaction with N5 of reference compound. One
H-bond was detected between the OG1 of Tr43 residue
and N3 of reference compound, while another H-bond
was formed between ND2 of Asn56 residue and O1 of
reference compound. OD1 atom of Asn56 additionally
participated in H-bond interaction with the H13 atom
of the ligand. Furthermore, it was observed, that all the
H-bonds displayed a distance of~2.8 Å.
Amoxicillin formed one hydrogen bond between N
atom of Tyr48 has formed the H-bond with the O2 atom
of the ligand with a distance of 2.9 Å, while another
H-bond was observed for NE22 of Gln51 and O2 of
amoxicillin with a bond distance of 2.9 Å (Fig. 7b).
Hydrogen bond interactions with key residues were
noticed with all the phytochemicals, however their
number varied signifcantly (Fig. 7). To further authen
ticate our results, we have done a comparison with the
co-crystal inhibitor and the known antibiotic. Te active
site and the key residues (Ala44, Tr43, Val46, and
Asn56) were defned 10.0 Å around the inhibitor. Te
prospective drug molecules were examined critically for
their interactions with these residues. Amongst them,
Fig. 4 Potential energy plots of ten systems during 100 ns. The plots appear to be well converged between −390,000 and
−395,000 kJ/mol. a
Reference, b amoxicillin, c gingerenone-A, d gingerol, e shogaol, f zingerone, g 6dehydrogingerdion, h paradol,
i trans-1,8-cineole-3,6-dihydroxy-3-
O-β-d-glucopyranoside, j trans-3-hydroxy-1,8-cineole-O-β-d-glucopyranosidePage 8 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
phytochemicals, shogaol, gingerol, trans-1,8-cineole-
3,6-dihydroxy-3-O-β-d-glucopyranoside and trans-1,8-
cineole-3,6-dihydroxy-3-O-β-d-glucopyranoside have
displayed 2 H-bonds with protein, 6-dehydrogingerdione
and zingerone have formed one H-bond each while gin
gerenone-A displayed 4 H-bonds (Fig. 7).
Further delineating, it was noted that one H-bond was
observed between the N of Val46 and O2 of gingerenone
A with a bond distance of 2.3 Å. Te NE2 of Gln51 has
interacted with the O4 of the ligand with a distance of
2.9 Å. Another H-bond was noticed between the H44
of the gingerenone-A and OD1 of Asn56 with a distance
of 2.0 Å, while the other H-bond was detected between
NH2 of Arg121 and O3 of gingerenone-A with bond
distance of 1.3 Å, (Fig. 7c). Gingerenone-A also formed
water-mediated interaction with Asp95 of SaHPPK. On
the other hand, gingerol formed two hydrogen bonds
with Val46 and Gln51 each. Te O atom of the Val46 has
interacted with H37 of gingerol with a distance of 1.9 Å.
Gln51 has participated in the H-bond formation with
its NE2 atom and the O2 of the ligand with a distance of
2.9 Å (Fig. 7d). Moreover, shogaol formed two H-bond
with Val46 and Tyr48, respectively. Te H41 atom of the
ligand has formed a bond with the O of Val46 with a dis
tance of 2.9 Å, while the N atom of Tyr48 has interacted
with the O3 of the ligand with a distance of 2.9 Å (Fig. 7e).
Additionally, a water-mediated bond with Asp95 stabi
lized shogaol. Zingerone has also generated an H-bond
between N of Val46 and O3 of ligand with a bond dis
tance of 2.9 Å, (Fig. 7f). When 6-dehydrogingerdion was
evaluated for H-bond analysis, it was observed that O3 of
the inhibitor and N of Val46 formed a single H-bond with
a bond length of 2.5 Å, (Fig. 7g). Phytochemical paradol
did not render any hydrogen bond; however, it had shown
van der Waals interactions Table 1. Phytochemical trans-
1,8-cineole-3,6-dihydroxy-3-O-β-d-glucopyranoside
demonstrated two H-bonds with Val46 and Gln51. Te
O atom of Val46 has involved in the hydrogen bond with
0
0.1
0.2
0.3
0.4
0.5
0.6
0 20 40 60 80 100
120 140
160
Residue number
0
0.1
0.2
0.3
0.4
0.5
0.6
0
500
1000 1500 2000 2500 3000
Atoms
Inhibitor
Amoxicillin
Gingerenone-A
Gingerol
Shogaol
Zingerone
6 dehydrogingerdion
Paradol
trans-1,8-cineole-3,6-dihydroxy-3-O-β-D-glucopyranoside
trans-3-hydroxy-1,8-cineole 3-O-β-D-glucopyranoside
a
b
Fig. 5 RMSF plots during 100 ns. Blue box denotes the variations notices in the profles. The RMSF profle of amoxicillin is found to be relatively
deviated. a The RMSF of the residues. b The RMSF of the corresponding fuctuating atoms
RMSF (nm)
RMSF (nm)Page 9 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
H48 of the ligand with a distance of~1.8 Å. Te second
bond was formed between the HE22 of Gln51 and O20
of the ligand represented by a bond distance of 2.1 Å,
(Fig. 7h). Te compound trans-3-hydroxy-1,8-cineole-O-
β-d-glucopyranoside has rendered 2 H-bonds anchored
by Val46 and Gln51, respectively. Te O atom of Val46
and H47 of the ligand joined by a H-bond displaying a
bond length of 1.8 Å. Te second H-bond was formed
between the HE22 atom of Gln51 and O19 of the ligand
with a distance of 2.1 Å, (Fig. 7i).
Focusing on the importance of water molecule in
SaHPPK stability and augmenting its reactivity, it was
speculated that water molecule at active site plays a cru
cial rule (Fig. 7). Te water mediated bond with Asp95
was noticed in the presence of all the phytochemicals as
was observed with the reference molecule, however this
interaction was absent with the antibiotic amoxicillin,
(Fig. 7b). Further details of the interactions are recorded
in Table 1. Te resultant docked poses were validated by
MD simulation analysis and it was confrmed that the
binding stability of the selected poses remained unaltered
during the simulation.
Te interactions of the cofactor and the Mg2+ with the
protein were additionally evaluated that the benzene ring
of the adenine group has interacted with three hydro
gen bonds formed by Ile98 and Ser112 residues. Leu71
additionally holds the adenine group by the hydropho
bic interactions. Te ribose moiety has interacted with
Lys110 demonstrated by a hydrogen bond. Arg121 has
interacted with O1G of the cofactor by electrostatic
bond. Furthermore, the electrostatic bonds hold the
Mg2+ ions represented by Glu78 and Asp97. Addition
ally, the exposed O atoms of the cofactor frmly hold the
Mg2+ ions (Additional fle 1: Figure S2).
Inhibitor
Amoxicillin
Gingerenone-A
Gingerol
Shogaol
Zingerone
6 dehydrogingerdion
Paradol
trans-1,8-cineole-3,6-dihydroxy-3-O-β-D-glucopyranoside
trans-3-hydroxy-1,8-cineole 3-O-β-D-glucopyranoside
Fig. 6 Binding pattern of the co-crystal and the ginger phytochemicals. Only polar carbons are shown for clarity. Figure on the left depicts the
superimposition of the ligands and fgure right is its enlarged structure. The protein is represented in steel and the ligands in stick. The water mol
ecule is denoted in blue and the Mg2+ ions in greenPage 10 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
Discussion
Despite tremendous progress in medical sciences, the
efective treatment against infectious microorganisms
remains a major challenge. Te primary reason behind
this failure is the ability of the microorganisms to gain
resistance against antibiotics, which is conferred by a
variety of mechanisms. Consequently, it is essential to
develop new drugs that can efectively combat the micro
organisms and to overcome their pathogenicity. Staphy
lococcus aureus is one of the widely known pathogenic
microorganisms that has gained resistance against sev
eral antibiotics. In this study, ginger phytochemicals
were employed to evaluate their inhibitory efects when
challenged against microbial pathogenicity. Since, HPPK
plays a key role in microbial folate pathway and hence,
SaHPPK might be an ideal target for novel inhibitors.
Additionally, an ideal target should possess the following
attributes, such as having no homolog in humans, should
exist in large range of bacteria performing characteristics
role, should be specifcally druggable and should possess
a low cross-resistance potential (https://www.ncbi.nlm.
nih.gov/books/NBK200811/#sec_17). Because SaHPPK
represents all these characteristic features, we have relied
on SaHPPK for our current investigation.
Besides, the selection of the phytochemicals have
been performed based upon the literature search taking
into consideration that the phytochemicals portray and
are embedded with therapeutic activities [46–50]. We
aimed at understanding how these compounds that have
a similar structure act when challenged against SaHPPK
and further which phytochemical is potential against
the targeted protein. Such a study was conducted earlier
Fig. 7 Molecular interactions and the binding mode conformation of the reference and the phytochemicals with the protein target. Green dashed
lines demonstrate the hydrogen bonds between the protein and the ligands. The blue dashed lines represent the binding of the water molecule
and Asp95. The protein is represented in orange stick. The water molecule is denoted in blue and the Mg2+ ions in green. a Reference, b amoxicillin,
c gingerenone-A, d gingerol, e shogaol, f zingerone, g 6dehydrogingerdion, h paradol, i trans-1,8-cineole-3,6-dihydroxy-3-O-β-d-glucopyranoside, j
trans-3-hydroxy-1,8-cineole-O-β-d-glucopyranosidePage 11 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
considering diferent phytochemicals against diferent
targets and diseases [74–77]. Te study was conducted in
the presence of Mg2+-AMPCC to determine the efect of
the chosen phytochemicals.
Our investigation of eight ginger phytochemicals
against SaHPPK demonstrated that two candidate phy
tochemicals gingerenone-A and shogaol have higher
inhibitory efects. Furthermore, highest dock score, sta
ble orientation of selected phytochemicals in SaHPPK
active site and stable interactions with key residues,
support our investigation. Furthermore, to validate the
dock results, we have performed the in silico investiga
tion employing second docking tool, CDOCKER. Tese
docking results reafrm the superiority of gingerenone-A
and shogaol rendered by highest CDOCKER interaction
energy (Additional fle 1: Table S1) and the interaction
with key residues located at the active site of the protein.
Additionally, diferent interactions also afrmed that gin
gerenone-A and shogaol might be potential scafolds to
be developed has novel SaHPPK inhibitors.
In the current study, all the phytochemical have dis
played a score greater than the reference, however,
amongst them, since, gingerenone-A and shogaol pro
jected remarkable scores by both the molecular docking
programmes and further displayed stable MD results, we
therefore have considered only the top two dock scored
compounds for further evaluation and subsequently for
the in vitro experiments. Delineating on in vitro results,
it can be observed that the lead candidates have exhibited
remarkable inhibition at all the concentrations includ
ing at 25 µg/ml. However shogaol has demonstrated an
overall greater inhibition at 50 µg/ml while gingerenone
A rendered a marginally higher degree of inhibition at
75 µg/ml. Nevertheless, both the phytochemicals have
conferred with the inhibitory activities in par with the
antibiotic thus; afrm the inhibitory potential of ginger
phytochemicals against SaHPPK.
Since, it is widely accepted that S. aureus is resist
ant to existing antibiotics; we speculate that none of
these antibiotics have potential to inhibit SaHPPK. One
of the possible reasons for this failure is the missing of
H-bonding of amoxicillin with Val46. Valine at position
46 of SaHPPK has been reported as key catalytic residue
and forms stable H-bond with reference compound [26].
Our fndings showed that the phytochemicals as well as
the reference compound formed stable H-bond with
Val46 after 100 ns MD simulation, while such a binding
pattern was not observed for amoxicillin. Delineation on
Val46 and its signifcance, we scrupulously monitored
the interaction with their respective atoms of the ligands
throughout the MD run. Val46 was seen to anchor with
the ligands within the acceptable hydrogen bond length
of <3 Å. Conversely, amoxicillin failed to represent the
substantial interaction. Tis comparison and MD exam
ination led us to the conclusion that Val46 is crucial in
any SaHPPK targeted small molecule therapy. Apart from
that, water molecule plays very important role in increas
ing the accuracy and feasibility of chemical reactions [78,
79]. Te co-crystal structure of SaHPPK also proclaimed
a crucial role of the water molecule [26]. SaHPPK crystal
structure revealed that the reference compound is stabi
lized in enzyme’s active site by a water-mediated bond
Table 1 Molecular interactions between the protein and the compound
Compound
H-bond (<3.0 Å)
van der Waals interactions
π-Alkyl
Inhibitor
Ala44, Val46, Thr43, Asn56
Gly9, Pro45
Val46
Amoxicillin
Tyr48, Gln51
Gly9, Thr93, Asn11, Ile12, Thr43, Pro45, Gly47, Tyr48,
Thr93
Leu57
Gingerenone-A
Val46, Gln51, Asn56, Arg121 Gly9, Ala44, Gly47, Pro45, Phe54, Asn56, Asp97,
His115, Glu12, Phe149, Val154, Asp151, Ser153
Ala122
Gingerol
Val46, Gln51
Ala44, Asn56, Asp95, Arg121, Ala122, Pro127,
Ser153
Phe123, Val154
Shogaol
Val46, Tyr48
Gly7, Leu8, Gly9, Thr43, Ala44, Asn56, Phe54, Val96,
Asp95, Asp97, Leu99, Glu120, Ala122, Phe149,
Val154, Asp151, Ser153, His115
Val46,
Arg121,
Val124,
Phe 123
Zingerone
Val46
Gly7, Ser10, Asp95, Val96, Arg121
Val46
6-Dehydrogingerdion
Val46
Leu8, Ser10, Ala44, Asn56, Arg121, Arg151
Ala122
Paradol
–
Gly7, Leu8, Gly9, Thr43, Ala44, Pro45, Asn56, His115,
Glu120, Val124, Asp151, Ser153, Val154
Val46, Ala122
Trans-1,8-cineole-3,6-dihydroxy-3-O-β-d
glucopyranoside
Val46, Gln51
Pro45, Gly47, Tyr48, Phe54, Phe123
–
Trans-3-hydroxy-1,8-cineole-O-β-d
glucopyranoside
Val46, Gln51
Pro45, Gly47, Tyr48, Phe54, Phe123
–Page 12 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
present at Asp95 of the enzyme. Our results also showed
the same pattern of water-mediated stability for all phy
tochemicals as well as the reference compound (Fig. 7).
Conversely, amoxicillin could not gain water-mediated
stability in SaHPPK active site. Based on our results
we emphasize that the interaction of Val46 residue and
water-mediated interaction of Asp95 are the pre-requi
site for SaHPPK functional exploration and/or inhibition.
Te crystal structure of protein SaHPPK PBD code:
3QBC, has three loops loop 1, loop 2, loop 3; comprising
of 12–14, 45–51 and 82–94 residues, respectively [26]. As
in the crystal structure, the co-crystal substrate 8MG has
been sandwiched between aromatic residues Phe54 and
Phe123. Our results also confrmed similar π–π stacked
interactions between the ligand molecules and the Phe54
and Phe123 of the crystal structure.
Inhibitor
Amoxicillin Gingerenone-A
Shogaol
Gingerol
Zingerone
6-dehydrogingerdion Paradol
trans-3-hydroxy-
1,8-cineole -O-β-D
glucopyranoside
trans-1,8-cineole-3,
6-dihydroxy-3-O-β-D
glucopyranoside
Fig. 8 Diferent conformations exhibited by loop 1 (residues 1–9, in brown loop 2 (residues 43–53, denoted in olive green) and loop 3 (residues
82–92, represented in bottle green). Loop 1 and loop 2 remained semi-closed and closed in all the complexes, while the conformational changes
were noticed with loop 3. The protein is represented in steel and the ligands in stick. The water molecule is denoted in blue and the Mg2+ ions in
green
Table 2 Table depicting diferent conformational changes of the loops
Compound name
Loop 1
Loop 2
Loop 3
Inhibitor
Semi closed
Closed
Closed
6-Dehydrogingerdione
Semi closed
Closed
Closed
Gingerol
Semi closed
Closed
Closed
Zingerone
Semi closed
Closed
Closed
Amoxicillin
Semi closed
Closed
Semi-closed
Shogaol
Semi closed
Closed
Closed
Gingerenone-A
Semi closed
Closed
Closed
Paradol
Semi closed
Closed
Closed
Trans-1,8-cineole-3,6-dihydroxy-3-O-β-d-glucopyranoside
Semi closed
Closed
Closed
Trans-3-hydroxy-1,8-cineole-O-β-D-glucopyranoside
Semi closed
Closed
ClosedPage 13 of 15
Rampogu et al. Ann Clin Microbiol Antimicrob (2018) 17:16
We further investigated the structural topology and the
behaviour of the three loops upon the interaction with
the phytochemicals and the evaluation was based upon
the fndings of Kaifu et al. [80]. Loop 1 showed the semi
open conformation, while loop 2 and loop 3 demon
strated the closed conformations as represented in (Fig. 8
and Table 2). From the results, it was evident that loop
2 predominantly displayed closed conformation and loop
3 on the other hand showed closed conformation except
for amoxicillin. Tese results further impart informa
tion that the presence of AMPCC in the cofactor site has
induced the closed conformation of loop 3. We further
speculated that Asp95 that lies in close proximity with
loop 3 might also have played a role in providing closed
conformation while it shows semi-closed conformation
with amoxicillin, in which case the Asp95 binding was
absent (Fig. 8).
We further assessed to comprehend the binding ability
of the phytochemicals across diferent homologues. It is
well reported that the HPPK structures are highly con
served with six-strands in α-β-α fold with Mg2+ ions that
recognize the ATP and HMDP substrates. Additionally,
among the present HPPK proteins, the S. aureus homo
logue shares the identities with E. coli and Y. pestis by
39%, H. infuenza and S. cerevisiae by 37 and 34% with S.
pneumonia [36, 81]. It can be speculated that, since the
organisms share a conserved active site and high struc
tural similarity of the ternary complexes, the identifed
phytochemicals could also exert their antimicrobial efect
on its homologues as was reported earlier [36].
Based upon the results obtained from molecular dock
ing, MD simulation and in vitro studies it can be indi
cated that gingerenone-A and shogaol might be more
efective against SaHPPK. Consequently, we suggest
these two ginger-phytochemicals as foundation scafolds
for the development of novel SaHPPK inhibitors.
Conclusion
Te best way to increase the antibiotic activity especially
for the multidrug resistant bacteria is the inclusion of
natural products into the drug formulation, as they ofer
a plethora of advantages. In the present investigation,
ginger phytochemicals were evaluated for the prospec
tive drugs. Out of the eight phytochemicals, shogaol, and
gingerenone-A were potential drug candidates demon
strating highest dock scores and strong active site residue
interactions. Furthermore, the MD simulation results
confrmed their stable orientation and strong interactions
with catalytic active residues of SaHPPK catalytic pocket.
We therefore speculate that the two phytochemicals can
be efective against SaHPPK.
Authors’ contributions
SR and KWL conceived the idea of the project. SR, AB have conducted the
computational works. SR, AB and AZ wrote the manuscript. RGG performed
the in vitro experiments, RSB and RK performed the MD simulations of phyto
chemicals. SR, YSK, YJK and KWL approved the analysis of the manuscript. All
authors read and approved the fnal manuscript.
Author details
1 Division of Applied Life Science (BK21 Plus Program), Systems and
Synthetic
Agrobiotech Center (SSAC), Plant Molecular Biology and
Biotechnology
Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeo
ngsang National University, Jinju 52828, Republic of Korea. 2 Primer Biotech
Research Center, Jaipuri Colony, Nagole, Hyderabad, Telangana 500068, India.
3
Department of
Science Education, Kyungnam University, Changwon
51767,
Republic of
Korea.
4 Department of Chemical Engineering, Kangwon National
University, Chunchon 24341, Republic of Korea.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All the data is available with the manuscript and online.
Consent for publication
Yes.
Ethics approval and consent to participate
Not applicable.
Funding
This research was supported by Pioneer Research Center Program through the
National Research Foundation (NRF) funded by the Ministry of Science, ICT
and Future Planning (NRF-2015M3C1A3023028). Next-Generation BioGreen
21 Program (PJ01106202) from Rural Development Administration (RDA) of
Republic of Korea also supported this work. This material is based upon work
supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea) under
Industrial Technology Innovation Program (No. 10038744), ‘Establishment of
Drug Development System Using the Drug Repositioning Technology’.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub
lished maps and institutional afliations.
Received: 2 October 2017 Accepted: 9 March 2018
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Additional fle
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