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Bacterial siderophore mimicking iron complexes as
DNA targeting antimicrobials†
Sunil Kumar Boda,‡
a
Subhendu Pandit,‡
a
Aditya Garai,
b
Debnath Pal
c
and Bikramjit Basu*
ad
Microbial secretion of siderophores for iron uptake can be employed as an efficient strategy to smuggle in
bactericidal agents by conjugation to iron. Three iron complexes: complex 1 [Fe(L1)
2
]Cl
2
L1 ¼3-(pyridin-2-
yl)dipyrido[3,2-a:20,30-c]phenazine (pydppz or ligand 1), complex 2 [Fe(BHA)(L2)Cl]Cl$H
2
0 (BHA ¼
benzohydroxamate) L2 ¼pyrenyl-dipicolylamine (pydpa or ligand 2) and complex 3 [Fe(BHA)(L3)Cl]
Cl$H
2
0L3¼phenyl-dipicolylamine (phdpa or ligand 3) were synthesized. The ligands were docked for
binding to DNA and DNA polymerase I. The antibacterial efficacy of the iron complexes were evaluated
against three pathogenic bacteria –Staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas
aeruginosa as well as cytotoxicity assessed in C2C12 mouse myoblast cells. In silico docking and
molecular dynamic simulations of the ligands revealed stable and non-specific binding to DNA and DNA
polymerase I, in the order: L1 > L2 > L3. The bactericidal effect of the iron complexes against MRSA
predominantly occurred by bacterial DNA fragmentation as analyzed from gel electrophoresis and
comet assay. The extent of DNA damage followed the order: complex 1 > complex 2 > complex 3, in
commensurate with docking. Siderophore production elicited preferential bactericidal action of the iron
complexes against MRSA. Both, complex 1 and complex 2 were 5–10 fold less toxic in C2C12 cells
compared to MRSA. Taken together, a combination of DNA targeting and siderophore mimicking ligands
conjugated to iron can be deployed as Trojan horse for the entry of antimicrobials into pathogenic
bacteria. Thus, DNA targeting antimicrobials offer a promising solution to persistent bacterial infections.
Their selectivity towards microbes can be promoted via siderophore uptake pathways.
1. Introduction
Iron, in its soluble form, is an essential nutrient for most
bacteria. Iron is a co-factor for iron–sulphur clusters and
haem proteins along with enzymes, like superoxide dis-
mutase, peroxidase, glutamate synthase, and nitrogenase.
These enzymes facilitate cellular processes in bacteria, such
as oxidative phosphorylation, nitrogen xation, electron
transport and so on.
1
Further, iron is a coenzyme or enzyme
activator of ribonucleotide reductase, which converts ribo-
nucleotides to deoxyribonucleotidides.
2
However, most of
the iron in aerobic environments exists as insoluble Fe-
oxides or hydroxides. The solubility of ferric iron is 10
17
M at neutral pH, while bacteria require 10
5
to 10
7
Mofiron
for optimal growth.
3
Hence, most pathogenic bacteria secrete
high affinity Fe scavenging organic molecules called side-
rophores, which facilitate iron acquisition by forming
soluble iron–chelate complexes. These iron–chelates are
actively transported across the cell membrane and such
transport is mediated by periplasmic binding proteins and
cytoplasmic membrane transporters.
4
Fe is a hard Lewis acid
and hence most of the chelating siderophore molecules
coordinate to ferric ions through Lewis bases with hard
donor atoms, like neutral oxygen and nitrogen atoms or
negatively charged oxygen. In general, three types of iron
coordinating motifs are present in most microbial side-
rophores. These include hydroxyl groups from catechol
moieties like in enterobactin secreted by E. coli,hydroxamate
as in coprogen B and citrate derived a-hydroxy carboxylate
motifs as in petrobactin or carboxylates in rhiozoferrin.
5
The
bidentate chelating siderophores have a greater affinity to
ferric iron and form more stable hexacoordinate complexes,
resulting from a smaller entropy change as compared to
monodentate ligands. The secretion of siderophores under
iron –decient conditions is implicated in bacterial
a
Laboratory for Biomaterials –Materials Research Centre, Indian Institute of Science,
Bangalore –560012, India. E-mail: bikram@mrc.iisc.ernet.in
b
Department of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore –560012, India
c
Department of Computational and Data Sciences, Indian Institute of Science,
Bangalore –560012, India
d
Centre for Biosystems Science and Engineering, Indian Institute of Science, Bangalore
–560012, India
†Electronic supplementary information (ESI) available: See DOI:
10.1039/c6ra02603f
‡Equal contribution.
Cite this: RSC Adv.,2016,6, 39245
Received 28th January 2016
Accepted 10th April 2016
DOI: 10.1039/c6ra02603f
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virulence and pathogenecity.
6,7
Under infection of host by
bacterial pathogens, the microbes secrete siderophore
molecules and assimilate iron from mammalian iron-
binding proteins, such as transferrin and lactoferrin.
8
One of the antimicrobial strategies being employed is the
synthesis of articial siderophores conjugated to antibiotics
that are up taken by bacteria along with iron–chelates of the
antibiotic-conjugated siderophores. Another less explored
option is DNA targeting antimicrobials with specicity or
selectivity for inducing bacteriotoxicity. The iron–chelates of
siderophores can be used as ‘Trojan horse’to circumvent
permeability-mediated drug resistance and selective DNA
targeting for bactericidal action against most pathogenic
bacteria.
9
In the present study, the iron complexes: complex 1
[Fe(L1)
2
]Cl
2
L1 ¼3-(pyridin-2-yl)dipyrido[3,2-a:20,30-c]phena-
zine (pydppz or ligand 1), complex 2 [Fe(BHA) (L2)Cl]Cl$H
2
0
(BHA ¼benzohydroxamate) L2 ¼pyrenyl-dipicolylamine
(pydpa or ligand 2), and complex 3 [Fe(BHA)(L3)Cl]Cl$H
2
0
L3 ¼phenyl-dipicolylamine (phdpa or ligand 3), are tested for
their antibacterial and antibiolm efficacy against pathogenic
Gram-positive bacteria –Staphylococcus aureus (MRSA, USA
300) and Gram-negative bacteria –E. coli K12 (MG 1655) wild
type and Pseudomonas aeruginosa.Undersimilardosage,the
cytotoxicity of the above complexes is assessed in the dosage
window of antimicrobial action using C2C12 mouse myoblast
cells as a representative non-cancerous cell line. This is in the
light of earlier studies reporting comparable photo-
cytotoxicity elicited by identical iron complexes in HeLa
cancer cells, MCF-7 breast cancer cells and human keratino-
cyte cells HaCaT (non-tumorogenic).
10,11
One can, therefore,
argue that such similar iron complexes could be potent
bactericidal agents under non-photodynamic conditions, and
presents a strong case for in silico and in vitro investigations
of the bacteriotoxicity as envisaged in the current study.
Hence, we have employed docking and molecular dynamic
simulations to correlate in silico predictions and in vitro
bacteriotoxicity results, in a unique approach to corroborate
theory and experiments. Further, the therapeutic safety of
such metal complexes as antibacterial agents is also assessed
by TOPKAT, a computer assisted program that predicts
chemical toxicity.
12
Summarizing, we have deployed a novel
approach via bacterial siderophore mimicking iron
complexesasaTrojanhorsestrategyforbacterialuptakeand
eliciting bactericidal action via DNA intercalation and
fragmentation.
2. Materials and methods
2.1 Ligand exchange of labile iron complexes
The three iron complexes were synthesized following earlier
reported protocols.
10,11
The detailed synthesis procedures are
presented in the ESI†along with the reaction schemes and
characterization of the ligands and complexes. Here, the labile
nature of the synthesized iron complexes is characterized by
ligand exchange reaction with a strong metal chelator
8-hydroxyquinoline (8-HQ).
13
Incidentally, 8-HQ has been re-
ported as a potential phytosiderophore.
14
It is also known to be
a chemical moiety present in quinolobactin, a natural side-
rophore produced by Pseudomonas uorescens
15
as well as an
integral component of oxinobactin and sulfaxinobactin, both
synthetic analogues of enterobactin siderophore secreted by
E. coli.
16
The ligand exchange of the iron complexes with an
excess of 8-HQ was studied by monitoring the absorption
spectral changes recorded using UV-vis spectrophotometer
(Eppendorf). For the study, 2 : 1 equivalents of 8-HQ : complex
1 and 1 : 1 equivalents of 8-HQ : complex 2/complex 3 in DMSO
were mixed and the absorption spectra were recorded imme-
diately and aer a time interval. Further absorption spectra of
Fe
3+
: 8-HQ in 1 : 3, 1 : 2 and 1 : 1 mole ratios were recorded
using 0.4 mM of Fe(NO
3
)
3
as the iron precursor. These
measurements were made to compare the absorption spectra of
the iron complexes aer ligand exchange with 8-HQ.
2.2 DNA binding and fragmentation
Virtual screening of potential DNA and/or protein binding drug
candidates has become a common and standard technology in
modern drug discovery.
17
The AutoDock
18
and similar sowares
have been widely used for virtual screening of such drug
molecules. Here, we have used Autodock Vina
19
and AutoDock
tools
17
to calculate and analyze the binding of the ligands of the
labile iron complexes with few random nucleic acid sequences,
B-DNA Dickerson dodecamer and DNA polymerase I. The
molecular coordinates were taken from the Protein Data Bank
(PDB; http://www.rcsb.org). Discovery Studio
20
was used to
visualize and generate graphical images of interactions. The
default parameters were used for the molecular docking simu-
lation. Binding energy, number of hydrogen bonds, number of
electrostatic interaction and number of hydrophobic interac-
tion in the docking mode with highest score has been taken as
a measurement of theoretical binding strength. The experi-
mental investigations of DNA binding and fragmentation were
carried out by performing DNA gel electrophoresis and comet/
single cell gel electrophoresis (SCGE) assays.
2.2.1 Docking and molecular dynamics simulation with
DNA. We have performed docking of ligands from our
complexes with a standard representative B-DNA Dickerson
dodecamer (PDB ID: 1BNA)
21
sequence and some randomly
generated DNA sequences of 10 or 15 base pairs (ESI Table S1†).
The randomly generated sequences are constructed using
Discovery Studio.
20
Prior to docking, the ligands were energy
minimized in Discovery Studio using CHARm forceeld. The
ligands were docked using AutoDock Vina and AutoDock Tools.
The results were analyzed using Discovery Studio for calculating
the number of interactions in the best binding mode of the
ligand with receptor molecule. Top ve binding mode energies
of docking ligand 1, 2 and 3 to Dickerson dodecamer are
tabulated (ESI Table S2†). The binding energy and the number
of interactions of the ligands with the receptor DNA are re-
ported. The best binding modes for interaction between Dick-
erson dodecamer and ligands were subjected to 10 ns of
Nanoscale Molecular Dynamics (NAMD) simulation with 2 fs
step-size in NVT model with CHARm force eld to conrm
stable binding of the ligand (ESI Fig. S3†).
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2.2.2 Docking and molecular dynamics simulation with
DNA polymerase I. DNA polymerase I was obtained from PDB
(structure ID: 3EYZ).
22
The ligands were docked using AutoDock
Vina and AutoDock Tools at the PDB dened binding site of the
protein. The best t model was analyzed using Discovery Studio
4.0 Client and the affinities along with the number of interac-
tions are reported. The best binding modes of ligands with
3EYZ were subjected to 10 ns of NAMD with 2 fs step-size in NVT
model with CHARm forceeld to conrm stable binding of the
ligand (ESI Fig. S3†).
2.2.3 DNA gel electrophoresis. For analyzing the extent of
bacterial DNA fragmentation by the iron complexes, DNA gel
electrophoresis of bacterial cell lysates was carried out following
the protocol described elsewhere.
23
In brief, the bacteria treated
with 100 mM of the iron complexes for 4 h were harvested by
centrifugation at 12 000 gfor 5 min. The cell pellets were
resuspended in 50 mL of lysostaphin (Sigma; 100 mgmL
1
) and
incubated at 37 C for 10 min. Subsequently, 50 mL of proteinase
K (100 mgmL
1
) and 100 mLof1TE buffer (10 mM Tris–HCl of
pH 8 and 0.1 mM EDTA) were added to each tube and incubated
for an additional 10 min at 37 C. Finally, the bacterial
suspensions were placed in boiling water for 5 min to complete
the lysis. 50 mL of the lysates were loaded into the wells of a 0.8%
agarose gel and the samples were run at 5 V cm
1
.
2.2.4 Single cell gel electrophoresis (SCGE)/comet assay.
Comet or alkaline SCGE assay was performed to ascertain
bacterial DNA strand breaks upon treatment with 10 mM of the
iron complexes following the protocol established by Olive.
24
In
brief, the iron complex treated MRSA were mixed with low
melting agarose and applied on a glass slide precoated with
1.5% agarose. Upon solidication of the gel, the embedded
bacteria were lysed by overnight incubation of the slides in
alkaline lysis buffer (1.2 M NaCl, 100 mM EDTA, 0.1% sodium
lauroyl sarcosinate and 0.26 M NaOH) at 4 C. Subsequently, the
slides were carefully washed in ice-cold rinse buffer (0.03 M
NaOH and 2 mM Na
2
EDTA). The slides were subjected to elec-
trophoresis at 0.6 V cm
1
, 10 mA for 25 min, removed and
immersed in DI water. Finally the slides were stained with
propidium iodide (10 mgmL
1
) and visualized under uores-
cence (Nikon) and Confocal (Carl Zeiss 700) microscopes.
2.3 Antimicrobial and anti-biolm testing
2.3.1 Bacterial strains and culture protocol. For evaluation
of the antimicrobial efficacy of the iron complexes, one Gram
positive bacterium –Staphylococcus aureus (MRSA, USA300) and
two Gram negative bacteria –E. coli K12 wild type and Pseudo-
monas aeruginosa were chosen as representative microbes for
the study. The freeze dried stocks of the above bacterial species
were revived on tryptone soya agar plates. Single colonies of the
bacteria were cultured overnight for 10–12 h in 5 mL of Luria
broth (LB –20 g L
1
) media and 50 mL was sub-cultured in 4 mL
of fresh LB for 2.5 h. The optical density of the seeding bacteria
was adjusted to A
600 nm
¼0.1 (10
7
to 10
8
bacteria per mL) and
used for the experiments.
2.3.2 Determination of minimum inhibitory concentration
(MIC). The minimum inhibitory concentrations (MIC) of the
iron complexes were determined by microbroth dilution
method in 96-well plates. The puried test compounds were
dissolved in DMSO (Merck) to prepare 1 mM of fresh stock
solutions. The working solution concentrations of 200, 100, 50,
25 and 12.5 mM were prepared by two-fold serial dilution with
LB media. 100 mL of the above working solutions were added to
100 mL of bacterial suspension with A
600 nm
¼0.1 (10
7
to 10
8
bacteria per mL). With the help of a microplate reader
(Eppendorf AF2200) equipped with a shaker and thermostat set
to 37 C, the bacterial growth curves were monitored over
a period of 18 h, in a real time kinetic cycle with readings taken
at 10 min intervals followed by orbital shaking. The minimum
concentration at which there was no rise or 99% decline in the
growth curves was designated as minimum inhibitory concen-
tration (MIC).
2.3.3 Bacterial viability in mature biolms. MRSA biolms
were grown in 96 well microtiter plates for a period of 48 h with
a media change aer 24 h. The mature biolms were washed
with 1phosphate buffered saline (PBS) and treated with 0, 25,
50 and 100 mM of the iron complexes for 24 h. The bacterial
viability in the control and treated biolms was determined by
resazurin dye reduction test, as reported in one of our earlier
study.
25
Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is
a blue-colored dye, which is enzymatically reduced to a pink
uorescent molecule, resofurin by viable bacteria. Aer the
treatment of the biolms with the iron complexes, the media
from the wells was aspirated and washed thrice with 1PBS to
remove the planktonic bacteria. Subsequently, 200 mL of fresh
LB was added to each well followed by 10 mL of resazurin (0.5 mg
per well; Sigma) and incubated for 45 min at 37 C. With the
help of uorimeter (Eppendorf AF2200) equipped with the dye
excitation lter at 535 nm, the emission uorescence intensity
of resofurin was measured at 590 nm.
2.3.4 Total biomass quantication. The total biomass of
the biolms comprising of live/dead bacteria along with the
secreted polysaccharide matrix were quantied by staining with
0.1% (w/v) crystal violet.
25
The control and treated biolms were
washed with 1PBS and stained with 200 mL of 0.1% (w/v)
crystal violet for 10 min. Subsequently, the biolms were care-
fully aspirated to remove the unbound stain and washed with
PBS to remove the excess dye. The bound crystal violet stain was
then solubilized with 150 mL of 33% acetic acid by incubation
for 15 min. From each well, 125 mL of the eluate was used to
measure the absorbance of the samples at 600 nm in a multi-
mode plate reader (Eppendorf AF2200).
2.3.5 Fluorescence microscopy of live/dead biolms. For
the live/dead visualization of biolms, MRSA biolms were
grown on tissue culture glass cover slips. Aer the experimental
duration, the biolms were washed carefully with 1PBS and
stained by the contents of live/dead Baclight kit (Invitrogen;
L7012). The kit contains a green uorescent dye, Syto9 which
binds to the DNA of all bacteria along with polysaccharide
matrix while propidium iodide (PI) intercalates with the DNA of
membrane compromised cells. Each sample was stained with
500 mL of 1 : 2000 dilutions of the two dyes and incubated for 20
min, as optimized in our earlier studies.
26,27
The samples were
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then washed twice with 1PBS and were visualized under
auorescence microscope (Nikon) with an oil immersion lens.
2.3.6 Bacterial siderophore detection assay. The produc-
tion of bacterial siderophores by MRSA under iron decient
conditions and when supplemented with different concen-
trations of freely available Fe
2+
as well as iron complexes was
investigated in planktonic cultures, following the method
provided by Neilands et al.
7
For the study, the MRSA strain
was cultured in normal LB media until the bacteria reached
the logarithmic growth phase. Subsequently, the bacteria
were harvested by centrifugation at 2400 gfor 5 min and
resuspendedinirondecient minimum salt
medium 9 (MM9). The recipe and preparation of MM9 media
was followed from an earlier work.
28
The MM9 media was
further supplemented with 0.3% casamino acids and 0.05%
glutamic acid as nitrogen source while glucose was the
carbon source.
The bacteria in the logarithmic phase were cultured for 48 h
in iron decient MM9 media and supplemented with 3.13, 6.25,
12.5 and 25 mM concentrations of Fe
2+
and iron complexes in
a 96 well microtiter plate. It is understood that these are
MIC/sub-MIC concentrations of the iron complexes can elicit
siderophore production. Aer 48 h, the bacteria were centri-
fuged at 9600 gand the supernatants were collected. 100 mLof
the supernatant was treated with 100 mL of Chrome Azurol
S (CAS) assay solution. The recipe and protocol for preparing the
CAS assay solution was followed from a previous study.
29
The
1 : 1 mixtures of the culture supernatant and CAS assay solution
were allowed to equilibrate in dark for 1 h at room temperature.
The absorbance of the yellowish brown solution was measured
at 630 nm in a multimode plate reader. Also, a wavelength scan
of the absorbance spectra in the UV-visible range was performed
in order to ascertain the type of Fe–siderophore species.
2.4 Cytotoxicity assessment
2.4.1 C2C12 mouse myoblast culture. The C2C12 mouse
myoblast cell line was procured from the National Centre for
Biological Sciences, Bangalore, India. The C2C12 cells were
revived from cryopreserved stock and grown in complete growth
medium containing DMEM (Dulbecco's modied Eagle's
medium; Invitrogen), 20% FBS (fetal bovine serum; Invitrogen),
1% antibiotic antimycotic solution (Sigma) and 2 mM L-gluta-
mine (Invitrogen). The cells were maintained in a CO
2
incubator
(Sanyo, MCO-18AC, USA) at 37 C, 95% humidity and 5% CO
2
.
Upon reaching 70–80% conuency, the cells were detached
using 0.05% trypsin–EDTA and harvested by centrifugation at
425 gfor 5 min. The cells were sub-cultured for further use as
required.
2.4.2 Cell viability (MTT) assay. The cytotoxicity of the
three iron complexes in C2C12 mouse myoblast cells was
assessed by a 96-well microtiter based assay. Around 5000 cells
per well were seeded, supplemented with 200 mL of complete
media and incubated for 24 h at 37 CinaCO
2
incubator. As
performed in the antimicrobial assays, the cells were treated
with 25, 50 and 100 mM concentrations of the iron complexes
for 24 h and 72 h. Aer the stipulated time intervals, the media
from the control and treated cultures was aspirated and fresh
media containing 15% MTT (5 mg mL
1
; Sigma) reagent was
added. Following incubation with the dye for 4 h, the resultant
formazan crystals were solubilized in DMSO and the absor-
bance was measured at 600 nm using a multimode plate reader
(AF2200, Eppendorf).
2.4.3 Cell apoptosis/necrosis analysis by ow cytometry.
The mode of cell death caused by the iron complexes in the
treated myoblasts was monitored by ow cytometry. The
disruption of mitochondrial membrane potential is an indi-
cator of early cell apoptosis. JC-1 is a carbocyanine dye, which
is sensitive to mitochondrial membrane potential.
30
JC-1
aggregates in the live cells show red uorescence, while
mitochondrial membrane depolarized cells exhibit green
uorescence due to JC-1 monomers in the mitochondria.
31
The
induction of mitochondrial membrane depolarization by the
iron complexes was evaluated using ow cytometry. In
a manner similar to the above, the C2C12 cells were treated
with 25, 50 and 100 mM concentrations of the iron complexes
for 24 h and harvested by centrifugation at 425 gfor 5 min.
For ow cytometry, the cell suspensions in PBS were incubated
with JC-1 dye (10 mgmL
1
)at37Cfor10min.Thedeadcells
(apoptotic/necrotic) cells were determined by staining with PI
(10 mgmL
1
) and incubation for 5 min. The samples were run
on BD FACS Canto II ow cytometry. The data were analyzed
usingBDFACSDivaVersion6.1.1soware.
2.4.4 TOPKAT predictions. TOPKAT was employed to
predict the comparative in vivo toxicity of the iron complex
ligands with some of the FDA approved molecules. TOPKAT
(TOxicity Prediction by Komputer Assisted Technology) is
a computational tool for quantitative structure activity rela-
tionship (SAR) based toxicity prediction performed with the
help of structural descriptors. The molecule is broken into
fragments by the soware, along with chemical descriptors
such as molecular weight, symmetry of the molecule of
interest, and the toxicity is predicted.
32
In the present study, we
apply TOPKAT to predict the toxicity of our ligands and
compare the same in reference to commercially available
photodynamic therapy (PDT) drug pormer (Drug Bank ID:
DB00707) and a DNA targeted drug doxorubicin (DB00997). We
used the Predictive Toxicology packages provided by Accelrys
through Discovery Studio platform and used two models,
TOPKAT_rat_inhalational_LC
50
and TOPKAT_rat_oral_LD
50
,
to predict the toxicity/safety of our ligands in relation to
DB00707 and DB00997.
2.5 Statistical analysis
The statistical analysis for all the presented data was carried
out using IBM SPSS Statistics 20 soware. All the experiments
were carried out in triplicate and the analysis of variance (one
way ANOVA) was adopted to determine the statistical signi-
cance between the iron complex treated bacteria/cells and the
untreated controls. For the data analysis, Tukey and Games–
Howell tests were employed to determine the statistical
signicance at p< 0.05, where pdenotes the probability that
there is no signicant difference between the means.
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3. Results
We investigated the DNA binding affinities of the iron complex
ligands in silico as well as antibacterial efficacy and cytotoxicity
of the labile iron complexes in vitro. The degradation of
bacterial DNA by the iron complexes is studied by gel electro-
phoresis methods. Further, the in vivo toxicity of the iron
complexes is predicted by TOPKAT, in comparison to FDA
approved drugs. Overall, the utility of in silico methods for
predicting chemical and molecular toxicity are demonstrated
in the present study.
3.1 Labile behavior of the iron complexes
The molecular structures of the iron complexes and their cor-
responding ligands are shown in Fig. 1. The labile behavior of
the iron complexes is established by ligand exchange reaction
with 8-hydroxyquinoline (8-HQ), a strong metal chelator.
Following the addition of excess of 8-HQ, an instantaneous
color change was observed for both complex 2 and 3. The
change, as recorded in the absorption spectra, indicated a rapid
ligand exchange reaction. However, in case of complex 1, we
observed a continuous decrease in the intensity of the absorp-
tion peak in the UV range. The addition of few drops of
hydrogen peroxide (H
2
O
2
) accelerated the ligand exchange
reaction, resulting in a quick change in the absorption spectra
(see ESI Fig. S2A†). The ligand exchange reactions of complex 2
and complex 3 with 8-HQ are shown in Fig. S2B and C,†
respectively. A clear correlation of the absorption spectra of the
iron complexes, following ligand exchange with 8-HQ (Fig. S2A–
C†) can be observed by comparison with the UV-visible
absorption spectra for iron complexes prepared using ferric
iron : 8-HQ in 1 : 3, 1 : 2 and 1 : 1 mole ratios (Fig. S2D†). The
absorption maxima at 450 nm and near 600 nm recorded for
complex 1, 2 and 3 aer ligand exchange with 8-HQ match well
with the ferric iron : 8-HQ in different mole ratios. The above
results qualitatively proves the labile nature of the three iron
complexes used further for molecular docking and elucidating
the bacterial/cell cytotoxicity, as described in the subsequent
sections.
3.2 In silico and in vitro analysis of DNA binding and
fragmentation
The docking results of the ligands from the labile iron
complexes have been presented in terms of the binding energies
and number of interactions between the ligand and the receptor
sequence of DNA/protein. The best binding modes and binding
stability of the ligands to DNA and DNA Pol I were determined
by docking studies and molecular dynamic simulations.
3.2.1 Docking with DNA. The binding energies of Dick-
erson dodecamer with the ligands 1, 2, 3 (which are ligands
associated with complex 1, 2, 3 respectively) are shown in Table
1. The binding energy data in Table 1 lists the order for binding
energy of the ligands to the B-DNA Dickerson dodecamer
sequence as ligand 1 > ligand 2 > ligand 3. The docking results
show the possible modes of binding of the iron complexes in
biological systems. The binding energy data (Table 1) show that
the ligands from the labile iron complexes can intercalate with
cellular DNA, thereby hindering DNA replication and cell
multiplication. Similar affinity values generated by docking
with random DNA sequences (10–15 basepairs) also exhibit the
same trend and those results are presented in ESI Table S1.†
Also, the number of different kind of interactions between the
ligand and receptor in the optimal binding conguration is an
important parameter to predict the goodness of the binding.
This is visualized using Discovery Studio. The number and types
of receptor–ligand interactions in case of B-DNA Dickerson
dodecamer is shown in Table 1. In contrast to the binding
energy pattern, the number of ligand receptor interactions
presents the following trend: ligand 2 > ligand 3 ¼ligand 1. This
is due to counting of the hydrophobic interactions, which
contribute marginally to the overall binding energy. But in
complex 1, two molecules of ligand 1 are present in one mole-
cule of iron complex. This implies that upon cellular internal-
ization and degradation of the complex, two molecules of ligand
1 will be released making the effective ligand concentration
double compared to the initial molar concentration of the iron
complex. To account for this, we can say that the effective
number of ligand–DNA interactions upon dissociation of
complex 1 is double i.e. 22¼4.
Fig. 1 Molecular structures of the siderophore mimicking iron complexes and associated ligands synthesized for the study.
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Binding energy is a better parameter to determine the
strength of interaction compared to counting number of inter-
actions, because there may be a handful of weak interactions of
very low signicance, though the total interaction is not very
strong. From the best ve docking mode energies of ligand 1, 2
and 3 binding to Dickerson dodecamer (shown in ESI Table
S2†), we can see the energies are very closely spaced. This
suggests that there is no preferential site and the ligands bind
to DNA in a non-specic manner. The representative graphical
image of interaction between Dickerson dodecamer (PDB ID:
1BNA) and ligand 1 is shown in Fig. 2A(a) while those with
ligand 2 and ligand 3 are shown in Fig. S3.†All such observa-
tions corroborate well with the fact that the ligands bind to the
DNA groove, as experimentally observed by Garai et al.
10
Further, the binding constants (K
b
) for the intercalation of the
iron complexes with calf-thymus DNA (determined by UV-vis
absorption titrations) have been listed in Table 5. In the
present work, we conrm that the previously reported experi-
mental data
10,11
(Table 5) are in excellent agreement with the
docking results of specic and randomly designed DNA
sequences with the aromatic ligands of the iron complexes
(Tables 1 and S1†).
The best ligand binding modes were subjected to 10 ns
molecular dynamics simulation to conrm the stability of the
complexes. Total energy versus time plot (ESI Fig. S4†) suggests
stable binding. We plotted distance between randomly chosen
atoms of the ligand and binding site with time, and the plot also
indicates stable binding (Fig. 2B). The ligands bind to DNA in
a non-specic manner and such binding is stable, as suggested
by the molecular dynamics simulation.
3.2.2 Docking with DNA polymerase I. The binding energy
of the ligands to DNA Pol I is presented in Table 2. A similar
order of ligand affinity to the DNA Pol I enzyme like that towards
DNA was observed i.e., as ligand 1 > ligand 2 > ligand 3. Further,
the number of interactions for the ligands with DNA Pol I, in the
optimal binding conguration is listed in Table 2. Here too, the
same phenomenon is observed of ligand 1, as in case of docking
with DNA, where we see a different trend for number of inter-
action: ligand 2 < ligand 1 < ligand 3. Obviously, the hydrogen
bonds for ligand 1 appear to contribute more towards binding
energy than the electrostatic interactions for ligand 2. Due to
this, the binding energy trend diverges from the trend for
number of contacts. It can be reiterated here that complex 1 will
release two ligand 1 molecules upon cellular uptake, making its
effective molar concentration double compared to the intake of
the other two complexes. From the best ve docking mode (see
ESI Table S2†), we can see the best binding energy for ligand
1 with DNA Pol I is > 1 kcal mol
1
than the next best binding
candidate, suggesting specic binding of ligand 1 to the active
site of the protein. In case of ligand 2 with DNA Pol I, the
difference between the best binding energy and the next best
candidate is less, but the molecular dynamics simulation (ESI
Fig. S4D–F†) suggest tight binding, implying a fairly specic
binding. However, in case of ligand 3 with DNA Pol I, the
Table 1 Binding energies and number of interactions between ligand 1, 2 and 3 with Dickerson dodecamer (DD-DNA) at best binding mode
Ligand
Binding energy for DD-DNA
(1BNA) (kcal mol
1
)
Types and no. of ligand–DNA interactions
H-bond (conventional) Electrostatic Hydrophobic
a
Total
Ligand 1 9.9 2 0 0 2
Ligand 2 8.1 1 1 1 3
Ligand 3 6.3 2 0 0 2
a
Contacts include p–shydrophobic contact from DNA and ligand.
Fig. 2 (A) Interaction of ligand 1 with (a) B-DNA Dickerson dodecamer
(1BNA) and (b) DNA Pol I (3EYZ). All the three ligands bind to the minor
groove of DNA in a non-specific manner. For DNA Pol I, ligand 1 bind
to the active site specifically, ligand 2 exhibits moderate specificity and
ligand 3 has a non-specific binding to the active site. The binding is
stable in all cases irrespective of the specificity, as confirmed by the
molecular dynamics calculations. (B) Average distance between
centroid of the ligand and three randomly chosen atoms at the binding
site of the receptor molecule has been calculated in an interval of 1 ns
of the 10 ns molecular dynamics (MD) calculation. The average
distance between centroid of ligand and randomly chosen atoms at
the binding site with time has been plotted. It shows stable binding of
the ligands to DNA (1BNA)/DNA Pol I (3EYZ).
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binding energies of all top candidates are close to each other,
suggesting non-specic binding of ligand 3 at the active site
of DNA Pol I. A representative image showing the interaction
ofligand1withDNAPolIisshowninFig.2A(b),whilethose
with ligand 2 and ligand 3 are shown in ESI Fig. S3.†Further,
ligand 1 has a pyrene ring moiety similar to that of drug
molecule 1,2,3-trihydroxy-1,2,3,4-tetrahydrobenzo[A]pyrene
(Drug bank ID: DB07435). This molecule is reported to act by
binding to DNA polymerase I as one of its target receptor.
33
The best binding modes of ligands with 3EYZ were subjected
to 10 ns MD simulation to conrm the stability of the
complex. Stable binding is conrmed by total energy versus
time plot (Fig. S4D–F†). In Fig. 2B, we have plotted average
distance between the centroid of the ligand and three
randomly chosen atoms of the binding site of receptor
molecule in every 1 ns of the 10 ns MD simulation. The plot in
Fig. 2B importantly suggests stable binding of the ligands to
receptor molecules.
3.2.3 Bacterial DNA fragmentation. The DNA gel electro-
phoresis bands of cell lysates from bacteria treated with 100 mM
of the iron complexes are shown in Fig. 3B. Beside the DNA
ladder in lane 1, distinct bands corresponding to the genomic
(1000 base pairs) and plasmid DNA (800 bp) can be seen in
the control (lane 2) and complex 3 (lane 5) treated samples. In
lane 3, it is evident that complex 1 caused a near complete
degradation of the bacterial DNA leading to the absence of
genomic and plasmid DNA bands and the formation of faint
band from extra small sized DNA fragments (<50 bp). In lane 4,
the genomic and plasmid DNA bands are very faint indicating
their susceptibility to degradation. In the lanes 2, 4 and 5, RNA
bands can also be seen at close to 50 bp. Overall, complex 1 and
2 induced degradation of complete and partial degradation of
genomic and plasmid DNA at a concentration of 100 mM.
Comet assay or SCGE was carried out to ascertain damage to
the bacterial genomic DNA by the iron complex ligands at lower
dosage of 10 mM. Fig. 3A present low and high magnication
images of control and iron complex treated bacterial DNA
captured under a uorescence and confocal microscope
respectively. Using the image analysis tools provided by Image J
soware, the average circularity of bacterial DNA was deter-
mined from a minimum of n¼6 images, excluding the
blotched signals. It can be clearly seen from Fig. 3C that average
circularity of the DNA decreased from spherical in the control
towards convex/ellipsoid shape in the iron complex treated
samples. Further analysis of the DNA comets was performed
with the help of the OpenComet plugin in Image J. An
automated scoring and analysis of the comets was carried out
following the established guidelines.
34
The parameters used to
estimate DNA damage were the tail DNA% and tail moment of
the comets. Fig. 3D shows that the DNA damage parameters of
the iron complex treated bacteria were signicantly greater than
their respective control parameters. Importantly, the order of
DNA strand breakage is commensurate with the order of DNA
binding affinities of the iron complex ligands.
3.3 Antimicrobial testing of iron complexes
The iron complexes 1, 2 and 3 were tested against Gram positive
S. aureus and Gram-negative E. coli and P. aeruginosa, for their
antibacterial and anti-biolm activity. All the protocols used for
the testing of the iron complexes are in consonance with stan-
dard microbial practices and methods, as mentioned in the
guidelines by the National Committee for Clinical Laboratory
Standards (NCCLS).
35
3.3.1 MIC assay. The Minimum Inhibitory Concentration
(MIC) was determined by continuous monitoring of the bacte-
rial growth curves for a period of 16 h. The minimum concen-
tration at which the exponential growth phase is absent has
been designated as the minimum inhibitory concentration. The
MIC values for the iron complexes against three bacterial
strains are shown in Table 3. From the observation of the
growth curve patterns shown in Fig. 4, it is clear that complex 1
is the most potent antibacterial agent among the iron
complexes. The antibacterial effect is more pronounced in case
of S. aureus, wherein the observed MIC was as low as 3mM. In
E. coli, a distinct exponential phase was not observed for any
dose of complex 1, leading to a plateau in their growth (Fig. 4). It
is probable that these bacteria became dormant and entered
into an early stationary phase for survival. For P. aeruginosa too,
complex 1 led to an extended lag phase for upto 8 h, which is
followed by multiplication of the resistant cells as seen from the
exponential growth phase. The detailed growth curves of the
three bacterial strains treated with various doses of complex 2
and 3 are presented in the ESI as Fig. S5 and S6,†respectively.
The growth curves in Fig. S5†for complex 2 revealed a MIC of 25
mM against MRSA, no inhibition against E. coli and an elonga-
tion of the lag phase in P. aeruginosa, recorded for $6.25 mM.
On the contrary, Fig. S6†shows the no growth inhibition by
complex 3 for any dosage against the three bacterial strains. It
may be noted that we have not assayed for ferrous/ferric ion
related bacteriotoxicity. This is in the light of earlier literature
reports which indicate negligible toxicity of iron towards
bacterial growth. No toxicity was recorded even at
Table 2 Binding energies and number of interactions between ligands 1, 2 and 3 with DNA Pol I at best binding mode
Ligand
Binding energy for
DNA Pol I (3EYZ) (kcal mol
1
)
Types and no. of ligand–DNA Pol I interactions
H-bond (conventional) Electrostatic Hydrophobic Total
Ligand 1 10.6 4 2 0 6
Ligand 2 9.6 2 6 0 8
Ligand 3 6.4 2 2 0 4
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concentrations of 20 mg L
1
(350 mM),
36
which is 3 times greater
than the maximum concentration (100 mM) of the iron complex
used in the current study.
3.3.2 Anti-biolm evaluation. Two quantitative biochem-
ical assays were carried out in order to assess the effect of iron
complexes on the viability and stability of mature biolms of S.
aureus. The 48 h cultured biolms in microtiter plates were
treated with the iron complexes for 24 h and evaluated by
resazurin and crystal violet assays. The resazurin dye reduction
test indicates around 80% reduction in the viability of S. aureus
biolms, following treatment with iron complex 1 and 2 at 25–
100 mM concentration for 24 h (Fig. 5A). In case of complex 3,
a maximum reduction of 50% in bacterial viability was observed
at 25–100 mM dosage of the complex. This suggests the higher
anti-biolm efficacy of the complex 1 and 2 in comparison to
complex 3, which is commensurate with the molecular docking
predictions. On the other hand, the crystal violet assay revealed
Fig. 3 Representative comet/SCGE images visualized under (A) fluorescence microscope (left column) –scale bar ¼10 mm and confocal
microscope (right column) –scale bar ¼1mm. (B) DNA gel electrophoresis of bacterial cell lysates showing DNA ladder –lane 1, control –lane 2,
complex 1 –lane 3, complex 2 –lane 4 and complex 3 –lane 5; (C) average circularity of the nucleoid of the control and iron complex treated
bacteria determined from a minimum of n¼6 images using Image J; (D) comet DNA parameters of the control and iron complex treated bacteria
determined using OpenComet plugin in Image J. The data shown are mean sd of n$6 images analyzed from two independent experiments
that yielded similar results. *indicate statistically significant difference (p< 0.05) of the iron complex treated samples w.r.t control.
Table 3 MIC of the iron complexes against representative bacterial
strains
Complex
MIC (mM) of iron complexes against pathogenic
bacteria
S. aureus (MRSA) E. coli P. aeruginosa
Complex 1 3.13 >100 >100
Complex 2 25 >100 >100
Complex 3 >100 >100 >100
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around 60%, 70% and 50% decrease in the total mass of the
biolms as compared to the control for complex 1, 2 and 3,
respectively (Fig. 5B). Fig. 5A and B are the line graphs depicting
the anti-biolm effect of the iron complexes. Further, uores-
cence microscopy data show a gradual increase in the
percentage of PI stained (dead) bacterial populations with
increasing concentration of the iron complexes. Fig. 6 shows
representative images of biolms stained with live/dead Bac-
light kit aer treatment with different doses of the iron
complexes. It is evident from the images that both complex 1
and 2 cause destruction of the biolm and the bacteria exhibit
greater planktonic character compared to the dense biolms in
the control. However, in case of complex 3, the biolm pheno-
type is persistent even in the treated MRSA biolms. Neverthe-
less, the co-localization of Syto9 and PI in the complex 3 treated
biolms indicates partial bacterial injury. Thus, the uores-
cence micrographs provide complementary evidence on the
reduced viability and stability of the biolms upon treatment
with iron complexes.
3.3.3 Siderophore production under iron limited culture
conditions. The production of bacterial siderophores by MRSA
under iron limited culture was assessed by CAS assay. The
bacteriainthelogarithmicgrowthphaseweretreatedwith
sub-MIC doses (3.13, 6.25, 12.5 and 25.0 mM) of the iron
complexes for 48 h. Fig. 7A comprises of line graphs, which
show the relative production of siderophores, measured as
absorbance at 630 nm, for different concentrations of Fe
2+
and
iron complexes. Upon treatment with Fe
2+
,thesiderophore
production increasedmildlyupto6.25mMandwascompletely
absent at 25 mM. In earlier studies, 20 mM has been reported to
be the threshold concentration of soluble iron for siderophore
production by bacteria.
37
In the case of iron complexes, a spike
in the biosynthesis of bacterial siderophores can be noted at
3.13 mM dosage, while higher concentrations of the complex
appeared to retard or inhibit siderophore production. This
maybeexpectedashigherconcentrationsofironcomplexes
also inhibit the metabolic activity in bacteria in nutrient
decient cultures. However, the most striking feature of the
siderophoreproductionassayisthattheironcomplexestrig-
gered a much greater synthesis of siderophores at 3.13 mM
thanthefreeFe
2+
ions. In summary, the production and
transport of bacterial siderophores opens up some interesting
prospects for the entry and transport of chemotherapeutic
agents into bacteria.
Furthermore, the UV-visible absorption spectra of the CAS
solution and 1 : 1 mixture of culture supernatants and CAS
shuttle solutions show marked differences in the absorption
maxima (Fig. 7B). The absorption spectrum of the CAS blue
Fig. 4 MIC determination of complex 1 against three representative bacterial strains. For each graph, the X-axis scale corresponds to time (0–18
h) and the Y-axis scale corresponds to optical density (OD @ 600 nm)
Fig. 5 Anti-biofilm efficacy of iron complexes evaluated by (A) resazurin assay and (B) crystal violet assay. All the experiments were performed in
triplicate (n¼3). Data shown are mean SD of replicates obtained from three independent experiments that yielded similar results. *indicate
statistically significant difference (p< 0.05) of the iron complex treated samples w.r.t control.
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assay solution exhibited absorption maxima at 338 nm and 518
nm along with a shoulder peak at 585 nm. In contrast, the 1 : 1
equilibrated mixtures of CAS blue and culture supernatants
containing siderophores exhibited absorption maximum at 418
nm, corresponding to Fe–CAS red complex and another low
intensity absorption maximum at 625 nm due to Fe–side-
rophore complex. The UV-visible absorption bands for the three
iron complexes (1, 2 and 3) were however not detected. This
suggests that the labile iron complexes have apparently under-
gone complete ligand exchange with the siderophore molecules
and transported into the bacterial cells via membrane
receptors.
3.4 Cell cytotoxicity assessment
In a manner similar to the treatment of suspension cultures and
bacterial biolms, cell monolayers of C2C12 mouse myoblasts
were treated with 25, 50 and 100 mM concentrations of the iron
complexes. The results were analyzed using MTT assay and ow
cytometry.
3.4.1 MTT assay. The cytotoxicity of the iron complexes
against C2C12 mouse myoblasts was determined in terms of
their IC
50
concentrations, which is the dose at which 50% of the
cells are viable. The IC
50
values determined aer 24 and 72 h of
treatment with the iron complexes are summarized in Table 4.
The dose-dependent toxicity proles of the iron complexes
against the C2C12 cells have been depicted as line plots in
Fig. 8A and B. The complex 1 was the most toxic with IC
50
values
of 41.3 and 14.5 mM, respectively aer 24 and 72 h of culture of
C2C12 cells. However, complex 2 and complex 3 exhibited
similar levels of cytotoxicity at both the timepoints. At 24 h, the
IC
50
values were around 60 and 50 mM for complex 2 and
complex 3, respectively. The values decreased identically to 20
mM for both complexes 2 and 3 aer 72 h. Thus, a time-
dependent cytotoxicity can be inferred from the myoblast
response towards iron complexes. However, it may be worth-
while to note that identical iron complexes were toxic at 2–10
times lower concentration towards bacteria than that of the
eukaryotic cells. This presents a therapeutic dosage window for
antibacterial application.
Fig. 6 Representative fluorescence microscopy images of S. aureus (MRSA) stained with live/dead Baclight kit after exposure to different
concentrations of the iron complexes. Green represents live bacteria stained by Syto9, while red represents dead bacteria stained with propidium
iodide (PI); scale bar ¼10 mm.
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3.4.2 Mode of cell death inferred from ow cytometry data.
In order to ascertain the mode of cell death, the cells treated
with the iron complexes were analyzed by ow cytometry. In an
earlier report, identical iron complexes were reported to
localize within the mitochondria of cancerous Hela cells.
10
Hence, in the current study, the alteration in the mitochondrial
membrane potential was used as an indicator of early
apoptosis. Also, the percentage of necrotic cells was detected by
propidium iodide (PI) staining. The panels in Fig. 9A and B
show bivariate ow cytometry dot plots, revealing the
percentage of necrotic (PI stained) cells and apoptotic (JC-1
green stained) cells, respectively, aer 24 h exposure to
complex 1. Similar ow cytometry data were recorded for
identical dosage of iron complexes 2 and 3 (data not shown).
Among the three complexes, ligand 2 and ligand 3 coordinated
to ferric iron in complex 2 and 3 appeared to trigger mild, but
signicant necrosis in C2C12 cells (Fig. 10A). On the contrary,
a dose-dependent increase in the percentage of early apoptotic/
mitochondrial membrane depolarized cells was observed for
all the iron complexes and notably in case of the complex 1
(Fig. 10B). Thus, it may be concluded that the iron complexes
predominantly caused cytotoxicity via cellular apoptosis. This
observation is consistent with an earlier study, wherein DNA
fragmentation was used as the marker for apoptosis when
cancer cells were treated with iron complexes under photody-
namic conditions.
10
3.4.3 TOPKAT toxicity predictions. For therapeutic safety
evaluation of our iron complexes in comparison to FDA
approved drug molecules, TOPKAT was employed to predict
acute systemic toxicity of the ligands via oral and inhalation
Fig. 7 (A) Staphylococcal siderophore production under iron limiting culture for 48 h determined by CAS assay. All the experiments were
performed in triplicate (n¼3). Data shown are mean SD of replicates obtained from three independent experiments that yielded similar results.
*indicate statistically significant difference (p< 0.05) of the iron complex treated samples w.r.t control. (B) UV-visible absorption spectra of the
blue CAS complex and 1 : 1 mixtures of CAS blue and culture supernatants after treatment with iron complexes for 48 h.
Table 4 MTT based cytotoxicity estimation of the iron complexes
against C2C12 mouse myoblast cells
Complex
IC
50
(mM) of iron complexes against
C2C12 mouse myoblasts
24 h 72 h
Complex 1 41.3 1.1 14.5 1.3
Complex 2 59.1 1.1 21.6 1.0
Complex 3 48.9 1.0 21.4 1.0
Fig. 8 Cytotoxicity of the iron complexes against C2C12 mouse myoblast cells after (A) 24 h and (B) 72 h of exposure. The data were fit by non-
linear regression method after normalization of the treated samples w.r.t untreated control (100% viability). All the data points are mean SD of
(n¼3) replicates obtained from three independent experiments that yielded similar results.
Table 5 Calf-thymus (ct) DNA binding data of the iron complexes
10,11
Complex
Equilibrium binding constants
(K
b
/M
1
) determined from UV-vis absorption titrations
Complex 1 3.4 (0.6) 10
6
Complex 2 6.4 (0.8) 10
5
Complex 3 2.6 (0.5) 10
5
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routes. The rat oral LD50 (LD –lethal dose) module of the
statistically-based TOPKAT program performs 19 regression
analyses using experimental values of approximately 4000
chemicals from the registry of toxic effects of chemical
substances (RTECS) database. Similarly, the rat inhalation LC50
(LC –lethal concentration) values were predicted by applying
regression analyses of experimental values from RTECS data-
base. Fig. 11 is a bar chart showing the percentage toxicity
(logarithmic axis) of our ligands for oral delivery and inhalation,
normalized to the PDT drug pormer (DB009977) taken as
100%. The TOPKAT predicted values of Rat Inhalational LC50
and Rat Oral LD50 for our ligands and reference molecules
(pormer and doxorubicin) is presented in Table 6. The values
clearly indicate the toxicity of all of our ligands are comparable
to the FDA approved PDT drug pormer and the DNA targeted
anticancer/antibiotic doxorubicin. Thus, the iron complexes
tested in our study are predicted to be safe for therapeutic
application.
4. Discussion
4.1 Iron complexes elicit bactericidal action by siderophore
mediated uptake and DNA fragmentation
Iron is an essential micronutrient for bacteria to replicate
and cause infection in vertebrate hosts. Recent works have
shownthatironiscriticalforbiolm formation by bacteria
Fig. 9 Mode of cell death detected by flow cytometry after exposure of C2C12 mouse myoblasts to different concentrations of complex 1. Panel
(A) shows the percentage of PI stained (necrotic) cells while panel (B) shows the percentages of red (live) and green (apoptotic) fluorescing
populations after staining with a mitochondrial membrane potential sensitive probe, JC-1.
Fig. 10 Flow cytometry analysis of mode of cell death after treatment with different concentrations of iron complexes –(A) bar chart showing
the percentage of necrotic cells by PI staining; (B) bar chart showing the percentage of live (JC-1 red stained) and apoptotic/mitochondrial
membrane depolarized cells (JC-1 green stained). All the experiments were performed in triplicate (n¼3). Data shown are mean SD of
replicates obtained from three independent experiments that yielded similar results. *indicate statistically significant difference (p< 0.05) of the
iron complex treated samples w.r.t control.
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on solid surfaces.
38,39
It was elucidated that iron chelators in
culture media prevented S. aureus attachment onto material
surfaces and reduced polysaccharide intercellular adhesion
(PIA) secretion.
39
In another study, the addition of gallium
(Ga) to the culture media was shown to prevent biolm
formation in Pseudomonas aeruginosa.Thechemicalsimi-
larity of Ga to Fe supposedly interfered in Fe uptake and
signaling leading to the anti-biolm effect.
38
The addition of
exogenous iron effect reversed the effectinbothcases.Under
iron starvation, bacteria are reported to secrete iron chelating
molecules called siderophores. Based on the chemical
moieties that coordinate to iron, siderophores are broadly
classied into cateocholate and hydroxamate types.
40
There
can be two possible antimicrobial strategies based on side-
rophore production and iron transport: (i) the synthesis of
iron chelating molecules with strong binding affinity to iron
and thus deprive bacteria with essential iron necessary for
biolm formation. (ii) Siderophore transport also makes
such iron chelate producing bacteria more susceptible to the
entry of lethal molecules/drugs and antimicrobial payloads
conjugated to labile iron complexes.
In the present study, the latter of the above two strategies
has been applied successfully against MRSA in planktonic and
biolm cultures. The iron complexes containing identical
hydroxamate siderophore moieties and/or different DNA
intercalators were 5–10 times less toxic in C2C12 cells as
compared to MRSA. Especially in the case of complex 1, a MIC
value of 3mM was recorded against MRSA while the IC
50
doses
against C2C12 cells were 40 mMand15 mMaer 24 h and 48
h of treatment, respectively. These results are promising in the
light of earlier works showing similar photocytotoxicity of the
iron complexes in both cancerous and non-cancerous cell
lines.
10,11
The mechanism of bactericidal action involves side-
rophore mediated uptake of iron complexes and bacterial DNA
fragmentation as testied by gel electrophoresis and comet
assay (Fig. 3). A high dose of 100 mMcausedcompletedegra-
dation of both genomic and plasmid DNA by complex 1, while
a much lower concentration of 10 mM led to the formation of
the formation of distinct comet tails. A comparison of the DNA
fragmentation induced by the iron complexes is shown in
Fig. 3D.
4.2 DNA as the primary target for the development of next
generation antimicrobials
In the light of the emergence of multidrug bacterial resis-
tance, DNA is of late being explored as the primary target in
the development of antibacterial drugs.
41
In this regard,
a host of DNA groove binding molecules in the form of
transition metal complexes with aromatic p-acceptor ligands
are also being developed.
42
Such metal complexes can
interact with DNA by –(i) direct metal atom/ion coordination
to the DNA bases, (ii) non-covalently by van der Waals or p-
interactions of the aromatic residues with DNA, (iii) through
heteroatoms such as O, N and S by hydrogen bonding
between the heterocyclic aromatic ligands and DNA.
43
In the
current study, the antimicrobial potential of iron complexes
comprising of DNA intercalating ligands has been success-
fully elucidated against Gram-positive MRSA. Further, an
excellent correlation between in silico docking predictions
and in vitro antibacterial results arising from bacterial DNA
fragmentation is evident from the data presented in Tables
1–3.
However, one of the problems posed by DNA groove
binding molecules is their potential host cytotoxicity and
lack of specicity/selectivity to the target organisms. One of
Fig. 11 TOPKAT in vivo toxicity predictions for the ligands of the three
iron complexes and a photodynamic therapy (PDT) drug, porfimer
(DB00707) normalized to anticancer/antibiotic, doxorubicin
(DB00997). The toxicity of doxorubicin was set to 100%.
Table 6 TOPKAT in vivo toxicity predictions for the ligands of iron complexes
a
Molecule/drug
Rat_inhalational_LC
50
(mg m
3
h
1
) Rat_oral_LD
50
(g kg
1
of body weight)
TOPKAT Experimental TOPKAT Experimental
Ligand 1 5985.149 ND 0.29 ND
Ligand 2 7017.849 ND 2.583 ND
Ligand 3 4218.606 ND 1.461 ND
Pormer (DB00707) 0.004 NA 0.227 0.065*
Doxorubicin (DB00997) 55.258 NA 0.227 0.570–0.698*
a
ND –not determined, NA –not applicable, *information from material safety datasheets MSDS.
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the methods of achieving selectivity towards bacterial path-
ogens is the conjugation of antimicrobials/antibiotics to
siderophores.
44
In the present study, the cytotoxicity of the
iron complexes towards mammalian cells have been cir-
cumvented considerably by the design of the iron complexes
sporting benzhydroxamate ligands. These ligands have
a structural similarity to the hydroxamate class of bacterial
siderophores, which promoted greater bactericidal than
cytocidal action, as suggested by the data in Tables 3 and 4
Further, TOPKAT in vivo toxicity analysis of our iron
complexes predicts them to be comparable to FDA approved
drug molecules (Table 6). Taken together, we demonstrate in
this study the design of iron complexes conjugated to DNA
intercalators and siderophore mimicking chelators as
a potent strategy for the development of DNA targeting
antimicrobial agents.
5. Conclusions
The design of labile iron complexes with a combination of
ligands, which possess structural similarity to siderophores
for bacterial uptake and those eliciting bactericidal action by
DNA intercalation and fragmentation is demonstrated as an
efficient strategy to overcome the pathogenic MRSA in the
present work. Such iron complexes are 5–10 fold less toxic in
C2C12 mouse myoblasts and this presents a therapeutic
dosage window for their application as DNA targeting anti-
microbial agents. Further, TOPKAT in vivo toxicity predic-
tions conrm the therapeutic safety of the complexes in
comparison to representative FDA approved drugs. The
experimental bacteriotoxicity and bacterial DNA fragmenta-
tion elicited by the iron complexes are commensurate with
the molecular docking determined binding affinities of the
ligands to DNA and DNA polymerase I. Thus, DNA targeting
in conjunction with siderophore mimics can be deployed as
an efficient antimicrobial strategy to address persistent
bacterial infections.
Acknowledgements
The authors acknowledge the major funding support from
the Department of Science and Technology (DST) and
Department of Biotechnology (DBT), Govt. of India under
‘Centers of Excellence and Innovation in Biotechnology’
scheme through the center of excellence project –'Trans-
lational Center on Biomaterials for Orthopedic and Dental
Applications'.Prof.A.R.Chakravarty from Department of
Inorganic and Physical Chemistry, IISc is acknowledged for
his guidance and suggestion for synthesis and characteriza-
tion of the complexes used in this study. Supercomputer
Education and Research Centre (SERC), IISc is deeply
acknowledged for providing the necessary soware and
computational tools for performing the Docking and Molec-
ular Dynamics studies. The authors thank the IISc FACS
facility for helping with the ow cytometry experiments. One
of the authors, Sunil Kumar B. (09/079 (2501)/2011-EMR-I dt.
16-11-2011) acknowledges the Council for Scienticand
Industrial Research (CSIR) for providing scholarship during
theperiodofstudyandSubhenduPanditacknowledges
Department of Science and Technology (DST) for his KVPY
undergraduate scholarship.
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