Synergistic interactions of eugenol-Tosylate and its congeners with fluconazole against candida albicans

Article abstract of DOI:10.1371/journal.pone.0145053

We previously reported the antifungal properties of a monoterpene phenol "Eugenol" against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and t

Full text of DOI:10.1371/journal.pone.0145053

RESEARCH ARTICLE
Synergistic Interactions of Eugenol-tosylate
and Its Congeners with Fluconazole against
Candida albicans
Aijaz Ahmad1☯, Mohmmad Younus Wani2☯, Amber Khan3, Nikhat Manzoor4,5*,
Julitha Molepo1*
1 Department of Oral Biological Sciences, School of Oral Health Sciences, Faculty of Health Sciences,
University of the Witwatersrand, 7 York Road, Parktown, Johannesburg, South Africa, 2 Departmento de
Quimica, FCTUC, Universidade de Coimbra, Rua Larga, Coimbra, Portugal, 3 Department of Internal
Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown,
Johannesburg, South Africa, 4 College of Applied Medical Sciences, Taibah University, Al-Madinah Al-
Munawarah, KSA, 5 Department of Biosciences, Jamia Millia Islamia, New Delhi, Delhi, India
☯ These authors contributed equally to this work.
* Julitha.molepo@wits.ac.za (JM); nikhatmanzoor@yahoo.co.in (NM)
Abstract
We previously reported the antifungal properties of a monoterpene phenol “Eugenol”
against different Candida strains and have observed that the addition of methyl group to
eugenol drastically increased its antimicrobial potency. Based on the results and the impor-
tance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners
(E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- sus-
ceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by deter-
mining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from
microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the
tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6,
with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC
exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however,
no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when
used in combination with the test compounds. Like their precursor eugenol, all the deriva-
tives showed significant impairment of ergosterol biosynthesis in all C. albicans strains cou-
pled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11.
The results were further validated by docking studies, which revealed that the inhibitors
snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain
their excellent inhibitory activity. Our results suggest that these compounds have a great
potential as antifungals, which can be used as chemosensitizing agents with the known
antifungal drugs.
OPEN ACCESS
Citation: Ahmad A, Wani MY, Khan A, Manzoor N,
Molepo J (2015) Synergistic Interactions of Eugenol-
tosylate and Its Congeners with Fluconazole against
Candida albicans. PLoS ONE 10(12): e0145053.
doi:10.1371/journal.pone.0145053
Editor: Chaminda Jayampath Seneviratne, National
University of Singapore, SINGAPORE
Received: May 5, 2015
Accepted: November 29, 2015
Published: December 22, 2015
Copyright: © 2015 Ahmad et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: AA and JM are highly thankful to Faculty
Research Committee Individual Research Grants/No.
001254 84511025121105 for finical support. Funding
agency has no role in the study design or any other
experimental work.
Competing Interests: The authors have declared
that no competing interests exist.
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Eugenol-tosylate and Its Congeners against C. albicans
Introduction
Natural products have been regarded as rich sources of potential chemotherapeutic agents.
However, due to high minimum inhibitory concentrations, most of them are often overlooked,
which results in a decline of investment in this area, despite the fact that natural products are a
source of new drugs [1]. In view of this, medicinal chemists started modifying the natural prod-
ucts to synthesize modified analogs and derivatives with improved potency and safety [2]. The
modified analogues, sometimes referred to as semi-synthetic drugs, are of great scientific focus
and a real advancement is the use of drug combinations, with known mode of action for the
treatment of life threatening diseases. Drug combinations have already been in use for the
treatment of diseases like cancer, HIV or cardiovascular diseases and it is believed that drug
combinations are better at controlling complex diseases with minimal resistance [2,3].
Although the potential of combining natural products or their semi-synthetic analogues with
synthetic drugs or introducing these into conventional treatment regimes are not yet systemati-
cally explored, the field is abound with opportunities [2].
In antifungal chemotherapy the heterocyclic compounds “azoles” have been immensely
researched and currently these are the most popular class of antifungals used in medicine.
These compounds bind as the sixth ligand to the heme group in CYP51, changing the structure
of the active site and acting as non-competitive inhibitors [4]. Several studies are devoted to
validate the potency of azoles as inhibitors of 14 α-demethylase [5]. Impairment of ergosterol
biosynthesis by these compounds has also been documented [4,6]. However, prolonged use of
some azoles as antifungal agents has resulted in the emergence of drug resistance among cer-
tain fungal strains [7–9]. Genetic mutations have been found to be possibly responsible for the
resistance to clinically used drugs especially fluconazole [10]. In addition, resistance to new
structurally related azoles such as voriconazole [11], and ravuconazole [12] has also been
observed. Consequently, the focus of azole research is beginning to shift towards identifying
new efficient drug molecules to circumvent this major obstacle. A novel strategy of generating
an effective treatment option is the combined use of the semi-synthetic analogues, with
enhanced potency, compared to the precursor natural product and an already established drug
[13,14].
Our previous findings on the bioactivity of eugenol revealed that eugenol exerts its anti-
fungal activity by targeting ergosterol biosynthesis pathway [15] and has the potential to con-
vert the fungistatic FLC into a fungicidal drug when used in combination with known
antibiotics [16]. Here we chose to synthesize eugenol-tosylate and its congeners and investi-
gated their mechanism of binding to Cytochrome P450 14α-sterol demethylase (CYP51),
which are essential enzymes in sterol biosynthesis in fungi. We further determined the anti-
fungal efficacies of these newly semi-synthetic derivatives E1-E6 against different FLC-sus-
ceptible and FLC-resistant Candida albicans species alone and in combination with
fluconazole. The mechanism of action was elucidated by quantifying the ergosterol content
in different Candida isolates following treatment with semi-synthetic eugenol derivatives
and the results were validated by docking studies. The differences in the ergosterol biosynthe-
sis are attributed to the differences in the gene expression patterns and in view of this; real
time-PCR (qRT-PCR) of one of the striking gene of ergosterol biosynthesis (ERG11) was also
included in the study.
Materials and Methods
Solvents and organic reagents were purchased from Sigma Aldrich and Merck (Germany) and
were used without further purification. Elemental analyses were performed on HeraeusVario
EL III analyzer. The results were within 0.4% of the theoretical values. FT-IR spectra were
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Eugenol-tosylate and Its Congeners against C. albicans
recorded using a Thermo Nicolet 380 instrument equipped with a Smart Orbit ATR attach-
ment. 1H and 13C NMR spectra were recorded at ambient temperature on a Bruker AVANCE
400 NMR spectrometer using standard parameters. All chemical shifts are reported in δ units
with reference to the residual peaks of CDCl3 (δ 7.24, 1H NMR; δ 77.0, 13C NMR) or DMSO-d6
(δ 2.50, 1H NMR; δ 39.52, 13C NMR). ESI-MS was recorded on a MICROMASS QUATTRO II
triple quadrupole mass spectrometer. Precoated aluminum sheets (silica gel 60 F254, Merck
Germany) were used for thin-layer chromatography (TLC) and spots were visualized under
UV light.
Synthesis of Eugenol-tosylate and its congeners
All the compounds E1-E6 were synthesized from 2-methoxy-4-(prop-2-en-1-yl)phenol (1) by
treating with different phenyl and substituted phenylsulfonyl chlorides in refluxing pyridine
for 18–24 h (completion of reaction was monitored by TLC) (Fig 1). After the completion of
reaction the reaction mass was quenched with distilled water and extracted with dichloro-
methane. Finally the combined organic layer was washed with distilled water again and dried
over anhydrous Na2SO4. All the compounds were obtained as pure oil after removal of the sol-
vent in vacuum.
2-methoxy-4-(prop-2-en-1-yl)phenyl benzenesulfonate (E1). Yield: 84%; Anal. Calc. for
C16H16O4S; C, 63.14; H, 5.30%, found; C, 63.30; H, 5.18%; IR maxcm-1: 3025 (C-H stretch),
Fig 1. Synthesis of eugenol-tosylate and its congeners E1-E6. (a) Sulfonyl chlorides, pyridine, reflux 18-
24h. (1) Eugenol
doi:10.1371/journal.pone.0145053.g001
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Eugenol-tosylate and Its Congeners against C. albicans
1595 (C = C, Ar), 1308, 1150 (S = O); 1H NMR (DMSO-d6) (ppm): 7.95–7.50 (4H, m, Ar-H),
7.35 (1H, t, J = 7.8 Hz), 6.84–6.65 (3H, m, Ar-H), 6.32 (1H, m), 4.85 (2H, m), 3.75 (3H, s,
OCH3), 3.15 (2H, d, J = 15.2 Hz); 13CNMR (DMSO-d6) (ppm): 149.8, 143.5, 138.6, 136.2,
134.6, 132.2, 130.1, 129.6, 128.7, 122.8, 118.9, 115.3, 58.6, 45.6; ESI-MS m/z [M+H]+ 305.10.
2-methoxy-4-(prop-2-en-1-yl)phenyl 4-methylbenzenesulfonate (E2). Yield: 85%; Anal.
Calc. for C17H18O4S; C, 64.13; H, 5.70%, found; C, 64.28; H, 5.56%; IR maxcm-1: 3033 (C-H
stretch), 1587 (C = C, Ar), 1315, 1158 (S = O); 1H NMR (DMSO-d6) (ppm): 7.87–7.59 (4H, m,
Ar-H), 6.68–6.52 (3H, m, Ar-H), 6.35 (1H, m), 4.78 (2H, m), 3.82 (3H, s, OCH3), 3.10 (2H, d,
J = 12.5 Hz), 2.25 (3H, s, CH3); 13CNMR (DMSO-d6) (ppm): 148.9, 144.6, 138.5, 137.0, 135.4,
133.5, 130.6, 129.3, 128.0, 122.5, 118.5, 117.0, 115.7, 56.4, 47.4, 24.8; ESI-MS m/z [M+H]+
319.10; [M+Na]+ 342.06.
2-methoxy-4-(prop-2-en-1-yl)phenyl 4-bromobenzenesulfonate (E3). Yield: 83%; Anal.
Calc. for C16H15O4BrS; C, 50.14; H, 3.94%, found; C, 50.26; H, 3.87%; IR maxcm-1: 3020 (C-H
stretch), 1572 (C = C, Ar), 1315, 1158 (S = O stretch); 1H NMR (DMSO-d6) (ppm): 7.89–7.71
(4H, m, Ar-H), 6.55–6.42 (3H, m, Ar-H), 6.33 (1H, m), 4.96 (2H, m), 4.50 (2H, t, J = 7.5 Hz),
3.98 (2H, t, J = 8.2 Hz), 3.72 (3H, s, OCH3), 3.22 (2H, d, J = 14.8 Hz); 13CNMR (DMSO-d6)
(ppm): 154.2, 149.2, 145.7, 143.6, 136.8, 129.0, 122.4, 118.0, 116.5, 115.4, 56.4, 48.4; ESI-MS m/
z [M+H]+ 382.98, [M+Na++H]+ 406.10.
2-methoxy-4-(prop-2-en-1-yl)phenyl 4-nitrobenzenesulfonate (E4). Yield: 85%; Anal.
Calc. for C16H15NO6S; C, 55.01; H, 4.33, N, 4.01%, found; C, 55.12; H, 4.26, N, 4.15%; IR maxcm-1:
3025 (C-H stretch), 1585 (C = C, Ar), 1380 (NO2 stretch), 1310, 1148 (S = O stretch); 1H NMR
(DMSO-d6) (ppm): 8.45–8.15 (4H, m, Ar-H), 6.85–6.72 (3H, m, Ar-H), 6.30 (1H, m), 4.93 (2H, m),
3.75 (3H, s, OCH3), 3.18 (2H, d, J = 15.5 Hz); 13CNMR (DMSO-d6) (ppm): 154.2, 149.2, 145.7,
143.6, 136.8, 129.0, 122.4, 118.0, 116.5, 115.4, 56.4, 48.4; ESI-MS m/z [M+H]+ 350.06; [M+Na]+
373.10; [M+K++H]+ 390.14.
2-methoxy-4-(prop-2-en-1-yl)phenyl 4-chlorobenzenesulfonate (E5). Yield: 80%; Anal.
Calc. for C16H15O4ClS; C, 56.72; H, 4.46%, found; C, 56.84; H, 4.38%; IR maxcm-1: 3033 (C-H
stretch), 1587 (C = C, Ar), 1308, 1152 (S = O), 730 (C-Cl); 1H NMR (DMSO-d6) (ppm): 7.85–
7.62 (4H, m, Ar-H), 6.75–6.55 (3H, m, Ar-H), 6.22 (1H, m), 4.83 (2H, m), 3.85 (3H, s, OCH3),
3.18 (2H, d, J = 12.5 Hz); 13CNMR (DMSO-d6) (ppm): 148.5, 145.2, 138.2, 136.8, 135.6, 133.5,
132.1, 129.0, 128.1, 122.4, 118.0, 116.5, 115.4, 58.5, 48.2; ESI-MS m/z [M+H]+ 339.07; [M+-
Na++H]+ 363.04.
2-methoxy-4-(prop-2-en-1-yl)phenyl 4-ethylbenzenesulfonate (E6). Yield: 80%; Anal.
Calc. for C18H20O4S; C, 65.04; H, 6.06%, found; C, 65.28; H, 6.02%; IR maxcm-1: 3028 (C-H
stretch), 1575 (C = C, Ar), 1318, 1143 (S = O stretch); 1H NMR (DMSO-d6) (ppm): 7.89–7.71
(4H, m, Ar-H), 6.55–6.42 (3H, m, Ar-H), 6.33 (1H, m), 4.96 (2H, m), 3.72 (3H, s, OCH3), 3.22
(2H, d, J = 17.5 Hz), 2.58 (2H, m), 1.29 (3H, s); 13CNMR (DMSO-d6) (ppm): 149.8, 144.5,
143.2, 136.1, 128.4, 122.1, 117.5, 115.4, 113.8, 56.3, 48.0, 32.6, 14.8; ESI-MS m/z [M+H]+
333.11; [M+Na]+ 356. 08.
Strains and media
Besides one standard laboratory strain C. albicans ATCC90028 used as a quality control, fifteen
clinical isolates (eight from South Africa and seven from India) of FLC- susceptible and nine
FLC- resistant C. albicans were used in this study (Table 1). Clinical strains were isolated from
HIV positive patients and other immunocompromised conditions from the Charlotte Maxeke
Johannesburg Academic Hospital, Johannesburg, South Africa and from the Department of
Microbiology, All India Institute of Medical Sciences, New Delhi, India. All strains were main-
tained on YPD (yeast extract, peptone dextrose agar plates). Prior to experiments, growth was
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Eugenol-tosylate and Its Congeners against C. albicans
Table 1. Minimum inhibitory concentrations (μg/ml) of E1-E6 and fluconazole (FLC) against different C. albicans strains.
Strains
EUG
E1
E2
E3
E4
E5
E6
FLC
ATCC 90028
500
16
16
4
8
2
31
4
074
072gr
072 big gr
079 small gr
002 B1
003 gr
004 gr
004 B1
177
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
31
31
31
31
8
31
31
62
31
16
32
62
31
16
31
31
16
8
4
4
16
16
31
31
2
2
2
4
4
1
4
4
1
2
8
4
2
1
4
1
8
8
4
8
2
8
8
2
2
62
62
62
31
31
62
62
62
31
62
31
31
16
62
62
62
62
62
62
31
62
62
31
62
8
16
16
16
8
4
4
2
16
31
8
4
16
16
4
16
16
8
8
1
4
4
8
8
2281
31
16
8
8
16
8
8
2323
8
8
2367
4
4
8
2427
8
2
4
8
2434
31
8
62
31
62
62
31
62
16
62
31
31
31
16
2
31
4
8
2508
8
3001
31
31
16
16
16
31
31
16
8
16
16
4
16
16
8
125
125
125
125
125
125
125
125
125
3048
3068
3087
8
8
3081
4
8
3140
16
8
16
16
8
2357
2261
4
3178
4
8
doi:10.1371/journal.pone.0145053.t001
initiated by growing one loopful of cells in YPD media up to mid-log phase. All the media and
other analytical grade chemicals were purchased from Oxoid, England and E. Merck, India.
Minimum Inhibitory Concentration (MIC)
Two-fold broth microbroth dilution method, as described by the CLSI, was used to determine
the MICs of all the newly synthesized derivatives E1-E6 and FLC against different FLC- suscepti-
ble and FLC- resistant isolates [17]. All the compounds were diluted using 1% DMSO to yield a
concentration of 1000 μg/ml. The positive control fluconazole (1000 μg/ml) and the negative
vehicle control (1% DMSO) were also included in every set of experiments. Culture and media
controls were also included in every set of experiments to confirm the viability and sterility,
respectively. Results were calculated as a mean of the two separate experiments done in triplicate.
Assessment of the Fractional Inhibitory Concentration Index (FICI)
To determine the interaction of compounds E1-E6 with FLC, microdilution assays were per-
formed as previously described [18]. Briefly, E1, E2, E3, E4, E5, E6 and FLC were added in 1:1
volumes together with 100 μl of media and were serially diluted. Interactions were assessed on
the basis of zero-interaction theory of Loewe additivity and FICI values were calculated as
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Eugenol-tosylate and Its Congeners against C. albicans
follows:
MICa in combination MICb in combination
FICI ¼ FICa þ FICb ¼
þ
MICa tested alone
MICb tested alone
Where, MICa is the MIC of the derivatives E1-E6 and MICb is the MIC of FLC. An FICI
value of ꢀ0.5 was interpreted as synergy whereas the FICI values between 0.5 and 1.0 were
interpreted as additive. FICI values >4.0 were considered as antagonism and FICI values
between 1.0 and 4.0 were considered as indifferent, following the conservative approach as
described by van Vuuren [19].
Varied ratio combinations and isobolograms
Based on the synergistic interactions in 1:1 ratios between E1, E2, E3, E4, E5, E6 and FLC, iso-
bolograms were constructed, where nine different ratios of E1-E6 and FLC were mixed in 9:1;
8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; and 1:9 ratios, respectively, and their MIC values were then deter-
mined. MIC values were also determined for the compounds E1-E6 and FLC, independently.
To represent the mean MIC values of these combinations isobolograms were plotted using
GraphPad Prism version 5-software as described previously [18]. On examining the data points
for each ratio in relation to the MIC values independently, isobolograms were interpreted as
synergistic or additive.
Ergosterol Biosynthesis Assay
Total intracellular sterols from the different C. albicans cells which were treated with MIC and
sub-MIC concentrations of E1 to E6 were extracted and quantified as reported previously
using spectrophotometric method (Beckman Coulter) [20]. In every set of experiments
untreated (negative control) and fluconazole treated (positive control) cells were also tested.
The characteristic four-peaked curve confirmed the presence of ergosterol and 24(28) dehy-
droergosterol (DHE) in the samples while the flat line depicted the absence of ergosterol in the
extracted samples. Ergosterol content was calculated as a percentage of the wet weight of the
cell by the following equations:
% Ergosterol þ % 24ð28Þ DHE ¼ ½ðA281:5=290Þ ꢁ Fꢂ=pellet weight
% 24ð28Þ DHE ¼ ½ðA230=518Þ ꢁ Fꢂ=pellet weight
% Ergosterol ¼ ½% Ergosterol þ % 24ð28Þ DHEꢂ ꢃ % 24ð28Þ DHE
Where F is the factor for dilution in ethanol and 290 and 518 are the E values (in percent-
ages per centimeter) determined for crystalline ergosterol and 24(28) DHE, respectively.
Docking
Homology modeling. The 3D model structure of cytochrome P450 lanosterol 14α-
demethylase of C. albicans was built using homology modeling. A flow chart diagram for
homology modelling is illustrated in S1 Fig. The amino acid sequence of the enzyme was
obtained from the Universal Protein Resource (http://www.uniprot.org) (Accession Code:
P10614) and sequence homologous was obtained from Protein Data Bank (PDB) using Blast
search. Based on the result of blast search, we found lanosterol of yeast bearing PDB ID: 4K0F
with highest homology of 65%. This was selected as a template for modelling our protein
(P10614). P10614 was submitted to I-TASSER [21], an online server for modelling and the best
model was selected on the basis of higher C-score and least Z score. In parallel we also mod-
elled it manually by modeler [22]. We selected the model with least DOPE score and then we
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Eugenol-tosylate and Its Congeners against C. albicans
Fig 2. Geometric evaluation of the modeled protein and the template shown as Ramachandran plots
for 4KOF (A) and modeled protein (B); 97% of residues were in the favor, 2.9% allowed regions, and
0.2% was in the outlier region as compared to 4KOF template (97.5%, 2.3%, and 0.2%).
doi:10.1371/journal.pone.0145053.g002
compared the two results with our template. The model with least RMSD was selected as the best
one and further used for docking studies. To have the heme group, necessary for the ligand mole-
cules to bind, in our modeled protein, we took the homology modeled structure and aligned it to
the templates (PDB code: 3LD6; 4LXJ), containing the heme in its active site using PyMol, then
we figured out the atoms that are interacting with it. Finally we incorporated the co-ordinates of
heme in our model and also wrote down the interactions as extracted from the template file.
Geometric evaluations of the modeled 3D structure were performed using Rampage as shown
in Fig 2A and 2B. The Ramachandran plot of our model showed that 97% of residues were in the
favor, 2.9% allowed regions, and 0.2% was in the outlier region as compared to 4K0F template
(97.5%, 2.3%, and 0.2%). The plot statistics are presented in Table 2. (Also see S2 and S3 Figs).
Validation was carried out using ProSA to obtain the Z-score value for the comparison of
compatibility (See S4 Fig). The Z-score plot showed spots of Z score values of proteins deter-
mined by NMR (represented in dark blue color) and by X ray (represented in light blue color).
The two black dots represent Z-scores of our model (-8.56) and template (-8.63). These scores
indicate the overall quality of the modeled 3D structure of lanosterol 14-alpha demethylase of
C. albicans. RMSD (0.35 Å) was calculated between the main chain atom of model and tem-
plate, which indicated close homology. This ensured the reliability of the model.
Active Site Prediction. The best model thus obtained was submitted to DogSite scorer
[23]. It predicted the existence of 10 different pockets. The one with highest P score was
selected as the reliable active site pocket and considered to have a potential active site residue.
Table 2. Ramachandran plot statistics for the modeled protein and the template#.
Structure
Number of residues in favored region
(%)
Number of residues in allowed region
(%)
Number of residues in outlier region
(%)
4KOF
97.5
97
2.3
2.9
0.2
0.2
MODEL
BUILT
#Done using RAMPAGE evaluation server
doi:10.1371/journal.pone.0145053.t002
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Eugenol-tosylate and Its Congeners against C. albicans
The 3D structure of ligands (compounds E1-E6 and FLC) were created by ChemDraw 8.0
and converted to the pdb file format after energy minimization (MOPAC). Ligand preparation
was conducted by assigning Gastegier charges, merging nonpolar hydrogens, and saving it in
pdbqt file format using AutoDock Tools (ADT) 4.2. The docking of the active molecules into
the lanosterol 14α-demethylase (CYP51) modeled protein was done by positioning within the
active site cavity. Molecular docking calculations were carried out with Autodock vina [24].
The conformation with the lowest binding free energy was used for analysis. All molecular
docked models were prepared using PyMOL [25].
RNA extraction and qRT-PCR
RNA extraction and gene expressions of ERG11 gene in C. albicans ATCC90028, FLC- suscep-
tible C. albicans 2281 and FLC- resistant C. albicans 3001 cells when exposed to MIC values of
the test compounds E1-E6 or FLC were done as described previously [26]. Briefly, C. albicans
ATCC90028, C. albicans 2281 and C. albicans 3001 were grown up to mid-log phase and were
then exposed to MIC values of E1-E6 or FLC for 2 h in the same media. After incubation cells
were washed and harvested by centrifuging at 3000g for 5 min. Pellets of approximately 1×106
cells were homogenized and lysed with hydrochloric acid-treated glass beads (diameters, 0.5 mm;
Braun Biotech, Melsungen, Germany) for 10 min, and total RNA was isolated using total Gene-
JET RNA kit (Fermentas Life Sciences, EU) following manufacturer’s instructions. Subse-
quently, the isolated RNA was digested with RNase free DNase I, and cDNA was synthesized
using using RevertAid™ First Strand cDNA Synthesis Kit; K1622. The housekeeping gene
ACT1 which was expressed to a similar extent under all conditions investigated was used as an
endogenous control. Untreated cells and 1% DMSO represent the culture and negative con-
trols, respectively.
Primer sets for ACT1 and Egr11 were respectively as follows: forward 5-TTTAAGAATT
GATTTGGCT-3; reverse 5-GAAGATTGAGAAGAAGTTT-3 and forward 5-ATTGGTATT
CTTATGGGTGGTCAACATAC-3; reverse 5- CCCAATACATCTATGTCTACCACCACC-3
The relative concentration of each transcript was determined using Real-Time 2xPCR ABI
Power Sybergreen on Real Time PCR (ABI-7500). Each PCR protocol consisted of a primary
denaturation step at 95°C for 20 s, followed by 30 cycles of denaturation at 95°C for 20 s,
annealing at 45°C and extension at 72°C for 15 s. Each product was checked for its specificity
in melting curve analyses and efficiency of the amplification was verified with standard curves
for both the gene. Results were analysed by LightCycler Software 4.0. All qRT-PCR experi-
ments were done in triplicate in two independent experiments for all the treated cell samples
and results are shown as Mean standard deviation.
Results and Discussion
Synthesis
Eugenol, due to its antifungal properties was chosen as a reasonable scaffold for the present
study and the hydroxyl group of the moiety on treatment with different sulfonyl chlorides
under basic conditions resulted in the synthesis of the desired products as shown in Fig 1. Since
the reaction of the -OH group of eugenol with p-toluenesulfonyl chloride is a tosylation reac-
tion, all the synthesized derivatives were named after eugenol-tosylate. The structure of the
synthesized compounds was established by elemental analyses, FTIR, 1H, 13C NMR and
MS-ESI+-spectral studies.
FTIR spectra showed bands which significantly proved the formation of eugenol-tosylate and
its congeners E1-E6. The absence of any free or H-bonded band at/or around 3200–3700 cm-1
corresponding to -OH and the presence of bands around 1143–1158 cm-1and 1308–1318 cm-1
PLOS ONE | DOI:10.1371/journal.pone.0145053 December 22, 2015
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Eugenol-tosylate and Its Congeners against C. albicans
corresponding to the sulfonyl group of the respective derivatives is an ample evidence. In the 1H
and 13C NMR the absence of any signal for–OH proton and the appearance of characteristic
peaks at expected chemical shifts and integral values for phenyl ring protons and methyl and
ethyl protons in E2 and E6 provide significant indication for the formation of the derivatives
E1-E6. The mass spectra of all the derivatives (E1-E6) showed [M+H]+ peak which is in accor-
dance with the molecular formula of the compounds thus further confirming their formation. All
the derivatives obeyed a similar pattern of fragmentation and in some derivatives the molecular
ion peaks were observed as [M+Na++H]+, [M+K++H]+, [M+Na+] (Metal adduct ions). The data
is presented in experimental section.
Antifungal activity
The MICs of eugenol and its derivatives E1-E6 against all the tested clinical FLC- susceptible
and FLC- resistant Candida albicans strains along with a reference strain of C. albicans ATCC
90028 are shown in Table 1. Evaluation of MIC values showed that eugenol has an MIC value
of 500 μg/ml against all the tested Candida strains, however, its derivatives E1-E6 showed more
potent in vitro activity with MIC values ranging from 1 to 62 μg/ml for clinical FLC- suscepti-
ble strains and 2 to 62 μg/ml for clinical FLC- resistant strains. The order of sensitivity on the
basis of MIC values of the eugenol-tosylates is E5 > E3 > E4 > E1 > E2 > E6. The MICs of
FLC against clinical FLC- susceptible Candida strains was 4–16 μg/ml and against clinical
FLC- resistant Candida isolates was 125 μg/ml. These results also demonstrate that modifica-
tion of eugenol decreases the MIC values drastically not only against FLC- susceptible Candida
isolates but also against FLC- resistant isolates.
Susceptibility in combination with fluconazole
The Fractional Inhibitory Concentration Indices (FICI) of E1-E6 combined with FLC were cal-
culated to determine their possible interactions against all the tested C. albicans strains using
the microbroth dilution method as previously described [18]. The data, shown in Table 3, for
E1-E6 in combination with FLC in a 1:1 ratio, indicates synergistic, additive or indifferent
interactions, while no antagonistic interaction was observed. Of the 150 combinations pro-
duced (Table 3), the FICI values against all the Candida strains ranged from 0.127 to 2.630.
Out of 150 combinations tested, 36% were found to be synergistic while 41% and 23% were
additive and indifferent, respectively (Table 3). Of all the combinations, the prominent syner-
gistic interaction was observed between E5 and FLC against a resistant C. albicans 3081
(FICI = 0.127). Most of the synergistic interactions were observed between E5 and FLC (72%),
while minimum synergistic interactions were observed between E4 and FLC (4%).
For 54 of the synergies in the 1:1 ratio, isobolograms were constructed, where nine different
ratios of E1-E6 and FLC were blended and antimicrobial efficacies were determined. Represen-
tative isobolograms for each tosylate (E1-E6) are shown in Fig 3. From these results, marked
synergistic interactions were observed with most of the combinations irrespective of the ratio,
while all other ratios exhibited additive interactions. Interestingly, none of the combinations
displayed indifferent or antagonistic effects. The combinations of E4 and FLC against C. albi-
cans 3068 and E3 and FLC against C. albicans 90028 showed synergistic interactions for all the
nine ratios examined. E5 and E6 in combination with FLC against C. albicans 3001 and C. albi-
cans 3048, respectively, showed synergy for eight of the nine ratios assayed. These synergistic
interactions are in agreement with the previous findings on synergistic combinations of antibi-
otics and newly synthesized compounds [27,28], and we also confirm the potential of these
derivatives to chemosensitize the action of FLC and other azole drugs.
PLOS ONE | DOI:10.1371/journal.pone.0145053 December 22, 2015
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Eugenol-tosylate and Its Congeners against C. albicans
Table 3. The fractional inhibitory concentration index of E1-E6 tested in 1:1 combinations with fluconazole against different C. albicans strains.
Strains
E1 + FLC INT E2 + FLC INT E3 + FLC INT E4 + FLC INT E5 + FLC INT E6 + FLC INT
ATCC 90028
∑FIC
0.630
ADD
0.750
ADD
0.255
SYN
1.130
IND
0.375
SYN
1.250
IND
Clinical fluconazole
susceptible strains
074
072 gr
072 big gr
079 small gr
002 B1
003 gr
004 gr
004 B1
177
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
1.260
2.500
1.520
1.520
2.000
1.000
1.520
0.500
0.375
0.630
0.750
0.255
0.500
0.630
1.000
IND
IND
0.380
0.531
0.760
0.760
0.625
0.255
1.000
0.750
0.375
0.750
1.000
0.193
0.750
0.630
0.750
SYN
ADD
ADD
ADD
ADD
SYN
IND
0.193
0.750
0.625
0.313
0.313
0.625
0.750
0.281
0.193
1.000
0.500
0.193
0.161
0.750
0.313
SYN
ADD
ADD
SYN
SYN
ADD
ADD
SYN
SYN
IND
0.564
1.268
1.268
1.520
1.260
0.630
1.260
1.130
2.520
1.130
2.438
0.630
1.500
2.260
0.564
ADD
IND
IND
IND
IND
ADD
IND
IND
IND
IND
IND
ADD
IND
IND
ADD
0.625
0.280
0.313
0.625
0.281
0.313
0.625
0.146
0.161
0.500
0.375
0.161
0.146
0.193
0.563
ADD
SYN
SYN
ADD
SYN
SYN
ADD
SYN
SYN
SYN
SYN
SYN
SYN
SYN
ADD
0.630
0.768
2.438
1.520
0.750
0.750
0.630
0.630
0.750
1.260
0.630
0.750
1.000
2.260
0.630
ADD
ADD
IND
IND
IND
IND
IND
ADD
ADD
ADD
ADD
ADD
IND
IND
IND
SYN
SYN
ADD
ADD
SYN
SYN
ADD
IND
ADD
SYN
ADD
IND
2281
2323
SYN
SYN
SYN
ADD
SYN
ADD
ADD
IND
2367
SYN
ADD
ADD
ADD
2427
2434
IND
2508
ADD
Clinical fluconazole
resistant strains
3001
3048
3068
3087
3081
3140
2357
2261
3178
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
∑FIC
0.322
0.322
0.282
0.282
0.282
1.248
0.322
0.564
0.532
SYN
SYN
SYN
SYN
SYN
IND
0.564
0.564
0.532
0.532
0.532
0.282
0.564
0.266
0.266
ADD
ADD
ADD
ADD
ADD
SYN
ADD
SYN
SYN
0.564
0.564
0.258
0.266
0.129
0.282
0.532
0.516
0.258
ADD
ADD
SYN
SYN
SYN
SYN
ADD
ADD
SYN
0.748
0.748
0.374
0.266
1.284
0.748
0.748
0.624
0.748
ADD
ADD
SYN
ADD
IND
0.266
0.266
0.516
0.266
0.127
0.532
0.266
0.254
1.016
SYN
SYN
ADD
SYN
SYN
ADD
SYN
SYN
IND
0.374
0.374
0.624
0.374
1.092
0.748
1.248
0.322
0.624
ADD
SYN
ADD
SYN
IND
ADD
ADD
ADD
ADD
ADD
IND
SYN
ADD
ADD
SYN
ADD
INT: Interpretation, ∑FIC is the sum of FICA and FICB
doi:10.1371/journal.pone.0145053.t003
Ergosterol Biosynthesis Assay and Docking Studies
Sterol quantification was done in various C. albicans isolates following treatment with E1, E2,
E3, E4, E5 and E6. The results obtained on ergosterol biosynthesis inhibition by test com-
pounds in different Candida strains are represented in Fig 4 and the %-age reduction in ergos-
terol biosynthesis with respect to control cells is summarised in Fig 5. Complete inhibition of
ergosterol biosynthesis was observed at MIC values of the test compounds, which are depicted
by the flat lines, whereas at ½ MIC values of E1-E6, a dose dependent decrease in ergosterol
biosynthesis was observed (Fig 4). Compared to the control cells (untreated), 18% to 64%
ergosterol biosynthesis inhibition was noted when laboratory strain C. albicans ATCC90028
was treated with ½ MIC values of E1-E6, while this figure reached 89 to 100% when cells were
treated with MIC values of E1-E6. For clinical fluconazole susceptible isolates ergosterol bio-
synthesis inhibition was observed to decrease in the range of 14% to 65% and 85% to 95%
when cells were treated with ½ MIC and MIC values of E1-E6, respectively. Total cellular
ergosterol content decrease was recorded as 14% to 42% and 84% to 99%, when clinical
PLOS ONE | DOI:10.1371/journal.pone.0145053 December 22, 2015
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Eugenol-tosylate and Its Congeners against C. albicans
Fig 3. Isobolograms depicting synergistic interactions of eugenol-tosylates (E1 to E6) with
fluconazole in nine different ratios against different Candida albicans isolates.
doi:10.1371/journal.pone.0145053.g003
fluconazole resistant isolates were respectively treated with ½ MIC and MIC values of the test
compounds E1-E6 (Fig 5). Fluconazole, as expected, inhibited 94% to 100% of ergosterol bio-
synthesis in the standard and clinical fluconazole susceptible strains of Candida whereas only
7% decrease was recorded for clinical fluconazole resistant strains. Ergosterol biosynthesis inhi-
bition at sub-MIC values of the test compounds reached up to 65%, indicating that these C.
albicans isolates belong to ergosterol tolerant class. Ergosterol tolerant class is a group of fungi
which are tolerant to ergosterol biosynthesis inhibition and can grow in the deficiency of 14α-
PLOS ONE | DOI:10.1371/journal.pone.0145053 December 22, 2015
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Eugenol-tosylate and Its Congeners against C. albicans
Fig 4. UV spectrophotometric sterol profiles of representative laboratory strain C. albicans ATCC 90028 (A), clinical fluconazole-susceptible C.
albicans 2281 (B) and Clinical fluconazole-resistant C. albicans 3001 (C) strains after treatment with MIC and ½ MIC values of compounds E1 to E6.
Isolates were grown for 16 h in sabouraud dextrose broth and sterols were extracted using alcoholic KOH and n-heptane from the treated and untreated cells
and spectral profiles between 240 and 300 nm were determined. Control represents the culture cells without any treatment and positive control represents
cells treated with 64 μg/ml of fluconazole.
doi:10.1371/journal.pone.0145053.g004
demethylation while intolerant class of fungi cannot survive in 14α- demethylation deficiency
[20, 29]. In the latter group, MIC values of a drug are equal to the concentrations required to
completely inhibit the ergosterol biosynthesis, whereas, for the tolerant class of fungi, MIC val-
ues are always higher than the concentrations required to inhibit the ergosterol biosynthesis.
PLOS ONE | DOI:10.1371/journal.pone.0145053 December 22, 2015
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Eugenol-tosylate and Its Congeners against C. albicans
Fig 5. Ergosterol biosynthesis inhibition in different Candida strains upon treatment with compounds
E1 to E6 at MIC and sub-MIC values. Ergosterol content was expressed by the percent reduction in the
mean cellular ergosterol content compared with that of negative control cells grown without any treatment.
FLC represents the positive control.
doi:10.1371/journal.pone.0145053.g005
To further validate the obtained results and to gain insight into the mechanism of action
and binding interactions, all the compounds E1-E6 and reference azole drug FLC were docked
into the active site of the modeled cytochrome P450 lanosterol 14α-demethylase of C. albicans
using autodock 4.2 and autodock vina software package. The active site pocket consists of
amino acid residues LEU139, LYS143, LEU150, ILE304, GLY308, THR311, PRO375, LEU376,
PHE463, HIS468, ARG469, CYS470, ILE471, GLY472, GLU473, PHE475, ALA476 and Heme
360. Binding affinity values of the docked compounds E1-E6 were found to be in the range of
-7.59 to -14.85 kcal mol-1 and that for FLC and EUG -16.5 kcal mol-1 and -9.54 kcal mol-1
respectively (Table 4). These results also suggest that FLC and the synthesized compounds
Table 4. Results of docking study shown as binding affinity values and hydrogen bonding interactions of E1-E6 with aminoacid residues of cyto-
chrome P450 lanosterol 14α-demethylase of C. albicans.
Compound
Binding affinity (kcal/mol)
H-bond interactiona
H-bondb
H-Bond length (Å)
E1
-9.72
-8.49
THR311
GLY308
1
1
1
2
1
2
4
4
3.2
2.9
E2
E3
-10.68
-10.54
-14.85
-7.59
GLY472
2.6
E4
THR311, ALA476
3.2
E5
GLY472
3.2
E6
ARG469, GLY472
LEU204, THR311, PHE475, PHE463
HIS377, TYR505, SER506, SER507
3.2–3.3
2.6–3.3
2.6–3.2
FLC
EUG
-16.54
-9.54
a = hydrogen bonds formed between the aminoacid residues of the modelled protein and the docked compounds.
b = Number of hydrogen bonds
doi:10.1371/journal.pone.0145053.t004
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Eugenol-tosylate and Its Congeners against C. albicans
Fig 6. Docking and binding mode of (A) FLC (B) E5 and (C) EUG in the active site pocket of modelled
CYP51 of Candida albicans. EUG showing interaction with other residues of the protein away from the
pocket and heme.
doi:10.1371/journal.pone.0145053.g006
interact with different residues of the protein and this interaction depends on the structure of
the compounds. The presence of different substituents on the phenyl ring of the sulfonyl pen-
dent also guides their interaction with the protein residues and heme. The docking result indi-
cated that synthesized compounds were held in the active pocket by forming combination of
hydrogen bonds, hydrophobic bonds and van der Waals interactions with the enzyme, besides
showing interaction with the heme group. FLC coordinates with the heme iron by triazole
nitrogen, besides showing other interactions within the pocket (Fig 6A). All the eugenol deriva-
tives (E1-E6) showed binding in the active site of the enzyme, where the least binding energy
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Eugenol-tosylate and Its Congeners against C. albicans
and best results were displayed by E5 showing hydrogen bonding interaction with GLY472 res-
idue of the protein (Fig 6B). Eugenol shows hydrogen-bonding interaction with HIS377,
TYR505, SER506 and SER507 residues of the protein away from the heme (Fig 6C). The dock-
ing study values are given in Table 4. On the basis of activity data and docking results, it was
found that all the compounds had the potential to inhibit 14α-demethylase of C. albicans and
therefore can be considered as lead structures for further optimization.
From the above discussion it is clear that the incorporation of sulfonyl group in place of the
hydroxyl group of eugenol in compounds E1-E6 enhances their activity; it could be because of
the property of the sulfonyl group forming hydrogen bonding interactions with the active site
residues of biological targets and also its incorporation into core ring structure with modified
conformation allows snuggly fitting into the active site. The oxygen of the >S = O group of E5
also coordinates with the heme group of the modeled protein and therefore mimics flucona-
zole, in a similar way, when it coordinates with the heme group through its triazole nitrogen
[4]. The presence of substituents on the phenyl ring of the sulfonyl pendent also has a marked
effect on the biological profile of the molecule as seen in the present study. To define the chem-
ical consequences of modifying the structure and explaining the biological results, it seems that
the presence of electron releasing groups like CH3 and C2H5 in E2 and E6 decreases the activity
and the presence of electron withdrawing groups in E3, E4 and E5, resulting in enhancement
of activity compared to the unsubstituted E1. The presence of groups like Cl, NO2 and Br in
E3, E4 and E5, however, resulted in disparity in activity, which could possibly be due the differ-
ent nature of the groups. The chloro group resembles methyl in size, but differs electronically,
considerably reducing ring Pka, compared to methyl or ethyl groups. Bromo is almost similar,
but less electronegative compared to chloro, and nitro is considerably more electronegative
and reduces ring pka drastically. Quite often, a change in the pKa has a strong effect on both
the pharmacokinetic properties of the molecule and its binding affinity. For example, a strongly
basic group may be required for binding within a certain lead series, but at the same time this
basic group may also be found to result in compounds with low bioavailability due to the lim-
ited ability of a strong basic group to pass through membranes. The presence of different
groups helps in finding an optimum between these conflicting effects. The effect of different
substituents on physicochemical, biophysical, and pharmacological properties of a compound
are also different. Also the substituents can interact directly with the protein, or indirectly by
modulation of the polarity of other groups of the compound that interact with the protein.
From the results of these preliminary SAR studies it can be inferred that optimum structural
properties and balanced physicochemical properties are essential for a compound to show
excellent activity. These results also suggest that the ergosterol biosynthesis inhibition is a fun-
damental mechanism of antifungal action of these test compounds (E1-E6), in a manner simi-
lar to that of fluconazole.
Quantitative real-time PCR analysis
Since all the tested semi-synthetic derivatives E1-E6, inhibited ergosterol biosynthesis in differ-
ent Candida isolates, we hypothesized changes in the corresponding gene expression levels in
cells treated with E1-E6 or FLC. Studies have already reported the role of protein product,
lanosterol 14α-demethylase, of ERG 11 gene in ergosterol biosynthesis pathway [30,31] and the
up regulation of this gene in azole resistance [31]. In view of this, one FLC- susceptible C. albi-
cans 2281 and one FLC- resistant C. albicans 3001 along with C. albicans ATCC90028 were
grown up to mid-log phase and were then exposed to MIC values of E1-E6 or FLC for 2 h in
the same media. After incubation cells were harvested and RNA was extracted. From the
extracted RNA, c-DNA was synthesized which was used as a template for a series of qRT-PCR
PLOS ONE | DOI:10.1371/journal.pone.0145053 December 22, 2015
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Eugenol-tosylate and Its Congeners against C. albicans
Fig 7. Relative expression of ERG11 of C. albicans ATCC90028, C. albicans 2281 and C. albicans 3001
exposed to different eugenol-tosylates (E1-E6) or FLC at their respective MIC values. Control represent
the compound free cells and had an average relative expression (of 3 independent recordings) with *3.124
0.124; **2.234 0.134; ***1.795 0.102
doi:10.1371/journal.pone.0145053.g007
experiments. The gene expression data observed by qRT-PCR is summarized in Fig 7. The
expression of the housekeeping genes ACT1 was also determined in every set of experiments.
The expression of ERG11 gene is shown as relative values in comparison to the control
(untreated) that were set to one. From the results, down regulation of the ERG11 gene in differ-
ent C. albicans isolates to varying extents were observed when cells were exposed to these deriv-
atives. As expected, FLC also down regulated the ERG11 gene in C. albicans ATCC90028 and
C. albicans 2281, while up regulation of ERG11 was observed when FLC- resistant C. albicans
3001 cells were exposed to the FLC. The up regulation of ERG11 gene in resistant cells follow-
ing the exposure of FLC was congruent with the results observed previously and has been
shown to be induced by the transcription factor encoded by the UPC2 gene [32]. Up regulation
of ERG11 resulting in the increased production of ERG11p are often associated with the azole,
polyene and azole-polyene cross-resistances [33]. The variation in the expression of this gene
in the presence of FLC in different FLC- susceptible and FLC- resistant isolates in the present
study also confirms the role of this gene in azole resistance. Our results with gene expression
agree with observed changes in the inhibition of ergosterol biosynthesis as observed in sterol
quantification assay. In this study, we only focused on the ERG11 gene as a specific regulator
for ergosterol biosynthesis in C. albicans; however, several other genes are also involved in this
process. It is also believed that most of these specific gene regulations involve similar promoter
structure [34,35], suggesting the possibility of these semi-synthetic derivatives for influenced
regulation at their loci as well. Although resistance against available antifungals is very com-
mon, it has been extensively studied that eugenol showed antifungal activities with several dif-
ferent targets in fungal cells [15,36,37] and therefore the chance of resistance development
against these compounds is very minimal [37].
The in vitro hemolytic assay is a possible screening tool to gauge in vivo toxicity to host
cells [15]. With this assay, eugenol has already been reported to possess least or no cytotox-
icity against the human blood cells as compared to the known antifungals (amphotericin
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Eugenol-tosylate and Its Congeners against C. albicans
B and fluconazole). It has also been reported that concentration of eugenol when used in
combination with other drugs is far below its cytotoxic values [38]. However, these observa-
tions do not warrant that the congeners do not have any cytotoxicity and therefore further
cytotoxicity and in vivo studies are required to delineate the cytotoxic effects of these
compounds.
In conclusion, modification of eugenol structure into its semi-synthetic analogues and com-
bination with fluconazole is a promising strategy to treat azole resistant Candida strains on
account of their significant antifungal activity against different FLC- susceptible and FLC-
resistant strains by inhibiting ergosterol biosynthesis and also down regulating its related gene
at MIC values. The structure, however, further needs to be optimized by substituting the phenyl
ring of the sulfonyl pendent with electronically different groups and further studies are needed
to determine the underlying mechanisms of the antifungal enhancements and the synergistic
interactions. In addition the potential of combination therapy use in vivo warrants
investigation.
Supporting Information
S1 Fig. Flow Chart diagram for homology modelling
(TIF)
S2 Fig. Ramachandran plot of Modelled structure
(TIF)
S3 Fig. Ramachandran plot of 4KOF
(TIF)
S4 Fig. Plot of Z-score of 4K0F (A) and modeled structure (B)
(TIF)
Author Contributions
Conceived and designed the experiments: AA MYW AK. Performed the experiments: AA
MYW AK. Analyzed the data: AA MYW AK. Contributed reagents/materials/analysis tools:
NM JM. Wrote the paper: AA MYW JM.
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