Azole Based Acetohydrazide Derivatives of Cinnamaldehyde Target and Kill Candida albicans by Causing Cellular Apoptosis

Article abstract of DOI:10.1021/acsmedchemlett.0c00030

Opportunistic fungal pathogens including Candida albicans are responsible for the alarming rise in hospital acquired infections and millions of deaths worldwide. The current treatment modalities are not enough to handle this situation, and therefore, new treatment modalities and strategies are desperately needed. In this direction, we synthesized a series of azole based acetohydrazide derivatives of cinnamaldehyde and subjected it to antifungal activity evaluation. Preliminary antifungal activity evaluation revealed tremendous antifungal potential of some of the derivatives against fluconazole susceptible and resistant clinical isolates of Candida albicans. Although all the compounds in the series are structurally similar except for the presence of different substituents on the phenyl ring of the acetohydrazide pendent, they sharply differed in their activity profile. Further mechanism of action studies revealed that these compounds have an apoptotic effect on C. albicans confirmed via Annexin V-FITC staining and TUNEL assay.

Full text of DOI:10.1021/acsmedchemlett.0c00030

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Letter
Azole based acetohydrazide derivatives of cinnamaldehyde
target and kill Candida albicans by causing cellular apoptosis
Mohmmad Younus Wani, Aijaz Ahmad, Faisal Mohammed Aqlan, and Abdullah Saad Al-Bogami
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00030 • Publication Date (Web): 05 Feb 2020
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Azole based acetohydrazide derivatives of cinnamaldehyde
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target and kill Candida albicans by causing cellular apoptosis
Mohmmad Younus Wani†*, Aijaz Ahmad#,§, Faisal Mohammed Aqlan†, Abdullah Saad Al-
Bogami†
†University of Jeddah, College of Science, Department of Chemistry, Jeddah, Saudi Arabia
#Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, 2193, South Africa
§Infection Control, Charlotte Maxeke Johannesburg Academic Hospital, National Health Laboratory Service,
Johannesburg, 2193, South Africa
A cinnamaldehyde based acetohydrazone derivative showed remarkable anti-candida activity by
causing cellular apoptosis
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Azole based acetohydrazide derivatives of cinnamaldehyde target
and kill Candida albicans by causing cellular apoptosis
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Mohmmad Younus Wani†*, Aijaz Ahmad#,§, Faisal Mohammed Aqlan†, Abdullah Saad Al-
Bogami†
†University of Jeddah, College of Science, Department of Chemistry, Jeddah, Saudi Arabia
#Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, 2193, South Africa
§Infection Control Unit, Charlotte Maxeke Johannesburg Academic Hospital, National Health Laboratory Service,
Johannesburg, 2193, South Africa
ABSTRACT: Opportunistic fungal pathogens including Candida albicans are responsible for the
alarming rise in hospital acquired infections and millions of deaths worldwide. The current treatment
modalities are not enough to handle this situation and therefore new treatment modalities and strategies
are desperately needed. In this direction, we synthesized a series of azole based acetohydrazide
derivatives of cinnamaldehyde and subjected it to antifungal activity evaluation. Preliminary antifungal
activity evaluation revealed tremendous antifungal potential of some of the derivatives against
fluconazole susceptible and resistant clinical isolates of Candida albicans. Although all the compounds
in the series are structurally similar except for the presence of different substituents on the phenyl ring
of the acetohydrazide pendent, they sharply differed in their activity profile. Further mechanism of
action studies revealed that these compounds have apoptotic effect on C. albicans confirmed via
Annexin V-FITC staining and TUNEL assay.
KEYWORDS: Azole; Acetohydrazide; Candida albicans; Antifungal; Apoptosis
The discovery of new and effective human therapeutic
agents is one of humanity’s most critical tasks.
Equitable access to essential medicines for priority
diseases is one of the most important requirements to
healthy life. It has been estimated that over 300 million
people are afflicted with a serious fungal infection and
about 25 million are at a high risk, ended dead or
doomed.1 Among the fungal infections Candida alone is
responsible for causing >350,000 deaths annually and
the number is expected to grow as the current
treatment modalities are not sufficient enough to
antifungals have greatly improved their activity and it
is our premise that the structural modifications of the
active scaffolds would help evade the fungal resistance
mechanisms although not for a long time, albeit, it
would at least augment the current treatment
modalities until a new drug with a different
mechanism of action would arrive.
Azoles are actually the most widely used drugs against
fungal infections and the most important azoles like
fluconazole,
voriconazole,
itraconazole
and
2, 3
posaconazole are used to treat invasive fungal
infections (Figure1). Azoles are considered to be
extremely well tolerated but at the same time they can
interfere with a number of other drugs due to their
ability to inhibit cytochrome P450.7 They inhibit the
synthesis of sterols in fungi by inhibiting cytochrome
P450-dependent 14α-lanosterol demethylase (P-
45014DM), which removes the methyl group on C-14 of
tackle the growing multi-drug resistance menace.
Development of a new antifungal drug or identifying
novel drug targets is challenging because there are
many similarities between fungal and human cells.
Despite of the fact, different approaches and strategies
have been pursued to prevent and combat this growing
problem.4-6
modifications of the chemical structure of traditional
Among
the
different
strategies,
1
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lanosterol, a key intermediate step in the formation of resistant C. albicans isolates reached to 0.5 µg/mL.
ergosterol in the fungal cell membrane.7, 8 Most of the
azole based antifungal drugs are fungistatic and their
protracted use has hugely contributed to the
development of drug resistance in C. albicans and
other species.7
Among all the target compounds 6a, 6b and 6c possess
moderate antifungal activity with a mean MIC value of
125, 64 and 64 µg/mL, respectively. On the basis of the
MIC and MFC results, order of potency of the seven
active compounds was 6f>6h>6i>6j>6g>6e>6d. As a
positive control, FLC was used because of its common
use in the treatment of Candida infections and as
expected the mean MIC values for FLC against
susceptible and resistant strains was 0.25 µg/mL and 16
µg/mL, respectively. The C. albicans strains with MIC
values of 8 µg/mL for FLC were considered as FLC
resistant strains following the CLSI interpretive
guidelines for in vitro susceptibility testing of Candida
species (CLSI 2012). 1% DMSO was used as a negative
control because all the test compounds were prepared
to different concentrations using this concentration of
DMSO and it was observed to have no inhibitory
activity against any of the tested isolates.
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Although huge efforts have been made to develop new
compounds with antifungal activity, only a few have
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shown the mechanism of cell death.9,
Candida
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albicans show different patterns of cell death when
responding to the external insults.11 Numerous
biochemical and morphological changes differentiate
apoptotic cell death with that of necrosis. Among
different mechanism, studying the movement of
phosphatidylserine and DNA fragmentation by
terminal deoxynucleotidyl transferace mediated dUTP
nick end labelling (TUNEL) assay are considered
standards to study apoptotic mode of cell death.
Understanding these mechanisms while developing
new antifungals can provide strong basis for effective
antifungal therapies.
Due to the poor or moderate antifungal activity of the
intermediates and compounds 6a, 6b and 6c, these
compounds were excluded for further studies.
N
N
N
<Figure 1>
HN
N
N
O
Figure 1. Structures of the most common azole
antifungal drugs.
R
(6a-6j)
Herein, we report on the synthesis and anti-candida
activity of a new series of azole based acetohydrizide
derivatives obtained from cinnamaldehyde by a multi-
step reaction procedure as illustrated in scheme S1 and
S2 (Supporting information) following our previously
reported reaction methods.12-14 The structures of the
intermediate compounds (1-5) and the target
derivatives (6a-6j) were established by different
physical and spectroscopic techniques (see supporting
information). The in vitro susceptibility of all the
eleven different isolates of C. albicans against the five
intermediates (1-5) and the target compounds (6a-6j)
were evaluated by determining MIC and MFC values.
Table 1 summarizes the in vitro susceptibilities (MIC
and MFC) of all the test compounds against 1 standard
laboratory C. albicans SC5314, 7 clinically obtained FLC
susceptible C. albicans and 3 FLC resistant C. albicans
isolates. MIC values of the intermediates (1-5) was
comparatively higher (>125 µg/mL) than the MIC
values of the target compounds (6a-6j) which ranged
from 0.125–125 µg/mL, while as MFC values varied from
1 – >125 µg/mL. MIC results reflected that compound 6f
exerted highest inhibitory activity against all the tested
C. albicans isolates with a mean MIC value of 0.125
µg/mL and MFC value of 1 µg/mL against FLC
susceptible isolates and these figures against FLC
Table 1: Minimum inhibitory concentrations and
minimum
fungicidal
concentrations
of
the
intermediates (1-5*) and target compounds (6a-6j)
against different Candida albicans
Comp.
R
Mean MIC/MFC (µg/mL)
FLC susceptible FLC resistant
(n = 8)
(n = 3)
1
2
3
4
--
--
--
--
--
H
>125
>125
>125
>125
>125
125
64
64
16
8
0.125
4
>125
>125
>125
>125
>125
>125
>125
>125
16
8
1
8
1
>125
>125
>125
>125
>125
125
125
64
>125
>125
>125
>125
>125
>125
>125
>125
16
16
1
8
1
5
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
FLC
2-Cl
4-Cl
4-CH3
4-OCH3
4-NO2
4-OC2H5
3-NO2
2-NO2
3,4-dimet
--
16
8
0.50
8
0.50
0.50
4
0.25
0.50
2
4
8
4
8
0.25
16
2
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*for structures see supporting information
results of annexin V-FITC and PI staining are shown in
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a cytogram (Figure 3) and the percentage of apoptotic
(early and late apoptosis) and necrotic cells were
calculated in Table 2. As protoplasts were used for this
assay, it is important to make sure that lyticase did not
cause false positive annexin V or PI staining. Therefore,
titration of lyticase was done and it was observed that
concentration of 1 µg/mL of lyticase did not cause any
apoptosis or necrosis (data not shown). Exposure of C.
albicans cells to ½ MIC values of the test compounds
for 3 hours resulted higher percentage of cells in late
apoptosis phase with a mean range of 15.60 % (for 6d)
to 48.30 % (for 6f). Among all the tested compounds,
6f, 6h,and 6i, being the most active antifungal
compounds, they exhibit higher percentage of
apoptosis (Table 2). These results also revealed that
these compounds induced early apoptosis in only 8 –
14 %, while as complete necrosis was induced in 2 –
12%. Cells incubated with 2.5 mM H2O2 (positive
control) showed mean early apoptosis of 27.5 %, late
apoptosis of 26.5% and necrosis of <3 % (Table 2).
To further confirm the antifungal activity of the most
active compounds (6d-6j), we quantified the effect of
these compounds on the cellular viability of C. albicans
SC5314 at their respective MIC values. Cells at a density
of 1 × 107 were exposed to MIC values of the test
compounds and cell viability was accessed by Muse
count and viability kit. In comparison to the negative
untreated cells, cellular viability decreased after the
exposure of the C. albicans cells to the test compounds.
The results showed that the decrease in cell viability
after exposure to test compounds was at varying
extends ranging from 55.8 – 22.8 % (Figure 2). The
results also depict that compound 6f is the most
effective among all the tested compounds with a cell
death of >75%. The viability results reflect the same
pattern as observed in the MIC determination, which
also advocates the reliability of this technique to be
used in antifungal studies. To further differentiate the
results between the cells and debris and/or non-
nucleated cells, cell size index was also done in the
same assay and it was confirmed that the results are
due to live and dead cells (Figure supplementary S1).
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<Figure 3>
Figure 3: Representative density plots showing
phosphatidylserine externalization, an early stage
apoptosis marker, shown by annexin V staining. The
plots represent the alterations of phosphatidylserine
externalization when cells were exposed to compounds
(6d-6j) at their respective ½ MIC values. Untreated
cells and cells exposed to H2O2 were used as negative
and positive controls respectively. Quadrant Q1 shows
necrotic, Q2 shows late apoptotic; Q3 shows early
apoptotic and Q4 shows live cells.
<Figure 2>
Figure 2: Cell Viability assay for C. albicans SC5314:
Cells at a density of 1 × 107were exposed to MIC values
of the test compounds (6d – 6j) and cell viability assay
was performed by the Muse Count & Viability kit
(Millipore) following the manufacturer’s protocols.
Percentage live and dead cells are shown in two
different subsets. Negative and positive controls
represent untreated and heat killed cells respectively.
Table 2: Mean percentage of cells showing early
apoptosis, late apoptosis and necrosis after exposure to
½ MIC values of the most active compounds for 3 h; 2.5
mM H2O2 was used as a positive control.
From the viability data shown above, it was clear that
the target compounds (6d-6j) were able to kill the C.
albicans cells within a maximum concentration of 16
µg/mL. To further assess if the killing occurred
through induction of apoptosis and/or necrosis,
phosphatidylserine externalization was studied using
annexin V-FITC labelling and PI staining, by flow
cytometry. The asymmetry of phospholipids varies
during normal physiological conditions and during
apoptosis. In the latter case, phosphatidylserine is
externalised to the outer leaflet of the plasma
membrane, which could be detected using annexin V-
FITC dye.15 On the other side, PI was used to detect
necrotic cells as this dye can only permeate into
necrotic cells with disintegrated membranes. The
Comp.
(MIC)
% cell population with different effects
after 3 h exposure
Annexin
V+/PI-
Annexin
V+/PI+
Annexin
V-/PI+
(early
(late
(necrosis)
apoptosis)
apoptosis)
Negative
control
3.47 0.26
27.60  2.76
10.18 0.58
0.06 0.01
26.43 0.54
15.60 0.65
0.150.03
Positive
control
2.31 0.01
3.70 0.22
6d
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<Figure 4>
6e
13.90 0.53
8.21 1.82
23.60 0.97
48.30 1.97
27.92 2.13
44.65 2.76
36.34 2.97
24.93 2.11
1.78 0.11
1
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Figure 4: Representative confocal scanning laser
fluorescence images of treated and untreated C.
albicans cells, revealing the presence of (A) live or dead
intact cells, stained by hoechst 33342 dye indicated by
blue fluorescence; (B) apopototic cells with fragmented
DNAby the test compound exposed cells, indicated by
green fluorescence; and (C) merged image of both live
and apoptotic cells. Negative control represents
untreated cells with no apoptosis and positive control
represents cells treated 0.125 µg/ml of amphoterecin B.
Wavelengths of Ex/Em: 488/515 nm and for Hoechst dye
Ex/Em: 350/460 nm were used. The bar represents 10
µm.
6f
6g
6h
6i
9.80 0.32
2.44 0.08
11.70 0.34
1.98 0.97
4.55 0.79
10.49 0.76
8.23 1.08
13.37 0.69
9.69 0.95
6j
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As observed in annexin V-FITC affinity labelling, it is
evident that these compounds showed more late
apoptosis than early apoptosis and therefore TUNEL
assay was performed after exposing the cells to ½ MIC
values of most active compounds. In late apoptosis,
due to proteolysis of nuclear proteins chromatin
damages which further results in DNA damage and
chromatin condensation.16 In TUNEL assay, these cells
with late apoptotic characteristics can be evaluated by
DNA fragmentation at single cell level which can be
visualized with the help of a TUNEL assay by labelling
3’-OH ends of nicked DNA with fluorescent dUTP.
DNA damage is the characteristic feature of late
apoptosis.17 The apoptotic cells showed green
fluorescence when treated with the test compounds,
whereas blue fluorescence was observed in all cell
populations (Figure 4). The C. albicans SC5314 cells
exposed to ½ MIC values of the active test compounds
exhibited a significant amount of nuclear DNA
damage. The extent of apoptosis in the treated cells
was approximately in the range of 25 – 55% cells. These
results can be corroborated with the results observed
with the phosphatidylserine externalization in flow
cytometry. Comparatively, cells exposed to positive
control (0.125 µg/mL amphotericin B) showed an
increase of >90% in TUNEL-positive nuclei, identified
as green fluorescence spots. It is important to note that
all the cells were only exposed to ½ MIC values and
not the MIC values of the test compounds in order to
avoid the killing effects of these compounds. It is
evident from the viability assay that these compounds
have rapid antifungal action. At sub-MIC values these
compounds, the percentage of cells that exhibited
TUNEL-positive nuclei was significantly greater than
the percentage of untreated controls cells. These
results are in corroboration with the previous findings
where antifungal drugs such as amphotericin B and
caspofungin were reported to cause late apoptosis.11, 16
In the same study, it was also reported thatnuclear
fragmentation associated with DNA damage was
caspofungin concentration independent.16
To ensure that the active compounds do not show any
cytotoxicity, hemolysis assay on horse red blood cells
was done. Red blood cells provide a handy tool for
toxicity studies of the compounds, because they are
readily available, their membrane properties are well
known, and their lysis can easily be monitored by
measuring the release of haemoglobin.18 The in vitro
haemolytic assay is a possible screening tool for
gauging in vivo toxicity to host cells. The %-age cell
lysis caused by these compounds at MIC and MFC
values is <10 and reached maximum of 40 % at higher
concentrations (Figure 5). These results confirmed
that all the test compounds are less toxic and can be
tested for in vivo studies using animal models.
<Figure 5>
Figure 5: % cell lysis caused by the test compounds
against horse red blood cells. Hemolysis was
determined by an absorbance reading at 450nm and
compared to hemolysis achieved with 1% Triton X-100
(reference for 100% hemolysis).
Despite the extensive studies in synthesising new
compounds to introduce new and effective antifungal
drugs for the treatment of invasive fungal infections,
only few have reached clinical trials. The biggest
challenge in developing novel antifungal agents is to
identify unique drug targets that are not shared with
human hosts. Therefore, new compounds are needed
to be identified or synthesised that can cause fungal
programmed cell death also called as apoptosis to
allow specific manipulation of cell death without
costing human counterparts. The target compounds
under study were obtained from a natural compound,
cinnamaldehyde with inherent biological activities,19-21
and the aldehyde functionality of this molecule was
converted to an azole which was further alkylated to
furnish the different acetohydrazide derivatives (6a-
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6j). The target compounds bear a similar ring skeleton results with the binding of annexin V, which was
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except for the presence of different substituents on the
phenyl ring of the acetohydrazide pendent. The
different substituents resulted in disparity in activity of
the otherwise similar derivatives. Without any
substituent 6a was the least active derivative, followed
by 6b, 6c and 6d which contain 2-Cl, 4-Cl, and 4-
methyl groups as substituents respectively on the
phenyl ring. The activity sharply increased (4 to 6-fold
increase) when these substituents were replaced by 4-
methoxy in 6e, 4-ethoxy in 6g and 2,4-dimethoxy in 6j
respectively. The activity further increased by 9-11 folds
when these substituents were replaced by a –NO2
group at position -4, -3 and -2 of the phenyl ring in 6f,
6h and 6i respectively. The effect of these substituents
on the activity of the derivatives could be attributed to
their different electronic effects. The nitro group can
undergo enzymatic reduction inside the biological
systems by both one- or two-electron mechanism. The
reduction of NO2 group by two-electron mechanism
detected by flow cytometer. It has been extensively
observed that fungicidal compounds at sub-MIC values
show programmed cell death.24, 25 Similarly, the most
active compounds in this study are fungicidal and at
sub-MIC values these compounds are able to cause
apoptosis as can be seen in flow cytometry and
confocal microscopy.
9
The gold standard drug for treating fungal diseases,
amphotericin B, has also been reported to have
fungicidal activity, causing apoptosis at sub-MIC
values.26 As from the results, it is evident that most of
the compounds cause late apoptosis than early
apoptosis, which could be due to the exposure time
and concentrations used. It can also be hypothesised
that at higher concentrations, these compounds show
more necrosis than apoptosis leading to the fungicidal
characteristics of these compounds. The other
significance of targeting apoptosis relies in the
metacaspases targeted in C. albicans during apoptosis.
It has already been established that the only caspase
identified as executing apoptosis in Candida is a
metacaspase, which is unique to fungi and are
represented by the other caspases in human hosts.27
Furthermore, induction of apoptosis at lower
concentrations can also be due to the oxidative insult
caused by these compounds and therefore there is a
scope for further studies identifying other
mitochondrial factors activated during the course of
mediating apoptosis in these cells. Like other lipophilic
compounds, these compounds (cLogP = 2.97-3.21) can
conjugate with reduced form of glutathione and
thereby disrupting the intracellular redox equilibrium
and leading to cellular apoptosis. This selective
targeting death processes in C. albicans by these azole
based acetohydrazide derivatives and their least off
target toxicity may help finding new antifungal agents
that could switch on endogenous cell suicide
mechanisms.
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generates amines through nitroso and hydroxylamine
22, 23
intermediates.
The radicals generated during the
process could react with certain biomolecules to
produce compounds having undesired effects.
Reduction by one-electron mechanism, on the other
hand generates a nitro radical anion, which is unstable.
This unstable radical gets oxidized back to the nitro
group by molecular oxygen, which in turn is reduced
to a very reactive superoxide. The compounds under
study were found to possess moderate to strong
antifungal activity against different FLC susceptible
and resistant C. albicans isolates. When the cells were
exposed to these compounds, the number of viable
cells decreased. Among all the tested compounds, only
7 (6d-6j) possessed high antifungal activity and were
further investigated for the mode of cell death in C.
albicans.
Induction of apoptosis in yeast is a complex process,
which involve many pathways. There are several
endogenous and exogenous factors which also regulate
apoptosis in C. albicansand has been regulated both at
biochemical and molecular levels.24 Activation of
endogenous factors results in apoptosis via increase in
ROS production, mitochondrial dysfunction including
mitochondrial depolarization, release of cytochrome c,
increase in intracellular Ca2+, DNA fragmentation etc.
Therefore, targeting these mechanistic switches may
lead to the development of novel antifungal agents
that switch on endogenous cell suicide mechanisms.
Flow cytometry analysis revealed that the C. albicans
cells exposed to sub-MIC concentrations of the test
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge
on theACS Publications website at DOI:
Details on preparation and characterization of compounds
1-5, and 6a-6j, in vitro assays, mechanism of action,
protoplast preparation, Annexin V and PI staining and
confocal microscopy.
AUTHOR INFORMATION
compounds
showed
the
movement
of
Corresponding Author
phosphatidylserine to the outer leaflet of the plasma
membrane. The externalisation of phospholipids
*Email: mwani@u.edu.sa
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https://orcid.org/0000-0002-1838-1337
(6) Terry, R.; Damian, J. K. Antifungal drug therapy:
challenges, and new approaches. Cold Spring
HarbPerspect Med. 2014, 4, a019703.
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Other Authors
Aijaz Ahmad - https://orcid.org/0000-0003-2845-0727
Faisal M. Aqlan – https://orcid.org/0000-0001-9424-3262
Abdullah S. Al-Bogami - https://orcid.org/0000-0002-
8670-2729
(7) Prasad, R.; Shah,A.H.; Rawal, M.K. Antifungals:
Mechanism of Action and Drug Resistance.Adv Exp Med
Biol. 2016, 892, 327-349.
Author Contributions
(8) Frank, C. O.; Alistair, J.P. B.; Neil, A.R. G, Antifungal
agents: mechanisms of action, Trends in Microbiol.2003,
11, 272-279.
The manuscript was written through contributions of
all authors. All authors have given approval to the final
version of the manuscript.
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(9) Çavuşoğlu, B.K.;Yurttaş, L.; Cantürk, Z. The synthesis,
antifungal and apoptotic effects of triazole-oxadiazoles
against Candida species. Eur J Med Chem. 2018, 20, 255-
261.
Funding Sources
This work was funded by the University of Jeddah, Saudi
Arabia, under grant No. UJ-45-18-DR. The authors
therefore, acknowledge with thanks the University technical and
financial support.
(10) Neto, J.B.; Silva, C.R.; Neta, M.A.; Campos,
R.S.;Siebra, J.T.; Silva, R.A.; Gaspar, D.M.;Magalhães, H.I.;
de Moraes, M.O.; Lobo, M.D.;Grangeiro, T.B.; Carvalho,
T.S.;Diogo, E.B.; da Silva, J.E.N.; Rodrigues, F.A.;
Cavalcanti, B.C.; Júnior, H.V. Antifungal Activity of
Naphthoquinoidal Compounds In Vitro against
Fluconazole-Resistant Strains of Different Candida
Species: A Special Emphasis on Mechanisms of Action on
Candida tropicalis. PLoS One. 2014, 9, e93698.
ACKNOWLEDGMENT
Authors are thankful to Mr. Shabir A. Lone and Dr. Arif
Mohammed for their valuable comments and suggestions
during the preparation of the manuscript. Authors are also
thankful to the Deanship of Scientific Research (DSR),
University of Jeddah, Saudi Arabia, for technical assistance.
ABBREVIATIONS
PI: Propidium Iodide, TUNEL: Terminal deoxynucleotidyl
transferase dUTP nick end labeling, DNA: deoxyribonucleic
acid, MIC: Minimum Inhibitory Concentration, MFC:
Minimum Fungicidal Concentration, FLC: Fluconazole, CLSI:
Clinical & Laboratory Standards Institute.
(11) Phillips, A.J.; Sudbery, I.;Ramsdale, M. Apoptosis
induced by environmental stresses and amphotericin B in
Candida albicans. Proc Natl Acad Sci USA. 2003, 25, 14327-
14332.
(12) Mohmmad, Y.W.; Abdul, R.B.; Amir, A.; Inho,
C.;Fareeda, A. Probing the Antiamoebic activity and
Cytotoxicity potency of some Tetrazole and Triazine
derivatives. Eur. J. Med. Chem.2012, 48, 313-320;
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FIGURES
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Figure 1: Structures of most common azole antifungal drugs.
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Negative control
Positive control
6d
6e
6f
6g
6h
6i
6j
Figure 2: Cell Viability assay for C. albicans SC5314:Cells at a density of 1 × 107were
exposed to MIC values of the test compounds (6d–6j) andcell viability assay was performed
by the Muse Count & Viability kit (Millipore) following the manufacturer’s protocols.
Percentage live and dead cells are shown in two different subsets.Negative and positive
controlsrepresent untreated and heat killed cells respectively.
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Negative control
Positive control
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Figure 3: Representative density plots showing phosphatidylserine externalization, an early
stage apoptosis marker, shown by annexin V staining. The plots represent the alterations of
phosphatidylserine externalization when cells were exposed to compounds (6d-6j) at their
respective ½ MIC values. Untreated cells and cells exposed to H2O2 were used as negative
and positive controls respectively. Quadrant Q1 shows necrotic, Q2 shows late apoptotic; Q3
shows early apoptotic and Q4 shows live cells.
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6j
Figure 4: Representative confocal scanning laser fluorescence images of treated and
untreated C. albicans cells, revealing the presence of (A) live or dead intact cells, stained by
hoechst 33342 dye indicated by blue fluorescence; (B) apopototic cells with fragmented
DNA by the drug exposed cells, indicated by green fluorescence; and (C) merged image of
both live and apoptotic cells. Negative control represent untreated cells with no apoptosis and
positive control represents cells treated 0.125 µg/ml of amphoterecin B. Wavelengths of
Ex/Em: 488/515 nm and for Hoechst dye Ex/Em: 350/460 nm were used.The bar represents
10 µm.
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2 µg/mL
4 µg/mL
8 µg/mL
16 µg/mL
32 µg/mL
64 µg/mL
6D
6E
6F
6G
6H
6I
6J
PBS
Figure 5: % cell lysis caused by the designed test compounds against horse red blood cells.
Hemolysis was determined by an absorbance reading at 450 nm and compared to hemolysis
achieved with 1% Triton X-100 (reference for 100% hemolysis).
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