Identification of an indole-triazole-amino acid conjugate as a highly effective antifungal agent

Article abstract of DOI:10.1039/c5md00156k

Compounds constructed by the grafting of amino acid and triazole with an indole moiety were synthesized and investigated for antifungal activities wherein one of the compounds gave highly promising results. Although the compound has a high MIC₈₀ value in liquid medium, it shows significant antifungal activity against Candida albicans in solid plate assays. The compound is found to target chitin in the cell wall and lanosterol 14α-demethylase of the ergosterol biosynthesis pathway. The inhibitory effect of the compound becomes more pronounced in combination with azoles and CFW. The compound is also found to significantly affect chitin levels, cell morphology and cell viability in combination with ketoconazole.

Full text of DOI:10.1039/c5md00156k

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Identification of an indoleꢀtriazoleꢀamino acid conjugate as highly
effective antifungal agent
a,b#
a#
c
c
b
Kalpana Pawar , Anshuman Yadav , Parteek Prasher , Sahil Mishra , Balwinder Singh , Palwinder
c
a*
Singh * and Sneha Sudha Komath
c
5
Department of Chemistry, Guru Nanak Dev University, Amritsarꢀ143005 India
School of Life Sciences, Jawaharlal Nehru University, New Delhiꢀ 110 067 India
Uttarakhand Technical University, Dehradunꢀ 248 002 India
Equal contributing authors
a
b
#
*
To whom correspondence may be addressed (palwinder_singh_2000@yahoo.com; sskomath@mail.jnu.ac.in;
1
0
sskomath@yahoo.com)
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Compounds made up by the grafting of amino acid and
triazole with indole moiety were synthesized and investigated
in use include allylamines (terbinafine) and amorpholines
(nystatin) which inhibit ergosterol biosynthesis by targeting
for antifungal activities whereby one of the compounds gave 50 upstream enzymes, squalene epoxidase (Erg1) and sterol ꢁ ꢀ
1
2
2
5
0
5
1
4
reductase or ꢁ7ꢀ8 isomerase of the ergosterol biosynthesis
pathway, respectively, as well as amphotericin (polyene) which
binds directly to ergosterol in the cell membrane to form
cytotoxic pores for leakage of cellular content. More recently, the
fungal cell wall has begun to be targeted mainly with the help of
echinocandins (caspofungin, micafungin and anidulafungin)
which inhibit synthesis of β(1,3)ꢀglucan in the fungal cell wall,
and are gaining popularity in treatment of systemic infections of
highly promising results. Although the compound has high
MIC80 value in liquid medium, it shows significant antifungal
activity against Candida albicans in solid plate assays. The
compound is found to target chitin in the cell wall and
lanosterol 14αꢀdemethylase of the ergosterol biosynthesis
pathway, respectively. The inhibitory effect of the compound
becomes more pronounced in combination with azoles and
CFW. The compound is also found to significantly affect
chitin levels, cell morphology and cell viability in combination
with ketoconazole.
5
6
6
7
7
8
5
0
5
0
5
0
13–16
immunocompromised patients.
Nikkomycin z, a drug that
targets fungal cell wall synthesis by competitive inhibition of
chitin synthases, has shown promise in veterinary treatment and
1
8
is currently undergoing clinical trials for use on human subjects.
Introduction
What has become amply evident in recent years has been the low
rate of discovery of new antifungal drugs in stark contrast to the
variety of treatments available for bacterial infections.
Furthermore, the toxicity of the currently available drugs and
their rather limited mode of action (often fungistatic rather than
fungicidal) combined with increasing incidences of multidrug
resistance in the pathogen have led to the exploration of
combinatorial therapies as a viable option.
Fungal infections, varying from superficial ones to life
threatening systemic infections, have become a major challenge
in today’s medical world. Amongst pathogenic fungi, Aspergillus,
Candida and Cryptococcus species are responsible for causing
the maximum number of infections. Candida albicans is one such
species and is responsible for causing the maximum number of
3
3
4
4
0
5
0
5
1–4
systemic infections
in immunocompromised patients.
Using indole based compounds as possible candidates that could
both target the ergosterol biosynthetic pathway as well as
modulate response to currently available antifungal drugs; the
current manuscript explores the possibility of using these
compounds in combination antifungal therapies. The rationale for
this work is based on our previous results wherein we had
identified indole based compounds as possible ligands for
lanosterol14αꢀdemethylase using molecular docking studies and
had gone on to show that these compounds could act on the sterol
biosynthesis pathway as well as work in combination with azole
However, targeting these eukaryotic pathogens specifically
without harming the host has been a challenge.
Two major differences do exist between the pathogen and the
host that could be exploited. Unlike the human host, fungi
possess a cell wall. In addition, they use ergosterol as their major
cellular sterol and not cholesterol. Thus, the cell wall and
ergosterol or the sterol biosynthetic pathways are popular targets
of antifungal medication.
Prominent among the current antifungals are azoles such as
fluconazole,
itraconazole,
terconazole,
voriconazole,
17,18
drugs.
Keeping in mind the significance of the azole moiety in
ravuconazole, posaconazole, isavuconazole and albaconazole,
which target the cytochrome p450 (Erg 11) enzyme of the
several antifungal drugs, we introduced this moiety in the
previously optimized antifungal agents for our present study. We
5
ꢀ12
ergosterol biosynthesis pathway.
Older antifungals that persist
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demonstrate the effect of these chemical modifications on the
through column chromatography using ethyl acetate – hexane as
potential of the compound to inhibit fungal growth and induce 15 eluent (Scheme 1).
cell death in the absence or presence of antifungal drugs. Further,
we explore whether the compound itself can work at more than
one target site on the fungal pathogen.
Compound 3a (1 mmol) and NaH (1.2 mmol) (after washing with
dry hexane) were taken in dry ACN. Keeping the reaction
o
5
temperature 0 – 5 C, propargylbromide (1.2 mmol) was added
and the reaction mixture was stirred for 30 min. After filtration,
20 the filtrate was concentrated under vacuum and purified by
column chromatography to procure compound 4a (Scheme 2).
Similarly, compounds 4(bꢀd) were prepared from compounds
Synthesis
For introducing amino acid residue at Cꢀ3 position of indole, it
o
(
1) was dissolved in dry ether and keeping the temperature 0 C,
3
(bꢀd). Taking in ethanolꢀwater (9:1), compound 4a was treated
oxalyl chloride was added dropwise. A yellow colored solid (2)
was immediately formed which was filtered and dissolved in dry
ACN followed by the addition of K CO . Eventually, the
with sodium ascorbate (0.05 mmol). The reaction mass was
allowed to stir for 5ꢀ7 min and subsequently, CuSO .5H O (0.02
10
25
4
2
2
3
mmol) was added to the reaction mixture. After the addition of
neutralized Lꢀamino acid ester was added to the reaction mass.
After usual workup, 3ꢀsubstituted indole 3(aꢀd) were purified
N
H
1
i
O
O
H
O
H
N
Cl
N COOMe
NH
iii
ii
C
O
O
H
N
N
O
O
N
H
H
COOMe
3a
H
2
3
b
v
iv
N
COOCH3
H
O
H
H
N
NH
N
C
C
H
O
O
N
N
COOMe
COOCH3
H
H
3
c
3d
Reaction conditions:
i) (COCl) , dry ether, 0 C
o
2
o
ii) Dry ACN, K CO , glycine methyl esterHCl, 0ꢀ5 C
2
3
o
iii) Dry ACN, K CO , Lꢀtryptophan methyl esterHCl, 0ꢀ5 C
2
3
o
iv) Dry ACN, K CO , Lꢀhistidine methyl esterHCl, 0ꢀ5 C
2
3
o
v) Dry ACN, K CO , Lꢀglutamic methyl diesterHCl, 0ꢀ5 C
2
3
Scheme 1
30
phenylazide, the reaction mass was allowed to stir overnight at
room temperature which led to the formation of compound 5a.
Following the same sequence of steps, compounds 5(bꢀd) were
preliminary analysis, formation of a clear inhibitory zone around
the disc confirms the antimicrobial activity of the drug.
Compound 6d alone appeared to be a potential candidate since it
synthesized from compounds 4(bꢀd) (Scheme 2). Compounds 45 formed an inhibitory zone whose diameter increased with
5
(aꢀd) were treated with 1N NaOH in acetone – H O (1:1) to
increase in concentration of compound. No such effect was seen
in the presence of solvent which means that solvent itself does
not show any inhibitory effect on the growth of fungal cells
(Figure 1).
2
35
procure respective compounds 6(aꢀd) (Scheme 2). All the
compounds were characterized with NMR, IR and HRMS and
their optical purity was checked with LCꢀMS using chirobiotic
column.
50
Evaluation of Minimum Inhibitory Concentration
for Inhibition of Growth by 80% (MIC )
Antifungal Activity
8
0
40
Compounds 6a, 6b, 6c and 6d along with DMSO (solvent
MIC80, was determined for compound 6d by broth microdilution
control) were subjected to disc diffusion assay. In this
method (as described in experimental section). The concentration
2
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of compound that resulted in 80% inhibition of growth in
compound 6d was 312.5 ꢂg/ml.
comparison to control cells (of C. albicans strain BWP17) was
taken as MIC80 (Table 1). The MIC80 value determined for the
5
O
O
O
H
H
H
COOMe
COOMe
N
COOH
N
N
O
O
O
N
N
N
N
N
i
ii
iii
3
a
N
N
N
N
6a
4a
5a
O
O
H
N
H
N
O
NH
H
COOMe
NH
H
COOH
H
NH
H
COOMe
N
C
C
C
O
N
O
O
N
N
N
ii
iii
i
3
b
N
N
N
N
N
4b
5b
6b
N
N
H
N
O
O
H
H
N
N
H
N
N
O
N
H
N
C
H
C
H
H
O
N
O
C
H
N
N
COOMe
COOH
O
N
COOMe
i
ii
iii
3
c
N
N
N
N
N
O
5c
6c
4c
COOMe
COOH
COOCH3
O
H
N
H
N
O
H
N
C
C
H
COOH
C
H
O
H
O
N
O
N
N
COOMe
N
COOCH3
ii
iii
i
3
d
N
N
N
N
N
5d
6d
4d
Reaction conditions:
i) NaH, Dry ACN , propargylbromide, 0ꢀ5 C;
ii) Ethanol:water (9:1), CuSO .5H O (0.02 mmol), Sodium ascorbate (0.05 mmol), Phenylazide (1.2 mmol)
o
4
2
iii) 1N NaOH, acetoneꢀwater
Scheme 2
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Figure 2. Effect of compound 6d on cell growth in solid medium.
BWP17 cells were allowed to grow in the presence of compound 6d at
different concentrations and spotted on control SDꢀUra minimal plates.
Cells from the secondary culture (0.1 OD600nm) were taken and fiveꢀfold
serial dilutions were done. Cell suspensions from each dilution (5 ꢂl)
4
3
4
5
0
7
6
5
4
corresponding to cell numbers 1×10 , 2×10 , 4×10 , 8×10 and 1.6×10
were spotted from right to left on these plates. No difference in growth
o
was seen at any concentration of 6d used. Plates were incubated at 30 C
for 2 days. The experiment was done twice in duplicates and a
Figure 1. Disc diffusion assay showing antifungal activity of different
representative image is shown.
compounds. Compounds 6a, 6b, 6c and 6d was spotted on these disks
along with DMSO as a solvent control. Out of four compounds tested, 6d
alone showed the inhibitory effect by the formation of clear inhibitory
zone. The diameter of this inhibitory zone increased with increase in
concentration of compound. In the presence of solvent the diameter of
zone was found to be 0.6 cm. But in presence of compound 6d the
diameter was found to be 0.8cm, 1.2cm and 2.5cm at 0.5 mg/ml, 1.0
mg/ml and 2.0 mg/ml concentration of compound respectively. The
experiment was done twice and representative images are shown
5
0
1
Table 1. Growth inhibition for compound 6d
Concentration of compound 6d in
assay (mg/ml)
% inhibition observed
1
.25
84
84
81
33
18
6
Figure 3. Effect of compound 6d on growth in solid medium. BWP17
0
.6
45 was grown in minimal medium and was spotted on different
concentration plates of compound 6d. Cells from the secondary culture
(0.1 OD600nm) were taken and fiveꢀfold serial dilutions were done. Cell
suspensions from each dilution (5 ꢂl) corresponding to cell numbers
0
.3
0
0
0
.16
.08
.04
7
6
5
4
4
1×10 , 2×10 , 4×10 , 8×10 and 1.6×10 were spotted from left to right on
o
50
these plates. Plates were incubated at 30 C for 3 days and images were
taken. The experiment was done twice in duplicates and a representative
image is shown.
In order to look for the possible mode of inhibition, the following
two sets of experiments were done. In the first set, cells were first
grown in the presence of different concentrations of compound
15
20
25
30
Efficacy of compound 6d in combination with
azole drugs
6
d and spotted on SDꢀUra agar plates containing no 6d (Figure
2
). Very mild growth inhibition was observed with 400 ꢂg/ml of
compound 6d at 24 hours but not at 48 hours. It is possible that
the absence of the compound on the plate allowed the cells to 55 As explained in the Introduction, previous studies suggested that
recover with time. In the second experiment, compound 6d was
included in the plates on which the cells were spotted. Based on
the MIC80 value of the compound, culture plates containing
different concentrations of compound 6d were prepared. A
bisꢀindole derivatives act on the sterol biosynthesis pathway and
are significantly more potent in their action when combined with
azoles. Compound 6d, which is also an indole derivative but
additionally contains a triazole moiety, was monitored for its
control plate containing the solvent alone (DMSO) was also 60 ability to affect/alter azole drug response of BWP17 cells.
prepared. BWP17 cells (OD
~ 0.1) were spotted on these
00nm
Antifungal activity was monitored by spotting BWP17 cells on
SDꢀUra plates containing compound 6d as well as ketoconazole
at their respective MIC80 values (Figure 4i). Growth of BWP17
on control plates containing either solvent alone or compound 6d
6
o
plates and incubated at 30 C. No inhibition was seen in the
presence of solvent but a significant inhibition was observed in
the presence of compound 6d at its MIC80 or higher (Figure 3).
Thus, it appeared that the continued presence of compound 6d 65 alone or ketoconazole alone at the same concentrations were also
had deleterious effects on fungal cell growth in solid medium.
simultaneously monitored.
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3
4
4
5
5
6
6
5
0
5
0
5
0
5
inhibitor (Figure 4ii). Interestingly, when combined with
terbinafine, compound 6d exhibited only a mildly enhanced
nd
inhibitory effect, that was apparent only on the 2 Day but was
rd
not visible on 3 Day (Figure 4ii). When the concentration of
terbinafine was left intact and the concentration of compound 6d
was reduced, no significant effect of the latter could be observed
on the inhibitory effect of the former (Figure 4ii). Thus, it appears
that compound 6d may selectively affect the Erg11 catalyzed step
of ergosterol biosynthesis that the azole drugs also target but does
not target the Erg1 catalyzed step.
It may be noted that at low concentrations of 6d it appears that
the fungal cells grow better on plates containing a combination of
terbinafine and 6d as compared to growth on plates with
terbinafine alone. Amongst the several hypotheses to explain
these results, one possible explanation could be that 6d at low
concentrations inhibits the interaction of terbinafine with Erg1,
rendering the cells resistant to terbinafine. Alternatively, given
that it too has a triazole moiety, it is possible that 6d at low
concentrations acts like the azole drugs and inhibits Erg11. It is
well established that inhibition of Erg11 or accumulation of
lanosterol works as a feedback inhibitor for upstream steps of the
pathway, including the one catalyzed by Erg1 in yeast and
Figure 4i. Effect of compound 6d on fungal growth in combination
with azoles. BWP17 cells (0.1 OD600nm) were serially diluted five times.
Cell suspensions of each dilution (5 ꢂl) corresponding to cell numbers
7
6
5
4
4
5
1×10 , 2×10 , 4×10 , 8×10 and 1.6×10 were spotted from left to right on
plate containing either ketoconazole or 6d or both (concentrations
o
indicated in the figure). Plates were incubated at 30 C for 3 days and
photographed. The experiment was done twice in duplicates and a
representative image is shown.
1
0
19,20
Candida.
If indeed 6d inhibits Erg11, it would result in
accumulation of lanosterol and an inhibition of Erg1. This would
result in a loss in sensitivity of the cells to terbinafine. At high
concentrations of 6d (312.5 ꢂg/ml), however, cell wall damage
and necrosis become significant (as seen in PI staining described
below), making the cells grown on plates containing terbinafine +
6
d slightly more sensitive than those on terbinafine alone.
However, we also do not rule out the possibility of more complex
mechanisms of inhibition including those involving multidrug
resistance (MDR) transporters.
Cell wall defect
Cell wall damage and viability loss are often phenomena related
to growth defects. Since cell wall is a first point where pathogen
Figure 4ii. Effect of compound 6d on fungal growth in combination
with terbinafine. BWP17 cells (0.1 OD600nm) were serially diluted five 70 and host interacts with each other, cell wall targeting drugs
1
2
5
0
times. Cell suspensions of each dilution (5 ꢂl) corresponding to cell
21–23
which affect the cell wall integrity were designed.
Previous
7
6
5
4
4
numbers 1×10 , 2×10 , 4×10 , 8×10 and 1.6×10 were spotted from left
studies from our labs have shown that certain acridone and indole
to right on plate containing either terbinafine or 6d or both
concentrations indicated in the figure). Plates were incubated at 30 C for
o
24
(
derivatives can affect fungal cell wall assembly. Since we had
3
days and photographed. The experiment was done twice in duplicates
observed growth defects in cells grown in the presence of
compound 6d, we examined whether the cell walls of fungal cells
treated with compound 6d were also defective.
One commonly used assay to assess cell wall defects is to
examine the tendency of fungal cells to form clumps. As can be
seen from Figure 5, in comparison to control cells, higher cell
clumping was seen in cells grown either in presence of compound
and a representative image is shown.
7
8
8
5
0
5
As can be seen from Figure 4i, compound 6d showed a milder
inhibition than ketoconazole at the concentrations tested.
However, when compound 6d was used in combination of the
azole drug it caused a significantly greater inhibition of growth of
BWP17 cells. The effect persisted when the concentration of
compound 6d was decreased to oneꢀtenth of its MIC80 value.
Compound 6d was also checked in combination with terbinafine
for antifungal effects. Terbinafine, as explained in the
introduction, acts on squalene epoxidase (Erg1) upstream of
lanosterol14αꢀdemethylase (Erg11) in the ergosterol biosynthesis
pathway. At their respective MIC80 concentrations, compound 6d
as well as terbinafine could inhibit growth of BWP17 cells on
solid medium, although the latter appeared to be a better growth
2
5
6
d alone, in ketoconazole alone or in media containing a
combination of both ketoconazole and compound 6d at their
respective MIC80 values.
Next we examined whether the compound also altered chitin
levels of the cell wall. For this, we first treated the fungal cells
either with compound 6d alone or in combination with
ketoconazole and examined the cell wall by staining with
calcofluor white (CFW), a chitinꢀspecific dye. Compared to
control cells treated without or with DMSO, much higher CFW
3
0
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45
Figure 6i. CFW staining in BWP17 cells treated with compound 6d
alone or in combination with ketoconazole. Overnight grown cells in
the presence of solvent and drugs were stained with CFW. No difference
50 in staining was seen in control cells and in the presence of solvent which
confirmed that solvent does not affect the cell wall. The thicker cell walls
and higher staining was seen in the presence of 6d and ketoconazole
(Keto) at their MIC80 values. However, the staining was reduced at low
concentration of 6d. CFW staining was also monitored in the cells grown
Figure 5. Clumping assay showing the effect of compound 6d on cell
wall of fungal cells. Overnight grown BWP17 cells in the presence of
ketoconazole (Keto), 6d or a combination of both were monitored for
clumping. As a control, cells grown in solvent alone are also shown.
Images were taken on a confocal microscope. The scale bar represents
5
1
0ꢂm. Experiment was done twice in duplicates
staining was observed in cells treated with compound 6d or with 55 independently in presence of ketoconazole (Keto) and CFW as well as in
their combination with 6d. Compared to cells grown in presence of CFW
only, more staining was seen in the cells grown in presence of 6d with
CFW and ketoconazole (Keto). Confocal images were taken on an Andor
spinning disc microscope. The white scale bars in the figures represent 10
compound 6d in combination with ketoconazole (Figure 6i).
Quantification of the fluorescence intensity of CFW bound to a
1
1
2
2
3
0
5
0
5
0
1
00 cells in each set also confirmed this (Figure 6ii). In addition,
we grew the cells in CFW and in growth media containing both 60 ꢂm. The experiment was done twice in duplicates. A representative image
is shown.
compound 6d and CFW. Long term treatment with CFW (unlike
in staining cells for chitin estimation) is known to induce cell
wall defects due to inhibition of chitin polymerization. Further, in
response to the cell wall stress, fungal cells increase production
2
5ꢀ27
of chitin, resulting in higher chitin levels.
As can be seen
from Figure 6i and Figure 6ii, treatment with CFW alone or with
a combination of both CFW and compound 6d increases chitin
production in BWP17 cells, although the effect is a lot more
pronounced in the case of the latter.
In order to confirm the effect of compound 6d on cell wall stress
and growth defects, we also grew BWP17 cells on plates
containing both compound 6d and CFW. While sensitivity was
observed in presence of CFW or compound 6d alone, much
greater sensitivity was observed on plates containing
a
combination of the two (Figure 7). As can be seen from Figure 7,
the effect of compound 6d could be observed even at a
concentration that was 10ꢀfold below the MIC80 for the
compound. Thus, all in all we concluded that compound 6d
induces significant cell wall defects and in combination with
azole antifungals or CFW is able to significantly exacerbate cell
wall stress in C. albicans cells.
Figure 6ii. Quantification of chitin levels in BWP17 cells treated with
compound 6d alone or in combination with ketoconazole. Chitin levels
were estimated by quantifying the fluorescence intensity using NIS
Elements AR Version 4 software. In maximum chitin levels were seen.
The large error bar seen in the data for cells treated with a combination of
6d and ketoconazole is due to errors arising in identifying individual cells
in this sample owing to the tendency of these cells to form clumps. The
experiment was done twice in duplicates and the averages with standard
deviations are shown.
6
7
5
0
To monitor morphological alterations that could accompany the
cell wall defects, scanning electron microscopy (SEM) was done
35
(Figure 8). Compared to control cells significant alterations were
seen in the morphology of the drug treated cells. Compared to
ketoconazole treated cells, compound 6d, at the concentrations
tested here, appeared to cause a far greater damage. It is
noteworthy that compound 6d even at a concentration that was 5ꢀ
fold below its MIC , could significantly affect the morphology
40
alteration in morphology. Both cell shrinkage and deposition of
cellular content on the cell surface were visible, suggesting that
the compound could assist in killing fungal cells in combination
with azole drugs (Figure 8).
8
0
of Candida cells. Cells treated with
a combination of
ketoconazole and compound 6d showed significantly higher
7
5
6
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Loss of cell viability
Do growth and cell wall defects produce a fungicidal effect of
compound 6d on C. albicans cells? To test this possibility, cell
viability assays were done. We monitored the necrotic effect of
compound 6d on Candida cells using propidium iodide (PI), a
dye that enters inside and nonꢀspecifically stains nucleic acids of
inviable/ dead cells to yield a red fluorescence but is normally
impermeable in viable cells. We observed that in comparison to
control cells, where no necrosis occurs, the number of necrotic
cells was significantly higher upon treatment with compound 6d
alone or with a combination of compound 6d and ketoconazole
3
3
4
0
5
0
(Figure 9). It may also be noted that the extent of necrosis was
not very high in fungal cells treated with ketoconazole alone in
this assay as compared to that treated with compound 6d. As can
also be seen from Figure 9, the effect of compound 6d was
concentrationꢀdependent. A fiveꢀfold drop in concentration of
compound 6d reduces its necrotic effect on C. albicans cells
(Figure 9).
Figure7. Effect of compound 6d in combination with CFW on fungal
cell wall. BWP17 cells (0.1OD600nm) were serially diluted five times. Cell
7
suspension from each dilution corresponding to cell numbers 1×10 ,
6
5
4
4
5
2×10 , 4×10 , 8×10 and 1.6×10 were spotted on CFW plate and 6d
plates at its inhibitory and subꢀinhibitory concentrations. Spotting was
also done on combined plate of CFW and 6d to look for the additive
effect of two. In the presence of CFW and 6d alone a slight sensitivity
was seen which gets stimulated in the combination at their MIC80 values.
However, the sensitivity seen was found to be rescued in combination
when the concentration of 6d was decreased to 2, 5 and 10 fold of its
MIC80 value. Plates were incubated at 30˚C for 2 days, photographed and
representative images are shown. Experiment was done twice in
duplicates.
1
0
Figure 9. Propidium iodide (PI) staining showing fungal cell necrosis
caused by compound 6d.Cells were allowed to grow overnight in the
presence of solvent, ketoconazole (Keto) and 6d at their MIC80 values. In
comparison to cells treated with solvent, higher staining was seen in cells
grown in presence of ketoconazole (Keto) and 6d at their MIC80 values.
The staining was found to be enhanced in cells grown in combination of
ketoconazole and 6d. Images were taken in confocal microscope. The
scale bar represents 10ꢂm. One scale bar of 10ꢂm has been added from
outside. Experiment was done twice in duplicates
4
5
5
0
1
5
Conclusion
The ergosterol biosynthetic pathway and the cell wall are two
extremely important targets in the identification/ design of
antifungals. We have synthesized an azole containing indole
derivative with significant detrimental effect on both the cell wall
and sterol biosynthetic pathway of Candida albicans. This
compound also seemed to act in concert with currently available
azoleꢀbased antifungals to improve the efficacy of drug treatment.
Interestingly, the cell wall defects and therefore cell killing
induced by the compound is of particular interest in combination
therapies with current azole antifungals available which are
mostly fungistatic in their mode of action.
5
5
Figure 8. SEM images showing the effect of compound 6d on cell
morphology in combination with azoles. SEM was done on a Carl
60
Zeiss Microscope EVO40 at 10,000 X and 20,000 X magnifications to
monitor the morphological changes taking place in cell wall. Cells treated
with either ketoconazole (Keto) alone, compound 6d alone, or a
combination of the two were monitored. Untreated BWP17 cells and
solventꢀtreated control (DMSO) are also shown for comparison. The scale
bar shown inside the images corresponds to 2 ꢂm.
2
0
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Journal Name, [year], [vol], 00–00 | 7

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Page 8 of 9
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DOI: 10.1039/C5MD00156K
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E. M. Johnson, A. Szekely, D. W. Warnock, J. Antimicrob.
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Acknowledgements
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SSK and PS are grateful to a collaborative research grant from
Department of Science and Technology, India for funding
support. The funding agency had no role in the design of
experiments or choice of journal for data publication. KP
received a senior research fellowship through a Council for
Scientific and Industrial Research (India) ꢀfunded project of SSK
and AY a junior research fellowship through a Department of
Biotechnology (India) ꢀfunded research project of SSK. PP and
SM thank CSIR, New Delhi and DST, New Delhi respectively for
junior research fellowship. We thank Ms. Tripti and Ms. Ruchita
Pal for technical assistance with the confocal and SEM images
The confocal images were obtained at the central instrumentation
facility of School of Life Sciences, Jawaharlal Nehru University
(JNU) and the SEM images were taken at the advanced
instrumentation facility of JNU.
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XXXX; E-mail: xxxx@aaa.bbb.ccc
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