Inhibition of Yeast-to-Hypha Transition and Virulence of Candida albicans by 2-Alkylaminoquinoline Derivatives

Article abstract of DOI:10.1128/AAC.01891-18

A rapid increase in Candida albicans infection and drug resistance has caused an emergent need for new clinical strategies against this fungal pathogen. In this study, we evaluated the inhibitory activity of a series of 2-alkylaminoquinoline derivatives against C. albicans isolates. A total of 28 compounds were assessed for their efficacy in inhibiting the yeast-to-hypha transition, which is considered one of the key virulence factors in C. albicans. Several compounds showed strong activity to decrease the morphological transition and virulence of C. albicans cells. The two leading compounds, compound 1 (2-[piperidin-1-yl]quinolone) and compound 12 (6-methyl-2-[piperidin-1-yl]quinoline), remarkably attenuated C. albicans hyphal formation and cytotoxicity in a dose-dependent manner, but they showed no toxicity to either C. albicans cells or human cells. Intriguingly, compound 12 showed an excellent ability to inhibit C. albicans infection in the mouse oral mucosal infection model. This leading compound also interfered with the expression levels of hypha-specific genes in the cyclic AMP-protein kinase A and mitogen-activated protein kinase signaling pathways. Our findings suggest that 2-alkylaminoquinoline derivatives could potentially be developed as novel therapeutic agents against C. albicans infection due to their interference with the yeast-to-hypha transition.

Full text of DOI:10.1128/AAC.01891-18

AAC Accepted Manuscript Posted Online 22 January 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.01891-18
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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Inhibition of yeast-to-hypha transition and virulence of
Candida albicans by 2-alkylaminoquinoline derivatives
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Lili Meng1,2#, He Zhao3#, Shuo Zhao1,2#, Xiuyun Sun1,2, Min Zhang3*, Yinyue Deng1,2
*
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1Integrative Microbiology Research Centre, College of Agriculture, South China Agricultural University,
Guangzhou 510642, China
2School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou 510275, China
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3Key Laboratory of Functional Molecular Engineering of Guangdong Province, School of Chemistry and
Chemical Engineering, South China University of Technology, Guangzhou 510641, China
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#These authors have contributed equally to this work.
*Corresponding authors:
Yinyue Deng: yinyue_deng@hotmail.com
Min Zhang: minzhang@scut.edu.cn
Tel: (+86)2085286471; Fax: (+86)2085280203
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Running title: 2-Alkylaminoquinolines inhibit C. albicans virulence
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ABSTRACT
A rapid increase in Candida albicans infection and drug resistance has caused an
emergent need for new clinical strategies against this fungal pathogen. In this study,
we evaluated the inhibitory activity of a series of 2-alkylaminoquinoline derivatives
against C. albicans isolates. A total of 28 compounds were assessed for their efficacy
in inhibiting the yeast-to-hypha transition, which is considered one of the key virulence
factors in C. albicans. Several compounds showed strong activity to decrease the
morphological transition and virulence of C. albicans cells. The two leading
compounds, compound
1
(2-[piperidin-1-yl]quinolone) and compound 12
(6-methyl-2-[piperidin-1-yl]quinoline), remarkably attenuated C. albicans hyphal
formation and cytotoxicity in a dose-dependent manner, but they showed no toxicity to
either C. albicans cells or human cells. Intriguingly, compound 12 showed an excellent
ability to inhibit C. albicans infection in the mouse oral mucosal infection model. This
leading compound also interfered with the expression levels of hypha-specific genes
in the cAMP-PKA and MAPK signaling pathways. Our findings suggest that
2-alkylaminoquinoline derivatives could potentially be developed as novel therapeutic
agents against C. albicans infection due to their interference with the yeast-to-hypha
transition.
KEYWORDS:
Candida
albicans;
yeast-to-hypha
transition;
virulence;
2-alkylaminoquinoline derivatives; oral mucosal infection model
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INTRODUCTION
Candida albicans is known as an opportunistic pathogen of humans that may lead to
serious and even life-threatening diseases in immunocompromised patients, resulting
in an approximately 40% mortality rate (1). Normally, C. albicans cells first adhere to
the tissue surface of the host, and the yeast form of C. albicans cells has been shown
to play an important role in tissue surface adhesion (2). Morphological transitions from
yeast to filamentous forms are the major contributor to the pathogenicity of C. albicans
(3). These conversions depend on environmental cues, including living conditions,
nutrient substances and several signaling metabolites (4,5). Farnesol, the first
quorum-sensing system identified in eukaryotes, can mediate C. albicans dimorphism
(6). In contrast, tyrosol can accelerate C. albicans growth and germ tube formation
(7).
Previous studies have indicated that morphological changes in C. albicans depend on
a network including multiple signaling pathways. The two best-studied pathways are
the cAMP-protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)
pathways (8). Overexpression of EFG1, which encodes an essential transcription
factor that activates the PKA pathway, was shown to enhance the filamentous form of
C. albicans and stimulate the expression of hypha-specific genes (9-11). CPH1 is a
transcription factor in the MAPK pathway, and the cph1/cph1/efg1/efg1 double mutant
was restricted to the yeast form, while either the cph1/cph1 mutant or the efg1/efg1
mutant retained some ability to switch from the yeast to the filamentous form,
suggesting that morphogenesis is mostly controlled by these two pathways (12).
Some antifungal agents have been successfully used in therapeutic treatments
against C. albicans. Triazoles, such as voriconazole and fluconazole, have been
widely used to treat the infections caused by Candida spp. Under anaerobic and
aerobic conditions, fluconazole inhibits C. albicans cells by 99% and 90%,
respectively (13). Amphotericin B is an important antifungal agent against
deep-seated fungal infection despite the side effects (14,15). Drug combination is also
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used as a strategy to combat candidiasis. It was reported that lovastatin has
synergistic effect with itraconazole to inhibit biofilms of C. albicans; and curcumin
takes synergistic action with fluconazole to treat clinical isolates of C. albicans (16,17).
However, serious drug resistance has arisen rapidly in recent years, compromising
the use of these antifungal agents. Therefore, there is an emergent need to develop
new strategies and novel drugs to combat candidiasis.
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2-Alkylaminoquinolines, which are widely used for their pharmaceutical and biological
activities, have been attracting increasing attention in physiological and
pathophysiological studies. They were confirmed as the antagonist that modulated
native TRPC4/C5 ion channels in various cells and tissues (18,19). In our previous
report, we modified the synthesis of 2-alkylaminoquinolines by copper-catalyzed
dehydrogenative α-C(sp3)−H amination of tetrahydroquinolines with O–benzoyl
hydroxylamines under mild conditions (20). In this study, we demonstrate the
screening and evaluation of a series of 2-alkylaminoquinoline derivatives in the
inhibition of C. albicans yeast-to-hypha transition and virulence. The two leading
derivatives showed excellent efficacy in blocking the morphological transition and
virulence but did not obviously influence the growth rate of C. albicans cells. Overall,
our methods focus on evaluating pathogenesis-related functions using both in vitro
and in vivo models to promote the development of novel antifungal therapeutics
against C. albicans infection.
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RESULTS
2-Alkylaminoquinoline derivatives inhibit hyphal formation in C. albicans
The morphological transition is important for C. albicans to infect humans and cause
disease (12; 21-23). Thus, the influences of the 2-alkylaminoquinoline derivatives on
the C. albicans yeast-to-hypha transition were evaluated in vitro under hyphal
induction conditions at 37°C. After 6 h induction, the majority of C. albicans cells in the
control group had formed germ tubes, while hyphal formation was obviously inhibited
by the addition of many of the 2-alkylaminoquinoline derivatives (Fig. S1). At least 9
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compounds (Nos. 1, 2, 5, 6, 7, 11, 12, 13, 16) reduced hyphal formation in C. albicans
cells by more than 70% when present at a final concentration of 100 µM (Fig. 1).
Among them, compounds 7 and 11 inhibited hyphal formation by approximately 95%
(Fig. 1).
2-Alkylaminoquinoline derivatives attenuate C. albicans virulence
To evaluate the effects of 2-alkylaminoquinoline derivatives on the pathogenicity of C.
albicans, we then investigated whether these compounds influenced C. albicans
virulence in a human cell line. Cytotoxicity was measured by quantifying the release of
lactate dehydrogenase (LDH) into the supernatants of cultured A549 cells. Many
derivatives, such as compounds 1, 9, 11, 12, 14, 18, and 21-28, showed no toxic
effects on A549 cells at a final concentration of 100 µM (Fig. 2A). In addition, the
exogenous addition of some 2-alkylaminoquinoline derivatives led to a significant
reduction in C. albicans cytotoxicity to A549 cells (Fig. 2B). Compounds 1, 5, 6, 7, 9
and 12 were highly effective at attenuating C. albicans cytotoxicity by more than 80%
when present at a final concentration of 100 µM, and compounds 1, 9 and 12 also
exerted no toxic effects on the cell line at 8 h postinoculation (Fig. 2A, 2B). Given that
compound 9 did not efficiently inhibit hyphal formation in C. albicans (Fig. 1),
compounds 1 and 12 were then selected for further investigation.
Compounds 1 and 12 do not obviously affect C. albicans growth rate but alter
its morphology
Both
compounds
1
(2-[piperidin-1-yl]quinolone)
and
12
(6-methyl-2-[piperidin-1-yl]quinoline) (Fig. 3A, S2) exhibited high capacity to reduce C.
albicans cytotoxicity by more than 80% at a final concentration of 100 µM (equivalent
to 21.2 µg/mL and 22.6 µg/mL, respectively) (Fig. 2B) but did not obviously affect the
growth rate of the pathogenic cells (Fig. 3B). Given that compounds 1 and 12 showed
excellent inhibition of hyphal formation in C. albicans (Fig. 1, 3C), these compounds
might be good candidates for development as novel antivirulence agents against C.
albicans infection. We then further investigated the effects of compounds 1 and 12 on
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C. albicans morphology; in good agreement with their inhibition of hyphal formation,
compounds 1 and 12 also obviously affected the colony morphology of C. albicans.
After the addition of compound 1 or 12, the colonies changed from wrinkled to slippery
(Fig. 3D).
2-Alkylaminoquinoline derivatives inhibit C. albicans hyphal formation and
virulence in a dose-dependent manner
To determine whether the effects of 2-alkylaminoquinoline derivatives on C. albicans
are related to their dosage, different concentrations of compounds 1 and 12 were
assessed for their inhibitory activity on the C. albicans morphological transition and
virulence (Fig. 4). Both compounds 1 and 12 exhibited dose-dependent activity, in
which they reduced C. albicans hyphae formation by more than 70% at a final
concentration of 50 µM (equivalent to 10.6 µg/mL and 11.3 µg/mL, respectively) (Fig.
4A). The addition of compounds 1 and 12 at a final concentration of 50 μM decreased
C. albicans virulence by approximately 40% and 60%, respectively (Fig. 4B).
Compound 12 inhibited C. albicans virulence by more than 49% when present at a
final concentration of 25 μM (equivalent to 5.65 µg/mL) (Fig. 4B).
Compound 12 inhibits C. albicans infection in the oral mucosal infection model
In addition to the assays in vitro, we continued to test whether the
2-alkylaminoquinoline derivatives have antifungal activity against C. albicans in vivo
by using the mouse oral mucosal infection model. In the presence of compound 12,
the number of C. albicans cells aggregated in the pathological tissues was much less
than that in the tissues infected with only C. albicans, and addition of compound 12
restored the tissues to a state similar to that of the uninfected group (Fig. 5).
Compound 12 inhibits the C. albicans morphological transition by interfering
with the cAMP-PKA and MAPK pathways
To establish a putative model of the effects of 2-alkylaminoquinoline derivatives on the
C. albicans morphological transition, we continued to investigate whether compound
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12 interfered with the signaling pathways involved in hyphal development. Hyphal
formation in C. albicans is associated with two established signaling pathways:
cAMP-PKA (cyclic adenosine monophosphate/protein kinase A) and MAPK
(mitogen-activated protein kinase cascade) pathways. We then used real-time PCR
analysis to analyze the effects of the compounds on the hypha-specific genes.
Exogenous addition of compound 12 inhibited the expression of PDE2, CDC35 and
TEC1, which are regulators involved in the cAMP-PKA pathway (Fig. 6A, 6B) (8, 24).
In addition, some regulators of the MAPK cascade, such as HST7 and CPH1 (8),
were downregulated by the exogenous addition of compound 12 (Fig. 6A, 6B). The
expression of ALS3, which plays a crucial role in adhesion (25), was also obviously
repressed (Fig. 6A). In addition, the expression level of HWP1, a glucan-linked protein
with serine/threonine-rich regions that were forecast to function in extending a
ligand-binding domain into the extracellular space (26), was reduced dramatically (Fig.
6A). Taken together, these results demonstrated that compound 12 influenced
complex signal transduction pathways to interfere with the C. albicans filamentation
process.
Compound 12 inhibits hyphal formation and cytotoxicity in various clinical
Candida species
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To determine whether the efficacy of 2-alkylaminoquinoline derivatives on C. albicans
is widely conserved, we collected several different clinical Candida species, including
C. albicans ATCC 90028, C. albicans ATCC 10231, C. albicans ATCC 14053, C.
tropicalis ATCC 750 and C. glabrata ATCC 2001, and investigated the effects of
compound 12 on their morphological transition and virulence. Intriguingly, only the
cells of C. albicans ATCC 9008 and C. albicans ATCC 10231 formed hyphae under
this conditions, exogenous addition of compound 12 exerted strong inhibition on
hyphal formation in both C. albicans ATCC 9008 and C. albicans ATCC 10231 (Fig.
7A). Addition of 100 µM compound 12 (equivalent to 22.6 µg/mL) caused a reduction
in hyphal formation in C. albicans ATCC 90028 and C. albicans ATCC 10231 to
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approximately 30% and 50% of that untreated group, respectively (Fig. 7B), while it
reduced the cytotoxicity of C. albicans ATCC 90028, C. albicans ATCC 10231, C.
albicans ATCC 14053, C. tropicalis ATCC 750 and C. glabrata ATCC 2001 on A549
cells to 45%, 37%, 12%, 25% and 30% of that untreated group, respectively (Fig. 7C).
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DISCUSSION
Numerous studies have suggested that morphogenesis is an essential factor for the
pathogenicity of dimorphic fungi. In this study, 2-alkylaminoquinoline-derived
compounds were first evaluated for their inhibitory activity against C. albicans
morphogenesis. Our results indicated that some 2-alkylaminoquinoline compounds
are excellent agents against the yeast-to-hypha transition in C. albicans (Fig. 1, 3C).
Given the interaction between morphological transition and virulence in C. albicans,
our results also demonstrated the notable ability of 2-alkylaminoquinoline derivatives
to attenuate C. albicans virulence (Fig. 2B, 4B, 5). Moreover, these compounds
showed low toxicity to human cell line A549 (Fig. 2A). We also confirmed the efficacy
of the leading compound 12 against other Candida. spp on the hyphal formation and
cytotoxicity. These results suggested that 2-alkylaminoquinoline derivatives might be
good candidates for the development of new antifungal agents that block hyphal
formation.
The current clinical treatments for candidiasis caused by C. albicans or other Candida
spp. rely almost entirely on limited conventional antifungal agents, such as polyenes,
which usually kill the pathogenic cells directly, and azoles that inhibit
14α-demethylation of lanosterol in ergosterol biosynthetic pathway (27, 28). However,
the limitations of drug development has compromised the strategies currently used in
clinical treatment. Therefore, the development of new strategies and novel drugs to
treat Candida spp. pathogens is urgently required. As morphological transitions
between yeast cells and filamentous forms play a vital role in pathogenesis, some
recent studies have already focused on the inhibition of hyphal formation in C.
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albicans (29, 30). Our results showed that compound 12 obviously interfered with the
cAMP-PKA and MAPK pathways, which are widely employed by fungal pathogens to
control the morphological transition. In addition, compound 12 also inhibits the
expression of HWP1 and ALS3 (Fig. 6). HWP1 is a membrane-anchored protein that
plays an important role in biofilm formation, while ALS3 is an invasin of C. albicans (8).
Our study here provides an additional option for the design of antifungal drugs using a
functional approach. Given that compound 12 showed an excellent ability to inhibit C.
albicans infection in the mouse oral mucosal infection model (Fig. 5), compound 12
appeared to be highly promising in preventing C. albicans pathogenicity via inhibition
of hyphal formation rather than direct killing of pathogenic cells.
2-Alkylaminoquinolines were previously reported to exhibit extensive biological
functions and pharmacological activities, which inspired us to further exploration of
the pharmacological activity of 2-alkylaminoquinoline-derived compounds. In this
study, we report 28 2-alkylaminoquinoline derivatives for the first time and assess
their ability to inhibit the morphological transition and virulence in C. albicans. Some of
the derived compounds showed excellent efficacy in preventing the yeast-to-hypha
transition and reducing virulence in vitro and in vivo but did not obviously interfere with
the growth rate of C. albicans cells (Fig. 1, 2B, 3B, 5). Intriguingly, these compounds
were nontoxic or only slightly toxic to human cells (Fig. 2A). For these compounds, we
would like to continue to modify the structures and perform more assays on the animal
models. We also found that the compounds have a significantly synergistic effect with
fluconazole against the antifungal resistant isolate FLU-R in cell line model (Table S1,
Fig. S3), we would also continue to test the synergistic effect of these compounds with
different conventional antifungal agents on the treatment of C. albicans infection.
Overall, our findings focus on targeting the morphological transition instead of killing
pathogenic fungal cells to promote the development of a novel strategy against C.
albicans infection.
MATERIALS AND METHODS
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Strains and growth conditions
Candida albicans SC5314 (ATCC® MYA-2876TM), C. albicans ATCC 90028, C.
albicans ATCC 10231, C. albicans ATCC 14053, C. tropicalis ATCC 750 and C.
glabrata ATCC 2001 used in this study were maintained at 30°C in yeast peptone
dextrose (YPD, 2% peptone, 2% glucose, 1% yeast extract and 2% agar) agar plates.
Before the following assays, C. albicans was incubated at 30°C with shaking at 200
rpm in GMM (glucose minimal medium; 6.7 g Bacto yeast nitrogen base and 0.2%
glucose per liter) overnight. Human lung epithelial A549 cells were incubated in
DMEM (Dulbecco’s modified Eagle’s medium) with 10% FBS (fetal bovine serum) at
37°C with 5% CO2.
Chemical synthesis of 2-alkylaminoquinolines
Briefly, under oxygen atmosphere, a mixture of 1,2,3,4-tetrahydroquinoline (0.2 mmol),
1-benzoyloxy-piperidine (0.6 mmol), CuI (0.08 mmol), KOH (0.3 mmol), K2CO3 (0.3
o
mmol), BHT (0.45 mol %) and 3 mL distill THF was stirred at 80 C for 18 h. The
mixture was extracted with EtOAc three times, and the combined organic extracts
were washed with aq. NaCl three times and dried over Na2SO4 and concentrated in
vacuo. All compounds (Fig. S1B) were dissolved in dimethyl sulfoxide (DMSO) at an
original concentration of 20 mM.
Hyphal formation assays
The overnight-cultured C. albicans grown at 30°C was diluted to an optical density
(OD600) of 0.1 using fresh GMM. The yeast cells were incubated at 37°C for 6 h with
compounds at the concentration of 100 μM, and the same volume of DMSO was used
as a control. Cells were harvested by centrifugation at 5000 rpm for 10 min. Cell
suspensions were visualized directly under a Leica inverted fluorescence microscope
with 100× magnification. All the strains were treated following the same methods.
Cytotoxicity assays
Cytotoxicity was assessed by measuring the release of LDH from A549 cells. The
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A549 cells were routinely grown in DMEM supplemented with 10% FBS in a 96-well
tissue culture plate with 1.5 × 104 cells/well. Confluent A549 cells were washed and
incubated with DMEM containing 1% FBS before infection. Overnight-cultured C.
albicans cells were diluted to an OD600 of 0.1 with DMEM containing 1% FBS in the
absence or presence of the tested compounds at the final concentrations indicated.
A549 cells were infected with fungal cells for 8 h. The LDH level in the supernatant
was measured, and cytotoxicity was calculated by comparing the LDH level to that of
the uninfected control. Different strains and compounds were tested using the same
method.
Colony morphology. Spider medium agar plates (1% peptone, 1% mannitol, 0.2%
K2HPO4 and 1.5% agar) were supplemented with different concentrations of
compounds as indicated. C. albicans SC5314 cells were grown in these plates at
37°C for 24-30 h. Images of the colonies were obtained using a Leica DMi8
microscope and a Nikon Coolpix digital camera.
Cell growth analysis. For the cell growth assay, C. albicans cells were cultured in
YNB + 0.2% glucose and diluted in the same medium to an OD600 of 0.05 in the
absence or presence of the tested compounds at the final concentrations indicated for
48h at least. Three hundred microliters of inoculated culture were grown in each well
at 30°C in a low-intensity shaking model using a Bioscreen-C Automated Growth
Curves Analysis System (Oy Growth Curves Ab, Finland).
Mouse oral mucosal infection model
The protocol of the mouse oral mucosal infection was based on a published study with
minor modifications (31, 32). In this experiment, 20-22 g male BaLB/c mice (3 mice
per group) were subcutaneously injected with hydrocortisone (225 mg/kg) dissolved in
PBS (phosphate-buffered saline) containing 0.5% Tween-20 on the first day. The next
day, the overnight-cultivated cells were washed with Hank’s Balanced Salt Solution
(Biohao Biotechnology Co., Ltd, Wuhan, China) and then twice with PBS. The cells
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were resuspended in PBS to an OD600 of 0.1 in the absence or presence of compound
12 at a final concentration of 100 μM (equivalent to 22.6 µg/mL). Then, 10% chloral
hydrate (Yuanye Biotech, Shanghai, China) was injected into mice, the anesthetized
mice were placed on an isothermal mat maintained at 37°C, and cotton balls soaked
with pathogenic cells were placed under their tongues for 75 min. Mice were sacrificed
on the fifth day, and their tongues were dissected for further analysis using
pathological sections and scanning electron microscope observation.
Quantitative real-time PCR
Overnight cultures of C. albicans cells grown in YNB + 0.2% glucose at 30°C were
diluted in the same medium to an OD600 of 0.1 in the absence or presence of the
tested compounds at a final concentration of 100 µM. After incubation for 6 h at 37°C,
the cell samples were collected and washed with PBS. Total RNA was extracted using
TRIzol (Invitrogen, California, America) and quantified. cDNA was obtained through a
reverse transcription reaction using a reverse transcription kit (TaKaRa Biotechnology,
Dalian, China) with the primers shown in Table S2, and real-time PCR was performed
with a 7300Plus Real-Time PCR System (Applied Biosystems, America). The
expression level of each gene was normalized to that of GSP1, which is a
housekeeping gene in C. albicans cells (33). The relative expression levels of the
target genes were calculated using the comparative CT (ΔΔCT) method.
STATISTICAL ANALYSIS
For statistical analysis, the Excel data analysis package was used to calculate the
means and the standard deviation of the means. The data were analyzed using the
GraphPad Instate software package (version 7.0) according to the Tukey-Kramer
multiple comparison test at a p<0.05 or p<0.01 level of significance. All results were
calculated from the means of three separate experiments. The results were
expressed as the means ± standard deviation.
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CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
ACKNOWLEDGEMENTS
This work was supported financially by grants from the Guangdong Natural Science
Funds for Distinguished Young Scholars (No. 2014A030306015), the National Key
Project for Basic Research of China (973 Project, 2015CB150600) and the National
Natural Science Foundation of China (No. 31571969).
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FIGURE LEGENDS
FIG. 1 Effects of 2-alkylaminoquinoline derivatives on C. albicans SC5314 hyphal
formation. Each experiment was performed at least three times in triplicate, and each
time, at least 400 cells were counted for each treatment. Compounds were dissolved
in DMSO, and the amount of DMSO used as the solvent for the compounds was used
as a control. Data represent the means ± standard deviation of three independent
experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).
FIG. 2 Effects of 2-alkylaminoquinoline derivatives on C. albicans SC5314 virulence
using a cell line. (A) Analysis of the toxicity of compounds to A549 cells. The
compounds were dissolved in DMSO, and the amount of DMSO used as the solvent
for the compounds was used as a control. (B) Analysis of the effects of the
compounds on the cytotoxicity of C. albicans to A549 cells. Cytotoxicity was detected
and measured as LDH release. The LDH released by A549 cells after inoculation with
C. albicans in the absence of compounds was defined as 100% to normalize the LDH
release ratios of the other treatments. Data represent the means ± standard deviation
of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t
test).
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FIG. 3 Influence of 2-alkylaminoquinoline derivatives on C. albicans SC5314
morphology. (A) Structures of compounds 1 and 12. (B) Effects of compounds 1 and
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12 (100 µM, equivalent to 21.2 µg/mL and 22.6 µg/mL, respectively) on the growth
rate of C. albicans cells. (C) Effects of compounds 1 and 12 (100 µM, equivalent to
21.2 µg/mL and 22.6 µg/mL, respectively) on hyphal formation in C. albicans. C.
albicans cells were grown under noninduction conditions (30°C) or under induction
conditions (37°C). The photos were taken 6 h after induction. (D) Effects of
compounds 1 and 12 (100 µM, equivalent to 21.2 µg/mL and 22.6 µg/mL, respectively)
on the colony morphology of C. albicans.
515
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519
520
FIG. 4 Effects of different concentrations of 2-alkylaminoquinoline compounds 1 and
12 on C. albicans SC5314 hyphal formation (A) and virulence (B). The LDH released
by A549 cells in (B) after inoculation with C. albicans in the absence of compounds
was defined as 100% to normalize the LDH release ratios of the other treatments.
Data represent the means ± standard deviation of three independent experiments. *,
P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).
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FIG. 5 Efficacy of 2-alkylaminoquinoline compound 12 (100 μM, equivalent to 22.6
µg/mL) against C. albicans SC5314 in the mouse oral mucosal infection model.
Pathological sections were evaluated to determine the effect on C. albicans infection.
FIG. 6 Effect of 2-alkylaminoquinoline compound 12 on the signaling pathways
involved in the hyphal development process of C. albicans SC5314. (A) Comparison
of relative transcript levels of regulator-encoding genes between C. albicans cells with
and without the addition of the compound. qRT-PCR results were normalized using
the CT values obtained for GSP1 amplifications run in the same plate. The relative
levels of the gene transcripts were determined from standard curves. Data represent
the means ± standard deviation of three independent experiments. *, P < 0.05; **, P <
0.01; ***, P < 0.001 (unpaired t test). (B) Schematic diagram of the signaling pathways
that govern hyphal morphogenesis in C. albicans affected by compound 12.
FIG. 7 Analysis of the hyphal formation (A, B) and virulence (C) of different Candida
spp. isolates (C. albicans ATCC 90028, C. albicans ATCC 10231, C. albicans ATCC
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548
549
14053, C. tropicalis ATCC 750 and C. glabrata ATCC 2001) in the absence or
presence of compound 12 (100 μM, equivalent to 22.6 µg/mL). Data represent the
means ± standard deviation of three independent experiments. *, P < 0.05; **, P <
0.01; ***, P < 0.001 (unpaired t test).
19