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

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.

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 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.

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 cyclic AMP (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 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.

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.

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 of 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 (see Fig. S1 in the supplemental material). At least nine compounds (compounds 1, 2, 5, 6, 7, 11, 12, 13, and 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).

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 ± the standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).

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 to 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 and B). 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.

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 ± the standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).

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; see also Fig. S2) exhibited a high capacity to reduce C. albicans cytotoxicity by more than 80% at a final concentration of 100 μM (equivalent to 21.2 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 and 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 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).

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 12 (100 μM, equivalent to 21.2 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 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 and 22.6 μg/ml, respectively) on the colony morphology of C. albicans.

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 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).

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 panel 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 ± the standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).

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).

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. Arrows indicate C. albicans cells in hyphal form.

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 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 AMP/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. 6) (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. 6). 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.

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 or without the addition of the compound. qRT-PCR results were normalized using the C_T 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 ± the standard deviations 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.

Compound 12 inhibits hyphal formation and cytotoxicity in various clinical candida species.

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 these 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 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).

FIG 7.

Analysis of the hyphal formation (A and B) and virulence (C) of different Candida species isolates (C. albicans ATCC 90028, C. albicans ATCC 10231, C. albicans ATCC 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 ± the standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).

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 and 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, and 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. 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, and 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 and 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

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 of Bacto yeast nitrogen base and 0.2% glucose per liter) overnight. Human lung epithelial A549 cells were incubated in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) 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 mmol), butylated hydroxytoluene (0.45 mol %), and 3 ml of distilled tetrahydrofuran was stirred at 80°C for 18 h. The mixture was extracted with ethyl acetate three times, and the combined organic extracts were washed with aqueous NaCl three times, 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 at 600 nm (OD₆₀₀) of 0.1 using fresh GMM. The yeast cells were incubated at 37°C for 6 h with compounds at a concentration of 100 μM, and the same volume of DMSO was used as a control. Cells were harvested by centrifugation at 5,000 rpm for 10 min. Cell suspensions were visualized directly under a Leica inverted fluorescence microscope with ×100 magnification. All the strains were treated according to the same methods.

Cytotoxicity assays.

Cytotoxicity was assessed by measuring the release of LDH from A549 cells. The 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 the 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 to 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 (yeast nitrogen base; 6.7 g of Bacto yeast nitrogen base per liter) plus 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 48 h at least. A 300-μl portion of inoculated culture was grown in each well at 30°C in a low-intensity shaking model using the Bioscreen-C automated growth curve 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- to 22-g male BALB/c mice (three mice per group) were subcutaneously injected with hydrocortisone (225 mg/kg) dissolved in phosphate-buffered saline (PBS) containing 0.5% Tween 20 on the first day. The next day, the overnight-cultivated cells were washed with Hanks balanced salt solution (Biohao Biotechnology Co., Ltd., Wuhan, China) and then twice with PBS. The cells 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 day 5, 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 plus 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, Carlsbad, CA) 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). 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 C_T (ΔΔC_T) 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 (v7.0) according to a 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 are expressed as the means ± standard deviations.