Two New 1,3,4-Oxadiazoles With Effective Antifungal Activity Against Candida albicans

Abstract

Candida infections have become a serious public health problem with high mortality rates, especially in immunocompromised patients, since Candida albicans is the major opportunistic pathogen responsible for systemic or invasive candidiasis. Commercially available antifungal agents are restricted and fungal resistance to such drugs has increased; therefore, the development of a more specific antifungal agent is necessary. Using assays for antifungal activity, here we report that two new compounds of 1,3,4-oxadiazoles class (LMM5 and LMM11), which were discovered by in silico methodologies as possible thioredoxin reductase inhibitors, were effective against C. albicans. Both compounds had in vitro antifungal activity with MIC 32 μg/ml. Cytotoxicity in vitro demonstrated that LMM5 and LMM11 were non-toxic in the cell lines evaluated. The kinetic of the time-kill curve suggested a fungistatic profile and showed an inhibitory effect of LMM5 and LMM11 in 12 h that remained for 24 and 36 h, which is better than fluconazole. In the murine systemic candidiasis model by C. albicans, the two compounds significantly reduced the renal and spleen fungal burden. According to the SEM and TEM images, we hypothesize that the mechanism of action of LMM5 and LMM11 is directly related to the inhibition of the enzyme thioredoxin reductase and internally affect the fungal cell. In view of all in vitro and in vivo results, LMM5 and LMM11 are effective therapeutic candidates for the development of new antifungal drugs addressing the treatment of human infections caused by C. albicans.

Keywords: Candida albicans; antifungal activity; in vitro; in vivo; thioredoxin reductase.

FIGURE 1

Cellular viability (%) of Vero and HUVEC cells in the presence of different concentrations of LMM5 and LMM11. The experiment was carried out with two cell lines by a colorimetric assay (MTS) at 24 h, evaluating concentration range 0.5 to 256 μg/ml. The 50% cytotoxic concentration (CC₅₀) was defined as the compound’s concentration (μg/ml) required for the reduction of cell viability by 50%.

FIGURE 2

Quantitative and qualitative fungicide evaluation of LMM5 and LMM11 compounds against Candida albicans. Logarithm reduction of colony forming units (CFU) and minimum fungicidal concentration (MFC) after 24 h exposure to LMM5 (A) and LMM11 (B). C + (Control): inoculum under the same conditions but without compounds, including diluents. ^∗Values of p < 0.05 were considered statistically significant compared with control (C+).

FIGURE 3

Killing kinetics of the LMM5 and LMM11 against C. albicans. Time-Kill curve of compounds: (A) LMM5 and (B) LMM11. The killing ability of the compounds was plotted from log₁₀ CFU/ml versus time points. Standardized yeast cell suspensions were exposed to Sub-MIC (16 μg/ml), MIC (32 μg/ml) and 2xMIC (64 μg/ml) of LMM5 or LMM11. In time points 0, 2, 4, 6, 8, 12, 24, 28, and 36 h, aliquots were diluted and plated on SDA for CFU/ml determination. Absence of compounds was used as a positive control and the commercial antifungal fluconazole (0.25 μg/ml) was used for comparison. Data are representative of three independent experiments and each data point represents the mean ± standard deviation (error bars).

FIGURE 4

Scanning Electron Microscopy of Candida albicans reference strain after exposure to LMM5 and LMM11. Standardized yeast cells suspensions (2-3 × 10³ yeast/ml) were exposed to the LMM5 or LMM11 for 24 h/35°C in concentrations at Sub-MIC (16 μg/ml), MIC (32 μg/ml) and 2×MIC (64 μg/ml). Control: Absence of compounds. Magnification: 3000×.

FIGURE 5

Transmission Electron Microscopy of Candida albicans reference strain after exposure to LMM5 and LMM11. Standardized yeast cells suspensions were treated with LMM5/LMM11 at 32 μg/ml (MIC) for 24 h and then processed for transmission electron microscopy. Images were obtained on a Zeiss 900 TEM. Control: Absence of compounds. Magnification: 15000× and 30000×. Red arrow: cytoplasm appeared homogeneous with a nucleus, mitochondria, surrounded by a defined cell membrane and regular cell wall; yellow arrow: large number of membranous bodies; blue arrow: alterations in the cell membrane and dysfunctions of the organelles.

FIGURE 6

In vivo evaluation of antifungal activity of LMM5 and LMM11 compounds in murine systemic candidiasis by Candida albicans. For each compound 20 female BALB/c mice, at 6 weeks old were divided in four groups (n = 5) treated with: LMM5 (5 mg/kg), LMM11 (5 mg/kg), fluconazole (5 mg/kg) or diluent (PBS, DMSO, and 0.02% F-127). The systemic candidiasis model by C. albicans was established by administering 5 × 10⁵ yeast cells (ATCC 90028) through the lateral tail vein. After 3 h of infection, the respective treatments were administered intraperitoneally according to the group, twice a day for 5 days. (A) Colony Forming Units (Log₁₀ CFU) per g of kidney. (B) Colony Forming Units (Log₁₀ CFU) per g of spleen. The bars indicate the standard deviation. ^∗Values of p ≤ 0.05 were considered statistically significant compared with control. ^∗∗Values of p ≤ 0.05 were considered statistically significant compared with fluconazole.

FIGURE 7

Histological sections of kidney from mice treated with LMM5, LMM11 and fluconazole and stained with Gomori & Grocott and hematoxylin and eosin (H&E). (A–D) Gomori & Grocott. (E–H) Hematoxylin and Eosin. (A,E) Histological sections of kidney treated with diluent (control group) and in larger magnification the agglomerate of yeasts (red arrow) and exacerbated inflammatory infiltrate in the respective region where yeasts were found in the control group (green arrow). (B,F) Histological sections of kidney treated with LMM5. (C,G) Histological sections of kidney treated with LMM11. (D,H) Histological sections of kidney treated with fluconazole. The histopathological samples were observed and photographed using a binocular light microscope (Motic BA310- camera Moticam 5), at ×400 and ×600 magnification.