Anticancer drugs targeting topoisomerase II for antifungal treatment

Abstract

Fungal topoisomerase II (TopoII) has been identified as essential for viability. Thus, our research aimed to investigate the potential of fungal TopoII as a novel target for antifungal chemotherapy. We conducted studies on eleventh antitumor compounds targeting human topoisomerase II, either approved by the U.S. Food and Drug Administration (FDA) or currently under clinical trials to evaluate their potential for use in other therapeutic applications. While most of the compounds we analyzed are potent inhibitors of yeast TopoII, only a few exhibited antifungal activity. Idarubicin emerged as the most potent compound effectively inhibiting the growth of five reference fungal strains as well as clinical Candida glabrata fluconazole-resistant cells. Antifungal activity of this compound corresponded with its very high yeast TopoII inhibitory effectiveness. Additionally, idarubicin ability to be effectively accumulated into fungal cells is crucial for yeast TopoII targeting. Idarubicin, epirubicin, and bisantrene appeared to be even more effective inhibitors of yeast enzyme than its human counterpart. In fungal cells idarubicin exhibited a multifaceted mechanisms of action, including nuclear DNA fragmentation, disruption of mitochondrial network architecture and mitochondrial DNA aggregation as well as oxidative stress induction. Our results indicate that fungal topoisomerase II targeting is worth considering in antifungal treatment and the reported drugs may serve as a starting point for the reinnovation of a new molecule.

Fig. 1

Known human topoisomerase II inhibitors analyzed in this study.

Fig. 2

Inhibition of the catalytic activity of purified yeast (yTOPOII) and human (hTOPOII) DNA topoisomerase II by etoposide, teniposide, mitoxantrone, pixantrone, and idarubicin measured by relaxation. Supercoiled pBR322 plasmid DNA (- hTOPO II or –yTOPO II) was relaxed by purified topoisomerase II in the absence (+ hTOPO II or + yTOPO II) or presence of compounds at selected concentrations [µM]. The resulting topological forms of DNA were separated by gel electrophoresis. SC, supercoiled DNA; R, relaxed DNA; T, DNA topoisomers. The data shown are typical of three independent experiments. The gels were cropped to increase the clarity of the presentation. The unedited gels are shown in Supplementary Materials Figure S1 and Figure S2.

Fig. 3

Fluorescence microscopic analysis of uptake and accumulation of idarubicin; daunorubicin and doxorubicin in S. cerevisiae cells ATCC 9763. Cells were suspended in phosphate-buffered saline and incubated in the presence of fluorescent probes at 100 µM of compounds concentration for an appropriate period of time. Scale bars correspond to 5 μm.

Fig. 4

S. cerevisiae ATCC 9763 cells incubated for 1 h (A) or 3 h (B) with idarubicin 2 µg mL^− 1, 4 µg mL^− 1 and 100 µM H₂O₂ after staining with Hoechst 33342 and Mitotracker green. (A) White arrows indicate aggregated mitochondria, selected cells with fragmented genetic material undergoing the apoptotic process is marked by yellow arrows Under magnification in square frames, the fusion of mitochondrial DNA with nuclear DNA is visible. Scale bars correspond to 5 μm. Controls are indicated as Ctrl.

Fig. 4

S. cerevisiae ATCC 9763 cells incubated for 1 h (A) or 3 h (B) with idarubicin 2 µg mL^− 1, 4 µg mL^− 1 and 100 µM H₂O₂ after staining with Hoechst 33342 and Mitotracker green. (A) White arrows indicate aggregated mitochondria, selected cells with fragmented genetic material undergoing the apoptotic process is marked by yellow arrows Under magnification in square frames, the fusion of mitochondrial DNA with nuclear DNA is visible. Scale bars correspond to 5 μm. Controls are indicated as Ctrl.

Fig. 5

Detection of ROS in S. cerevisiae ATCC 9763 cells incubated for 1 and 3 h with idarubicin 2 µg mL^− 1, 4 µg mL^− 1 and 100 µM H₂O₂. Fluorescence images were obtained after staining with 2′,7′-dichlorofluorescin diacetate (H2DCF-DA) and Hoechst 33342. Controls are indicated as Ctrl. Bars represent 5 μm.

Fig. 6

Assessment of ROS generation in Saccharomyces cerevisiae following idarubicin treatment via flow cytometry. DMSO and H₂O₂ were used as negative and positive controls, respectively. (A) Representative histograms demonstrate ROS induction across different treatment conditions; (B) Quantitative analysis of ROS levels. Error bars represent the SEM from three independent experiments (n = 3). Statistical significance is indicated as **p < 0.001, ****p < 0.00001 compared to the vehicle control (two-way ANOVA with Dunnett’s post hoc test).

Fig. 7

Microscopy analysis of changes in the morphology of C. albicans ATCC 10231 cells following treatment with idarubicin at 1/4 and 1/8 MICs. The cells were subjected to light microscopy, and the scale bar represents 10 μm. The analysis was conducted after a 3-hour incubation period at 37 ◦C in RPMI + 10% FBS medium. The experiments were performed at least in three replicates. P – pseudohyphae, Y – yeast and M – mycelial form.