Targeting yeast topoisomerase II by imidazo and triazoloacridinone derivatives resulting in their antifungal activity

Abstract

Fungal pathogens are considered as serious factors for deadly diseases and are a case of medical concern. Invasive fungal infections also complicate the clinical course of COVID-19, leading to a significant increase in mortality. Furthermore, fungal strains' multidrug resistance has increased the demand for antifungals with a different mechanism of action. The present study aimed to identify antifungal compounds targeting yeast topoisomerase II (yTOPOII) derived from well-known human topoisomerase II (hTOPOII) poisons C-1305 and C-1311. Two sets of derivatives: triazoloacridinones (IKE1-8) and imidazoacridinones (IKE9-14) were synthetized and evaluated with a specific emphasis on the molecular mechanism of action. Our results indicated that their effectiveness as enzyme inhibitors was not solely due to intercalation ability but also as a result of influence on catalytic activity by the formation of covalent complexes between plasmid DNA and yTOPOII. Lysine conjunction increased the strength of the compound's interaction with DNA and improved penetration into the fungal cells. Triazoloacridinone derivatives in contrast to starting compound C-1305 exhibited moderate antifungal activity and at least twice lower cytotoxicity. Importantly, compounds (IKE5-8) were not substrates for multidrug ABC transporters whereas a derivative conjugated with lysine (IKE7), showed the ability to overcome C. glabrata fluconazole-resistance (MIC 32-64 µg mL^-1).

Figure 1

Inhibition of the catalytic activity of purified yeast topoisomerase II by selected compounds as measured decatenation of kinetoplast DNA (catenated). The influence of (a) IKE5, IKE13 at 1, 10, 50, 150 µM and (b) IKE1, IKE3, IKE7, IKE8, IKE9, IKE12 and IKE14 at 50, 150 µM concentration on the yeast topoisomerase II decatenation ability were tested. Kinetoplast DNA (-yTOPOII) was decatenated by yeast topoisomerase II in the absence (+ yTOPOII) or presence of etoposide (lane 3, 150 µM) or selected compounds. DNA was separated in a 1% agarose gel. The data shown are typical of three independent experiments. The gels were cropped to improve the clarity and conciseness of the presentation. Original gels are presented in Figure S4 (SI).

Figure 2

The influence of IKE3, IKE5, IKE7, IKE9, IKE12, IKE13, and IKE14 on the formation of covalent DNA-yeast topoisomerase II complexes. Supercoiled pBR322 plasmid DNA (lane 2, -yTOPOII) was incubated with purified yeast topoisomerase II in the absence (lane 1, + yTOPOII) or in the presence of 200 µM m-AMSA (lane 4) or with 0.25 µM or 10 µM of selected compounds (lanes 5–17). After forming DNA/yeast topoisomerase II complexes, proteinase K was used for digestion. Subsequently, the various topological forms of DNA were separated in a 1.2% agarose gel containing 0.1 µg mL⁻¹ ethidium bromide. The data shown are typical of three independent experiments. Lane 3, linearized pBR322 DNA. SC, supercoiled DNA; R, relaxed DNA; L, linear DNA; N, nicked circular DNA. The gel was cropped to improve the clarity and conciseness of the presentation. Original gels are presented in Supplementary Fig. S4.

Figure 3

DNA Unwinding Assay. Supercoiled (or relaxed) pBR322 plasmid DNA (lane 1,—Wheat germ TOPO I) was incubated in the absence (lane 2, + Wheat germ TOPO I) or presence of analyzed compounds IKE3, IKE5, IKE7, and IKE13 prior to incubation with wheat germ TOPO I. The ability to intercalate into DNA was investigated. m-AMSA was used to show the ability of intercalation, etoposide was used as an example of a non-intercalator. SC, supercoiled DNA; R, relaxed DNA; T, DNA topoisomers. DNA was separated in a 1% agarose gel. The data shown are typical of three independent experiments. The gels were cropped to improve the clarity and conciseness of the presentation. Original gels are presented in Supplementary Fig. S4.

Figure 4

Absorption spectra of IKE1 (a) and IKE8 (b) in the presence of increasing amounts of CT-DNA. Arrows indicate that absorbance changes upon increasing CT-DNA concentrations. Inset: plot of Ao/(A–Ao) = f(1/[DNA]) established as a result of both compounds’ interactions through titration with CT-DNA in Tris–HCl buffer (5 mM/50 mM NaCl; pH 7.43).

Figure 5

(a) Representative histograms and quantification of flow cytometry analysis of fungi labeled with 20 µg mL⁻¹ of propidium iodide to detect membrane permeability in S. cerevisiae ATCC 9763 cells after treatment with IKE7, IKE14, C-1305, and C-1311 at concentration 64 µg mL⁻¹. Error bars represent the mean ± (SD) of the data collected from three independent events. To determine the statistical significance, a one-way analysis of variance (ANOVA) test was conducted. (b) Fluorescence microscopic analysis of uptake and accumulation of IKE7 and C-1305 in S. cerevisiae ATCC 9763 cells. Cells were suspended in phosphate-buffered saline and incubated in the presence of compounds in 100 µM concentration for an appropriate period. Scale bars correspond to 20 µm. (c) Representative histograms and quantification of flow cytometry analysis for the accumulation of IKE7 and C-1305 at concentration 64 µg mL^-1 in S. cerevisiae ATCC 9763 cells. Incubation times of 15 and 60 min were considered. Error bars represent the mean ± (SD) of the data collected from three independent events. To assess statistical significance, a one-way analysis of variance (ANOVA) test was performed.