Antifungal activity of a maleimide derivative: disruption of cell membranes and interference with iron ion homoeostasis

Abstract

Fungal infections caused by Candida albicans have posed a persistent threat to human health. Existing clinical antifungal drugs are constrained by issues such as drug resistance and side effects. Compounds containing maleimide rings have been verified to possess antifungal properties, although the specific molecular mechanisms by which they exert this activity have yet to be fully understood. A total of 40 compounds containing maleimide rings were synthesised in the present study, and 12 derivatives that possessed antifungal properties were subsequently identified. The maleimide compound 5 (MPD) with the most potent activity demonstrated fungicidal action at a concentration that was twice as potent as the minimal inhibitory concentration and effectively prevented the formation of biofilms. Furthermore, the mechanistic studies revealed that MPD interfered with iron ion homoeostasis by reducing intracellular iron concentration inside cells, which led to the inhibition of ergosterol biosynthesis and increased cell membrane permeability, resulting in the leakage of intracellular trehalose. In addition, MPD was observed to perturb cell wall biosynthesis by reducing the activity of chitin synthase. Moreover, MPD was found to demonstrate therapeutical efficacy in vivo when assessed using a Caenorhabditis elegans-C. albicans infection model.

Keywords: Candida albicans; Maleimide derivatives; antifungal activity; cell membrane; iron ion homoeostasis.

Figure 1.

The structure of synthesised compounds with maleimide ring.

Figure 2.

The time-killing analysis of the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] against Candida albicans. SC5314 cells were incubated with different doses of MPD at 30 °C for 24 h. The MPD-free group served as the negative control. FLC (2 μg/mL) and AMB (2 μg/mL) served as positive controls. The data is represented as mean ± SD.

Figure 3.

Effects of the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] on biofilm formation in mixed cultures containing Candida albicans and non-albican fungal strains. SC5314 cells and other non-albicans cells were diluted in the RPMI1640 medium and incubated with different doses of MPD at 37 °C without shaking for 24 h. (a) The formation of biofilm was photographed by microscope. (b) The number of viable cells detected by the XTT Cell Proliferation Kit. The scale bar indicates 50 μm. The data is represented as mean ± SD. Asterisks represent statistically significant differences. * means P < 0.05; ** means P < 0.01.

Figure 4.

Transcriptional profiling of Candida albicans SC5314 in response to the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] treatment. (a) Volcano map of differentially expressed genes (DEGs). In the figure, the X-coordinate is the log2 fold change value, and the Y-coordinate is -log10 (padj). (b) Summary of gene enrichment analyses and the number of genes affected by C. albicans exposure to MPD. The X-coordinate denotes gene ontology (GO) representing biological processes, cellular components, and molecular functions, whereas the Y-coordinate represents the number of genes. (c) Heatmap of MPD on the expression of selected genes of C. albicans.

Figure 5.

Effects of the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] on iron homoeostasis. (a) Growth curves of Saccharomyces cerevisiae (BY4743) and heterozygous diploid deletion (aft2Δ/Δ). (b) Candida albicans SC5314 cells were cultured in RPMI 1640 medium with MPD for 10 h. The cells were stained with FeRhonox-1 and observed with a fluorescence microscope. The scale bar indicates 50 μm. (c) The OD₆₀₀ of SC5314 cells was detected after growing at 30 °C for 24 h under the action of different doses of FeSO₄ and MPD. (d) Effect of MPD on the expression of iron homeostasis-related genes. The data is represented as mean ± SD. Asterisks represent statistically significant differences. * means P < 0.05; ** means P < 0.01.

Figure 6.

Effect of the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] on the cell membrane. (a) Ultraviolet scanning spectrum of total sterol extracted after MPD treatment (200–320 nm). (b) The reductions of ergosterol levels induced by various doses of MPD. (c) The relative expression of ergosterol biosynthesis-related genes was determined by RT-qPCR. (d) The propidium iodide (PI) staining results of Candida albicans SC5314 after different treatments were observed by fluorescence microscope. The scale bar indicates 50 μm. (e) Treated cells were stained with PI and their staining rate was calculated. (f) C. albicans SC5314 cells were treated with various doses of MPD for 12 h, followed by DPH staining for detection using a fluorescence spectrophotometer. (g) Effect of MPD on the intracellular trehalose content. (h) Effect of MPD on the extracellular trehalose content. The data is represented as mean ± SD. Asterisks represent statistically significant differences. *means P < 0.05.

Figure 7.

Effects of the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] on the cell wall. (a) Effect of MPD on chitin synthase. (b) Showed the effect of MPD on the expression of chitin-related genes. (c) Treated cells were stained with calcofluor white (CFW) and spectrofluorophotometer detection. (d) The CFW staining results of Candida albicans SC5314 after different treatments were observed by fluorescence microscope. The scale bar indicates 50 μm. (e) The OD₆₀₀ of SC5314 cells was detected after growing at 30 °C for 24 h under the action of different doses of CFW and MPD. (f) C. albicans SC5314 cells were adjusted to 1 × 10⁵ cells/mL in SD medium and treated with 1 μg/mL MPD. After adding 0.8 mol/L sorbitol, it was incubated at 30 °C for 25 h. The growth curve was drawn by detecting OD₆₀₀. The data is represented as mean ± SD. Asterisks represent statistically significant differences.  *means P < 0.05; **means P < 0.01; ***means P < 0.005.

Figure 8.

The Caenorhabditis elegans-Candida albicans infection model. (a) Healthy nematodes were cultured with different concentrations of the maleimide compound 5 [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] or without it for 2 days. (b) Nematodes were infected with C. albicans SC5314 for 2 h and then transferred to a liquid medium with different concentrations of MPD; the group without MPD served as a negative control.