Evaluation of Antifungal Selective Toxicity Using Candida glabrata ERG25 and Human SC4MOL Knock-In Strains

Abstract

With only four classes of antifungal drugs available for the treatment of invasive systemic fungal infections, the number of resistant fungi is increasing, highlighting the urgent need for novel antifungal drugs. Ergosterol, an essential component of cell membranes, and its synthetic pathway have been targeted for antifungal drug development. Sterol-C4-methyl monooxygenase (Erg25p), which is a greater essential target than that of existing drugs, represents a promising drug target. However, the development of antifungal drugs must consider potential side effects, emphasizing the importance of evaluating their selective toxicity against fungi. In this study, we knocked in ERG25 of Candida glabrata and its human ortholog, SC4MOL, in ERG25-deleted Saccharomyces cerevisiae. Utilizing these strains, we evaluated 1181-0519, an Erg25p inhibitor, that exhibited selective toxicity against the C. glabrata ERG25 knock-in strain. Furthermore, 1181-0519 demonstrated broad-spectrum antifungal activity against pathogenic Candida species, including Candida auris. The approach of utilizing a gene that is functionally conserved between yeast and humans and subsequently screening for molecular target drugs enables the identification of selective inhibitors for both species.

Keywords: C. albicans; C. parapsilosis; C4-methyl sterol oxidase; antimicrobial resistance (AMR); drug resistant; methylsterol monooxygenase 1 (MSMO1); molecular target drug; non-albicans Candida (NAC); specific inhibitor; stealth infection.

Figure 1

Complementation with human SC4MOL and C. glabrata ERG25 in S. cerevisiae. (A) The growth curve in liquid culture of S. cerevisiae knock-in strains. Cells (5 × 10⁴/well) of three strains were grown in SD medium containing uracil and leucine at 28 °C, with turbidity measurements taken at OD₆₀₀ every 10 min. The data represent the average of three replicates, and the vertical axis is presented in log₁₀ notation. Statistical analyses, including t-tests and 1-way ANOVA (both two-tailed and unpaired), were performed. (B) Complementation assay by spotting. Starting with approximately 10⁴ cells, cells were diluted to 1/10 and spotted onto SD agar medium. The plates were subsequently placed in an incubator at 28 °C for two days.

Figure 2

Evaluation of growth inhibition by compounds targeting Erg25p. Relative growth inhibition against knock-in strains (A) with PF1163B and (B) with 1181-0519. Sc(hERG25) and Sc(CgERG25) strains were grown in the two drugs. In both plots, the x-axis denotes the concentration of the respective drugs, while the y-axis represents the “Relative growth”, calculated as the area under the curve (AUC) relative to the absence of the drug. The data are based on the average of three replicates, with error bars indicating the standard deviation.

Figure 3

WST-1 assay for evaluation of cytotoxicity of 1181-0519. The A431 (human cell line derived from epidermoid carcinoma) and HepG2 (human hepatoma) cell lines were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 0.1% (v/v) penicillin–streptomycin, and 0.1% (v/v) DMSO at 37 °C for 24 h in a 5% CO₂ incubator. Absorbance (A₄₅₀ − A₆₀₀) value represents the difference between the absorbance at 450 nm minus the absorbance at 600 nm. Cells treated with 100 μM of 1181-0519 are indicated by “−” or “+”, respectively. The bars in the graph represent the average and standard deviation.