Ergosterol-Dependent Membrane Stress and Calcineurin-HSP90 Signaling Govern Fluorinated Sulfone-Induced Cell Death in Candida albicans

The antifungal activity of halogenated methyl sulfones depends on both the type and number of halogen atoms in the halogenomethylsulfonyl moiety. In this study, 28 halogenomethylphenyl sulfones were evaluated for minimal inhibitory (MIC) and fungicidal concentrations (MFC) against Candida albicans. Active compounds were further tested for cytotoxicity in vitro using the Vero E6 cell line. Notably, 4-(4-chloro-3,4-dichlorophenoxy)-2-nitrophenyl difluoromethyl sulfone (compound 15) showed fungicidal activity while remaining non-toxic at effective doses. Additionally, no adverse effects were observed in Galleria mellonella larvae treated with 15 in vivo. Mechanistic studies with 15 at a sub-inhibitory concentration (2 μg/mL) showed predominant induction of necrosis in C. albicans cells (88.43%) and protoplasts (96.25%), along with increased reactive oxygen species (ROS) production (31.17%). Cell cycle analysis revealed arrest in the G0 phase (99%), consistent with a pro-necrotic mechanism. RT-qPCR indicated that HSP90 gene expression contributes to C. albicans resistance to 15. Confocal microscopy confirmed the compound's fungicidal activity against planktonic and sessile C. albicans cells in vitro. Modulation experiments with exogenous ergosterol showed that reducing sterol concentrations decreased MIC values, suggesting that the fungal cell membrane is the primary target of 15 and that its interaction with ergosterol is critical for activity. Overall, 15 demonstrated antifungal activity comparable to that of amphotericin B and is a promising lead candidate for the development of new antifungal agents for local and systemic candidiasis.