Coumarin derivatives targeting ergosterol and sphingolipid pathways to inhibit Candida albicans: Molecular, metabolomic, and Drosophila toxicity insights

초록

Background: The increasing resistance of Candida albicans to conventional antifungal agents and the protective nature of biofilms necessitate the development of alternative therapeutic strategies that target fungal virulence mechanisms rather than relying solely on direct fungicidal activity. Objectives: This study investigated the antivirulence activity and safety profile of two bis-coumarin derivatives, 3,3 '-((3-bromophenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (Compound 1) and 3,3 '-(thiophen-2-ylmethylene)bis(4-hydroxy-2H-chromen-2-one) (Compound 2), against the reference strain Candida albicans ATCC 90028. Methods: Antifungal antivirulence activity was assessed through adhesion, biofilm inhibition, morphogenesis, gene expression, reactive oxygen species (ROS), and mitochondrial membrane potential assays. Molecular docking and molecular dynamics simulations predicted favourable binding of both compounds to sterol 14-alpha-demethylase (CYP51) and demonstrated stable ligand-protein interactions, and untargeted metabolomics was employed to examine treatment-induced metabolic alterations. Safety was evaluated using a Drosophila melanogaster model. Results: Both compounds significantly inhibited adhesion, biofilm metabolic activity, and yeast-to-hypha transition in a concentration-dependent manner, with Compound 1 demonstrating greater potency. At 250 mu g/mL, biofilm metabolic activity was reduced by 64% and 68% following treatment with Compounds 1 and 2, respectively. In addition, both compounds significantly inhibited adhesion by 67% and 73%, respectively, compared with untreated controls.Furthermore, serum-induced hyphal formation decreased significantly compared with untreated controls. FESEM analysis revealed disrupted biofilm architecture, damaged cell surfaces, and loss of cellular integrity. Expression of virulence-associated genes, including ALS3, HWP1, and EFG1, was significantly downregulated 0.062-fold by compound 1 and 0.07-fold by compound 2. Treatment also altered intracellular ROS levels: droping to 1.812% by compound 1 and rising to 10.448% by compound 2, and induced mitochondrial membrane depolarization. Molecular docking and molecular dynamics simulations suggested favourable and stable interactions with CYP51, suggesting as a potential molecular target which is further supported by metabolomic perturbations in ergosterol biosynthesis, sphingolipid metabolism, and glyoxylate cycle intermediates. No major developmental, behavioural, or biochemical toxicity was observed in Drosophila melanogaster following continuous dietary exposure to the compounds at a concentration of 250 mu g/mL. Conclusions: The two bis-coumarin derivatives exert pronounced antivirulence effects against the reference strain of C. albicans by simultaneously disrupting adhesion, biofilm development, morphogenesis, oxidative homeostasis, mitochondrial function, and membrane-associated metabolic pathways. These findings support the further investigation of this bis-coumarin series as promising multi-target antifungal antivirulence agents.

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