A synthetic cationic thiopyrylium compound exploits preferential phosphatidylserine recognition to exert antifungal activity.

Invasive candidiasis poses a severe global health threat, with limited antifungal options and escalating drug resistance demanding novel therapeutic strategies. Here, we report IMB-G3, a cationic thiopyrylium-styryl compound, as a broad-spectrum antifungal agent that preferentially targets phosphatidylserine. IMB-G3 exhibited potent antifungal activity against multiple fungal pathogens, including fluconazole-resistant clinical isolates, with MIC values ranging from 0.25 to 4 μg/mL, and demonstrated synergistic interactions with amphotericin B and caspofungin. Meanwhile, IMB-G3 effectively promoted wound healing in a murine cutaneous infection model. For the mechanism of action, IMB-G3 accumulates at both the cell membrane and the cytoplasm without directly damaging the membrane, instead reducing membrane fluidity, inducing membrane depolarization, and triggering intracellular accumulation of reactive oxygen species (ROS). Convergent evidence from lipid feeding assays, isothermal titration calorimetry (ITC), molecular dynamics (MD) simulation, untargeted lipidomics, and nuclear magnetic resonance (NMR) spectroscopy indicates that IMB-G3 interacts with several major membrane phospholipids but shows a preference for phosphatidylserine (PS), disrupting PS metabolism, impairing vesicular structure, and triggering lipid homeostasis dysregulation. The cationic thiopyrylium moiety of IMB-G3 engages the anionic phosphate headgroup of PS via electrostatic interaction as determined by 31P NMR. These findings reveal a mechanism of action in which preferential binding to PS dysregulates fungal lipid homeostasis rather than lysing the membrane, and define IMB-G3 as a promising lead compound for a novel class of antifungal therapeutics.