Abstract

Invasive fungal infections (IFIs), predominantly caused by Candida albicans, are a significant threat to immunocompromised individuals. The emergence of drug-resistant strains has intensified the need for novel antifungal agents. Natural naphthoquinones, including 5,8-dihydroxy-1,4-naphthoquinone (DHNQ, also as PNP-02), have broad-spectrum antimicrobial properties, but their antifungal potential against C. albicans remains underexplored. This study evaluates the antifungal activity of DHNQ derivatives and elucidates their mechanisms of action. The antifungal properties of these compounds were evaluated using the Kirby-Bauer disk diffusion method, broth microdilution assays, and phenotypic screening. DHNQ was identified as the most effective compound, and further investigation focused on its effects on C. albicans growth, biofilm formation, hyphal development, and underlying mechanisms, including oxidative stress induction and mitochondrial dysfunction. In a murine candidiasis model, DHNQ significantly reduced the fungal burden in both the kidneys and the skin, with a minimum inhibitory concentration (MIC) ranging from 2 to 8 μg/mL, exceeding the activity of fluconazole against clinical isolates of fluconazole-resistant C. albicans strains by over 32 times. Mechanistic investigations revealed that DHNQ exerts its antifungal effects through a multi-pronged approach: inhibiting glycolysis, disrupting biofilm and hyphal formation, and inducing oxidative stress-mediated mitochondrial dysfunction. Notably, DHNQ exhibited low cytotoxicity in vitro and no observable toxicity in vivo. These properties make it a promising lead molecule for future optimization and development of treatments for C. albicans infections, pending crucial evaluations of its selectivity and safety in host environments.