Antifungal potential of naphthoquinone derivatives: screening of shikonin-based compounds and mechanistic insights into 5,8-dihydroxy-1,4-naphthoquinone against <i>Candida albicans in vitro</i> and <i>in vivo</i>

Invasive fungal infections (IFIs), predominantly caused by <i>Candida albicans</i>, 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 <i>C. albicans</i> 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 <i>C. albicans</i> 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 <i>C. albicans</i> 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 <i>in vitro</i> and no observable toxicity <i>in vivo</i>. These properties make it a promising lead molecule for future optimization and development of treatments for <i>C. albicans</i> infections, pending crucial evaluations of its selectivity and safety in host environments.IMPORTANCEIn summary, this study demonstrated that DHNQ exhibits potent and broad-spectrum antifungal activity, showing significant efficacy against <i>C. albicans</i> both <i>in vitro</i> and <i>in vivo</i>. Unlike conventional antifungals, DHNQ disrupted the virulence of <i>C. albicans</i> by inhibiting glycolysis, suppressing biofilm formation, and inducing oxidative stress-mediated mitochondrial dysfunction. These findings not only highlight the promising potential of DHNQ as a treatment for <i>C. albicans</i> infections but also provide critical insights that may facilitate the development of new antifungal agents.