Systematic Screening of a Glycolysis-targeting Small-molecule Library Identifies Novel Inhibitors of Fungal Morphogenesis in Candida albicans

Fungal infections are an increasing global health concern, with Candida albicans emerging as a leading cause of mucosal and life-threatening systemic infections. C. albicans relies on yeast-to-hyphae transition, to establish systemic infections in the host. Our recent work demonstrated that glycolysis is a key regulator of hyphal differentiation in C. albicans (Shah et al., 2025). Leveraging this knowledge, we screened a small-molecule compound library containing glycolysis inhibitors for their ability to block fungal morphogenesis. Our systematic screen of glycolysis-targeting compounds acting on distinct enzymatic steps of glycolysis identified multiple inhibitors of glycolysis that robustly block hyphal differentiation in C. albicans without compromising its overall growth. While early glycolysis pathway inhibitors showed moderate effects, two compounds (NPD10084 and PKM2-IN-6) targeting pyruvate kinase activity emerged as the most potent inhibitors of fungal morphogenesis, completely blocking hyphal differentiation in C. albicans, in multiple filamentation-inducing conditions. Notably, these inhibitors effectively impaired biofilm formation and exhibited synergistic activity with conventional antifungal drugs, including amphotericin B and fluconazole. Furthermore, administration of NPD10084 and PKM2-IN-6 significantly improved the survival of mice in a systemic model of murine candidiasis underscoring the in vivo efficacy of these compounds. Molecular docking analysis showed stable binding of both compounds to the ATP-binding pocket of pyruvate kinase, suggesting ATP-competitive inhibition. Collectively, our study has identified novel inhibitors of fungal morphogenesis in C. albicans that target pyruvate kinase activity to block a key virulence strategy of this human fungal pathogen.