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

Abstract

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. Leveraging this knowledge, we screened a small-molecule compound library containing glycolysis inhibitors for their ability to block fungal morphogenesis and identified multiple inhibitors of glycolysis that robustly block hyphal differentiation without compromising 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 multiple filamentation-inducing conditions. Comparative RNA-Seq analysis revealed that pyruvate kinase inhibition using PKM2-IN-6 resulted in the repression of multiple genes associated with fungal morphogenesis, biofilm formation, and virulence. These transcriptional changes were accompanied by a significant reduction in biofilm formation and increased sensitivity to conventional antifungal drugs, including amphotericin B and fluconazole, in the presence of PKM2-IN-6. Furthermore, administration of PKM2-IN-6 significantly improved the survival of mice in a systemic model of murine candidiasis underscoring the in vivo efficacy of this compound. Molecular docking analysis showed stable binding of both compounds to the ATP-binding pocket of pyruvate kinase, suggesting ATP-competitive inhibition as the mode of action. Collectively, our study has identified novel inhibitors of fungal morphogenesis in C. albicans that target pyruvate kinase activity to increase antifungal susceptibility and attenuate the virulence of C. albicans. ImportanceCandida albicans is a major fungal pathogen that causes fatal infections, particularly in immunocompromised individuals. The increasing prevalence of antifungal resistance and the limited availability of effective antifungal therapies underscore the need for new treatment strategies. In this study, we identify pyruvate kinase, a key glycolytic enzyme, as an important regulator of fungal morphogenesis and virulence. Pharmacological inhibition of pyruvate kinase blocked hyphal differentiation without affecting fungal growth, impaired biofilm formation, and enhanced the activity of clinically relevant antifungal drugs. Transcriptomic analysis further revealed broad repression of genes involved in filamentation, biofilm formation, cell adhesion, antifungal resistance, and virulence, providing a molecular basis for these phenotypes. Importantly, pyruvate kinase inhibition also improved host survival in a murine model of systemic candidiasis. Together, our findings identify pyruvate kinase as a promising therapeutic target and demonstrate the potential of targeting fungal metabolism to effectively combat C. albicans infections.