Exploiting Neosartorya ( Aspergillus ) fischeri antifungal protein 2-defined fungal targets for drug repurposing against Candida albicans

ABSTRACT Invasive fungal infections caused by Candida albicans remain major clinical challenges because of the limited therapeutic options, toxicity, and emerging resistance. Drug repurposing offers a promising strategy to accelerate antifungal discovery by leveraging existing compounds with known safety profiles. In this study, we applied a structure-based drug repurposing approach guided by the antifungal protein Neosartorya (currently Aspergillus ) fischeri antifungal protein 2 (NFAP2), which targets fungus-specific intracellular proteins, including glutamate decarboxylase, mitochondrial ATP synthase subunit alpha, and enolase. NFAP2-associated binding pockets were identified and used to virtually screen approved clinically investigated compounds. Selected candidates were further evaluated by molecular docking, followed by in vitro antifungal susceptibility testing and interaction analysis with conventional antifungal agents. In vivo toxicity and therapeutic efficacy were assessed using a Galleria mellonella infection model. Despite predicted binding affinities, most repurposed compounds exhibited limited antifungal activity in vitro , highlighting a disconnect between in silico predictions and biological efficacy. Among the tested compounds, dihydroergocristine mesylate (DHEC) and meclizine dihydrochloride exhibited measurable antifungal effects. Notably, DHEC demonstrated pronounced synergistic interactions with amphotericin B, resulting in near-complete growth inhibition in vitro. In vivo , DHEC + amphotericin B induced no detectable toxicity and significantly improved host survival compared with either drug alone. These findings indicate that NFAP2-guided structure-based drug repurposing can identify compounds targeting fungus-specific vulnerabilities. However, antifungal activity depends on factors beyond binding affinity. Our results highlight integrating computational screening with experimental validation and support repurposed compounds as adjuvants in combination therapies to enhance antifungal efficacy and translational potential. Invasive fungal infections caused by Candida albicans remain a major clinical challenge due to limited therapeutic options, toxicity, and emerging resistance. Drug repurposing offers a promising strategy to accelerate antifungal discovery by leveraging compounds with known safety profiles. A structure-based repurposing framework guided by the antifungal protein NFAP2, which targets fungus-specific intracellular proteins, is presented. Using these biologically informed targets, clinically relevant compounds with antifungal and adjuvant potential were identified. Predicted binding affinity alone did not reliably translate into antifungal activity, highlighting the need for integrated computational and experimental approaches. Notably, dihydroergocristine mesylate (DHEC) showed strong synergistic effects with amphotericin B, resulting in near-complete growth inhibition and improved host survival in vivo . These findings highlight the potential of repurposed compounds as adjuvants and support combination-based strategies targeting fungal-specific vulnerabilities. Furthermore, this work provides a framework for the future development of DHEC-inspired antifungal adjuvant strategies.