Chemical tools to probe antifungal targets

Abstract Chemical probes are selective, reversible small molecules that enable temporal control of protein function in cells and are central to target identification, pathway interrogation, and chemical validation in drug discovery. Compared with genetic approaches, they offer tunable target engagement, rapid perturbation, and direct assessment of druggability, thereby bridging basic biology and therapeutic development. Despite their widespread use in human biology, chemical probes that interrogate molecular machineries in pathogenic fungi remain scarce. This limitation is particularly significant given the increasing incidence of life-threatening fungal infections in immunocompromised patients and the urgent need for new antifungal targets and therapeutic strategies. In this review, we summarize 17 small-molecule chemical tools that probe 13 antifungal targets across Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, and the model yeast Saccharomyces cerevisiae. These compounds represent a foundational chemical toolkit for fungal research and provide a framework for developing next-generation chemical probes or antifungal agents in future studies.