Improved Broad Spectrum Antifungal Drug Synergies with Cryptomycin, a Cdc50-Inspired Antifungal Peptide

Fungal infections in humans are difficult to treat, with very limited drug options. Due to a confluence of factors, there is an urgent need for innovation in the antifungal drug space, particularly to combat increasing antifungal drug resistance. Our previous studies showed that Cdc50, a subunit of fungal lipid translocase (flippase), is essential for Cryptococcus neoformans virulence and required for antifungal drug resistance, suggesting that fungal lipid flippase could be a novel drug target. Here, we characterized an antifungal peptide, Cryptomycinamide (KKOO-NH₂), derived from a 9-amino acid segment of the C. neoformans Cdc50 protein. A fungal killing assay indicated that KKOO-NH₂ is fungicidal against C. neoformans. The peptide has antifungal activity against multiple major fungal pathogens with a minimum inhibitory concentration (MIC) of 8 μg/mL against C. neoformans and Candida glabrata, 16 μg/mL against Candida albicans and C. auris, and 32 μg/mL against Aspergillus fumigatus. The peptide has low cytotoxicity against host cells based on our hemolysis assays and vesicle leakage assays. Strikingly, the peptide exhibits strong drug synergy with multiple antifungal drugs, including amphotericin B, itraconazole, and caspofungin, depending on the specific species on which the combinations were assayed. The fluorescently labeled peptide was detected to localize to the plasma membrane, likely inhibiting key interactions of Cdc50 with membrane proteins such as P4 ATPases. Cryptococcus cells exposed to sub-MIC of peptide showed increased reactive oxygen species production and intracellular calcium levels, indicating a peptide-induced stress response. Decreased intracellular proliferation within macrophages was observed after 30 min of peptide exposure and 24 h coincubation with macrophages, providing a potential translational mechanism to explore further in vivo. In aggregate, the synergistic activity of our KKOO-NH₂ peptide may offer a potential novel candidate for combination therapy with existing antifungal drugs.