Rational Engineering of an Apis mellifera Royal Jelly Peptide Enhances Antifungal Activity against Candida spp.

Antimicrobial peptides (AMPs) represent a diverse and promising source of molecules for the development of new therapeutic strategies against antimicrobial-resistant microorganisms, particularly bacteria and fungi. These molecules exhibit diverse structural and physicochemical properties and can be isolated from natural sources or synthetically produced and modified to enhance their biological activity. Among arthropod-derived AMPs, Jelleine-I (Geleína-I), originally identified in the royal jelly of Apis mellifera, has demonstrated antifungal activity against several fungal species, including Candida spp., major etiological agents of vulvovaginal candidiasis (VVC). However, its therapeutic potential is limited by relatively low stability and weak fungicidal activity against certain yeast species. In this study, we proposed a rational structural modification of Geleína-I, replacing its phenylalanine residue at position 2 with a brominated tryptophan residue, generating the analog Jelleine-Wᴮʳ. Both peptides were evaluated against different Candida species, and the most promising analog was further characterized regarding its mechanism of action, activity against mature Candida albicans biofilms, stability in murine vaginal lavage fluid, and therapeutic potential in a murine model of VVC. Jelleine-Wᴮʳ exhibited substantially enhanced antifungal activity compared with its native counterpart, Jelleine-I, displaying Minimum Inhibitory Concentration values of 8 µg/mL against C. albicans and C. tropicalis, and 4 µg/mL against N. glabratus and P. kudriavzevii. Mechanistic assays indicated fungal membrane disruption associated with ergosterol interaction. Additionally, Jelleine-Wᴮʳ reduced mature C. albicans biofilm biomass at doses starting at 20 µg and remained stable for up to 12 hours in murine vaginal lavage fluid. In vivo, a single intravaginal administration of 80 µg reduced the fungal burden to a median of 1.60 log₁₀ CFU/mL after 24 hours, compared with 3.57 log₁₀ CFU/mL in the miconazole-treated group, achieving mycological cure rates of up to 50%. Overall, Jelleine-Wᴮʳ showed greater antifungal activity and stability than Jelleine-I, including against azole-resistant C. albicans. These findings contributed to the filing of a patent application and highlight the potential of rational peptide design as a strategy for developing novel antimicrobial agents.