Molecular Determinants on the Selectivity and Mechanism of Action for the cAMP Ctx(Ile21)-Ha towards Candida spp.

Antimicrobial resistance represents a significant threat to global health, necessitating the urgent exploration of alternative therapeutic strategies. Antimicrobial peptides have emerged as promising candidates due to their broad-spectrum activity against various pathogens. We investigated how chirality, conformational flexibility, hydrophobicity, and cationicity govern the activity and membrane interactions of the amphibian cationic antimicrobial peptide Ctx(Ile²¹)-Ha and five rationally designed analogues. Antifungal activity was quantified against Candida albicans, C. tropicalis, and C. krusei, and mammalian-cell compatibility was assessed in HEK293 cells, HaCaT keratinocytes, and dermal fibroblasts. Early cellular effects in C. tropicalis were examined using Sytox Green (cell permeabilization), H2DCFDA (endogenous reactive oxygen species), FITC-VAD-FMK (metacaspase activity), and propidium iodide (cell necrosis). Within 10 min, the lead analogue permeabilized ~79% of C. tropicalis cells and induced ROS in ~44%, with little evidence of metacaspase-like activation. Peptide folding, Trp local environment, and bilayer disruption were evaluated in fungal-like (PC/PE/PS, with or without ergosterol) and mammalian-like (PC/SM/cholesterol) large unilamellar vesicles. The parent peptide displayed low-micromolar activity (MICs of 3.12, 3.12, and 6.25 µM against C. albicans, C. tropicalis, and C. krusei, respectively) but substantial cytotoxicity. Its MFCs were equal to or one dilution above the MICs, linking growth inhibition to rapid loss of colony-forming ability. The all-D enantiomer retained antifungal activity without improving compatibility, whereas lysine-rich analogues increased potency at the cost of cytotoxicity. Introducing A13P reduced toxicity but weakened activity. Combining this conformational flexibility with balanced cationicity in Ctx(Ile²¹)-Ha_D4K-A13P restored activity against C. albicans and C. tropicalis (MICs of 3.12 µM) while yielding CC₅₀ values >50, >100, and >100 µM in HEK293, HaCaT, and fibroblasts, respectively. Fungal-like bilayers promoted stronger peptide structuring and a more hydrophobic Trp environment than the mammalian model, yet maximal alpha-helicity did not predict antifungal potency. The lead analogue retained marked leakage in fungal-like vesicles but caused substantially less disruption of mammalian-like vesicles. These findings support surface-associated bilayer destabilization rather than stable transmembrane pore formation and identify Ctx(Ile²¹)-Ha_D4K-A13P as a selective lead against C. albicans and C. tropicalis.

This work was supported by FAPERJ (grants 210.778/2021, 211.816/2021, and 204.310/2025), CNPq (grant 406990/2025-5), and CAPES (grants 88887.978846/2024-00 and 88881.125626/2025-01).