Pisum sativum defensin 2 (Psd2) is a small (4.7 kDa) antifungal peptide whose structure is held together by four conserved disulfide bridges. Psd2 shares the cysteine‐stabilized alpha‐beta (CSαβ) fold, which lacks a regular hydrophobic core. All hydrophobic residues are exposed to the surface, except for leucine 6. They are clustered in the surface formed by two loops, between β1 and α‐helix and β2 and β3 sheets. The observation of surface hydrophobic clusters reveals a remarkable evolution of the CSαβ fold to expose and reorganize hydrophobic residues, which facilitates creating versatile binding sites.

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Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.

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