The cytotoxicity of gomesin peptides is mediated by the glycosphingolipid pathway and lipid-cholesterol interactions.

Gomesins (AgGom and HiGom) are therapeutically promising spider-derived peptides that target a specific phospholipid composition (3PC:1PS:1Chol) to disrupt melanoma cell membranes and induce cytotoxicity. Their antiproliferative properties are interrelated to lipid metabolism, particularly glycosphingolipid biosynthesis. We used lipidomics, CRISPR/Cas9 knockout screening, molecular and biophysical experiments, followed by xenograft melanoma animal studies to demonstrate that gomesins target the glycosphingolipid pathway via inhibition of the ST3GAL5 gene. Notably, the addition of cholesterol reduced the cytotoxicity of gomesins, which may explain why melanoma cells with lower cholesterol levels than neonatal foreskin fibroblasts are more sensitive to gomesins. We propose that gomesins bind to melanoma CRAC domains, restricting intracellular cholesterol and thereby enhancing their cytotoxicity. In line with this hypothesis, cholesterol sequestration and the disruption of lipid raft microdomains enhanced the cytotoxic effects of gomesins in melanoma cells, whereas adding cholesterol in membrane permeability and proliferation assays reduced the effects of gomesin treatment. Taken together, this study highlights the specific role of cholesterol and the glycosphingolipid pathway in melanoma cells, paving the way for new strategies in targeted melanoma therapies.