N-terminal lipidation enhances membrane interaction and antifungal activity of a Trematocine-derived decapeptide

Invasive fungal infections are a growing global health threat, driven by the limited availability of antifungal therapies and the rapid spread of multidrug-resistant yeasts. This study explored the effects of N-terminal lipidation on Trem-4, a decapeptide derived from the antimicrobial peptide Trematocine-HSK, to enhance its antifungal potential. Trem-4 was modified with caprylic (C8) and myristic (C14) fatty acid chains, generating two lipopeptides: Cap-T4 and Myr-T4. Biophysical analyses, including fluorescence-based assays and circular dichroism, showed that lipidation significantly improved peptide-membrane interactions compared to the non-lipidated form. The two derivatives displayed distinct behaviors, with differences in aggregation and membrane selectivity depending on acyl chain length. Antifungal activity was assessed against 60 clinically characterized yeast isolates, including Candida spp., Candidozyma haemuli complex, and Cryptococcus neoformans, under both planktonic and biofilm conditions. Both lipopeptides exhibited broad-spectrum activity, against clinically relevant species, with conserved activity against antifungal-resistant strains. Importantly, both compounds remained effective against mature biofilms, causing metabolic disruption and structural damage. Cap-T4 demonstrated consistent activity across species, while Myr-T4 showed enhanced potency against selected isolates, particularly C. auris. Biocompatibility evaluation revealed a concentration-dependent cytotoxicity and hemolysis, more pronounced for Myr-T4. However, both compounds were well tolerated in vivo in the Galleria mellonella model. Overall, Cap-T4 emerged as the most promising candidate for further preclinical development. These findings support N-terminal lipidation as an effective strategy to improve the antifungal efficacy of short antimicrobial peptides and highlight its potential for developing new treatments against drug-resistant fungal pathogens. KEY POINTS: N-terminal lipidation enhances Trem-4 membrane interaction and activity. Myr-T4 and Cap-T4 show broad activity against susceptible and resistant yeasts. Cap-T4 retains activity against yeasts and mature biofilms.