Mechanistic Insights into the Antifungal Activity of the Synthetic Peptide FK18 Against Candida tropicalis

The increasing incidence of invasive fungal infections caused by opportunistic yeasts, particularly Candida tropicalis , poses a major clinical challenge due to rising antifungal resistance and the limited availability of effective therapies. Antimicrobial peptides (AMPs) have emerged as promising alternatives owing to their rapid mechanisms of action and low propensity for resistance development. In this study, we investigated the antifungal activity, mechanism of action, and structural properties of FK18, a rationally designed synthetic cationic peptide. FK18 exhibited broad-spectrum antifungal activity against Candida and Cryptococcus species, with minimum inhibitory concentration (MIC) values ranging from 5 to > 40 µM. Notably, C. tropicalis showed high susceptibility, with inhibition observed at 10 µM. Time-kill assays demonstrated rapid fungicidal activity, with FK18 at 10 µM (MIC) achieving complete elimination of C. tropicalis cells within four hours. Checkerboard assays revealed strong synergistic interactions between FK18 and fluconazole, significantly reducing the concentrations required for growth inhibition. Mechanistic studies using the fluorescent probe SYTOX Green demonstrated rapid membrane permeabilization. Additionally, supplementation with exogenous ergosterol resulted in a four-fold increase in MIC, suggesting that FK18 activity is influenced by sterol-containing membranes. Nuclear magnetic resonance analyses showed that FK18 adopts a stable amphipathic α-helical structure in membrane-mimetic environments, consistent with membrane-active AMPs. Molecular dynamics simulations further supported these findings, revealing stable interactions with POPC/ergosterol lipid bilayers. Furthermore, FK18 exhibited antioxidant activity, showing significant DPPH radical scavenging activity, whereas no significant ABTS radical scavenging activity was detected up to 128 µM. Collectively, these results highlight FK18 as a multifunctional peptide with rapid fungicidal activity, membrane-targeting properties, and synergistic potential, supporting its development as a promising antifungal candidate.