Antifungal Efficacy\nof Ultrashort β‑Peptides\nagainst <i>Candida</i> Species: Mechanistic Understanding\nand Therapeutic Implications

Candidiasis, a condition spurred by the unchecked proliferation\nof <i>Candida</i> species, poses a formidable global health\nthreat, particularly in immuno­compromised individuals. The emergence\nof drug-resistant strains complicates management strategies, necessitating\nnovel therapeutic avenues. Antimicrobial peptides (AMPs) have garnered\nattention for their potent antifungal properties and broad-spectrum\nactivity against <i>Candida</i> species. This study assessed\nthe antifungal effectiveness of ultrashort β-peptides against <i>Candida</i> strains, with a specific focus on peptide P3 (LAU-β3,3-Pip-β2,2-Ac6c-PEA).\nOur findings showed P3’s remarkable fungistatic and fungicidal\nactivities against <i>Candida albicans</i>, exhibiting an\nMIC of 4 μg/mL, comparable to those of standard antifungal drugs.\nThe MIC value remained unchanged in the presence of ADC and BSA, indicating\nthat serum albumin does not diminish the activity of P3. P3 demonstrates\nsynergistic effects when combined with Fluconazole (FLU), Itraconazole\n(ITR), and Nystatin (NYS) to the extent that it becomes effective\nat 0.125, 0.125, and 0.03125 μg/mL, respectively. Concentration\nversus time-kill kinetics showed its time-dependent activity up to\nthe first 12 h against <i>C. albicans</i>, and later concentration\nalso played a role; indeed, at 24 h the whole culture was sterilized\nat 8× MIC. Post-antifungal effect assays confirmed prolonged\nsuppression of pathogen growth after the removal of P3 from the media\nfor significant durations. More importantly, P3 inhibits hyphae formation\nand biofilm development of <i>Candida</i>, outperforming\nFluconazole with respect to these properties. Mechanistic insights\ndisplay P3’s potential to disrupt fungal cell membrane integrity\nand dose-dependent inhibition of ergosterol biosynthesis, essential\nfor fungal cell wall integrity. Using the Bradford assay, it was observed\nthat extracellular protein concentrations increased with higher doses\nof the compound, thereby validating the effect of P3 on membrane integrity.\nA comparative gene analysis using RT-PCR showed that P3 downregulates\nERG3, ERG11, and HWP1, which are crucial for the survival and pathogenicity\nof <i>C. albicans</i>. The impact of P3 on ERG11 and ERG3\nis more effective than that of Fluconazole. Molecular docking studies\nrevealed strong binding of P3 to various isoforms of lanosterol 14-α-demethylase,\na key enzyme in ergosterol synthesis. Furthermore, molecular dynamic\nsimulations validated the stability of the most promising docking\ncomplex. Overall, our findings underscore P3’s potential as\na leading candidate for the development of innovative antifungal therapies,\nwarranting further investigation and optimization.