Antifungal evaluation and mechanistic investigations of membrane active short synthetic peptides-based amphiphiles

Abstract

The quest for new class of peptide-based antibiotics has steered this research towards the design and synthesis of short sequences possessing modified amphiphilic histidine along with hydrophobic tryptophan residues. The new structural class of dipeptides Trp-His(1-Bn)-OMe/NHBn and tripeptides His(1-Bn)-Trp-His(1-Bn)-OMe/NHBn demonstrated promising antifungal and antibacterial activities with membrane lytic action. The illustration of desirable hydrophilic-lipophilic balance appeared in the dipeptide Trp-His[1-(3,5-di-tert-butylbenzyl)]-NHBn (13d) that produced the most promising antifungal activity with IC₅₀ value of 2.10 μg/mL and MIC = 3.81 μg/mL against C. neoformans and antibacterial activity against E. faecalis and S. aureus with identical IC₅₀ value of 4.40 μg/mL and MIC of 8.0 μg/mL. Peptide 13d did not exhibit cytotoxicity and hemolysis at the MIC value and above. This quintessence amphiphilicity was further corroborated by the mechanistic elucidations, which revealed that, peptide act by utilizing charge and hydrophobicity as the primary characteristic tools. Owing to their fundamental affinity, the negatively charged fungal membrane is enacted upon by the positively charged peptide, whereas the intrinsic hydrophobicity of the peptide allowed penetration into the lipophillic core of the fungal cell membrane. Consequently, the integrity of cell membrane is compromised leading to increased fluidity. The membrane eventually disintegrates thereby creating a hollow pore and appearance of a doughnut into the cell when visualized under SEM. The cell death mechanism and damage to the cell wall and intracellular organelles have been elucidated with the help of flow cytometry, TEM and CLSM studies.

Keywords: Antifungal peptides; C. neoformans; Membrane disruption; N-1-Benzyl-l-histidine; Peptide-based antibiotics; Pore formation.