Brilacidin's Antifungal Mechanism: Insights from Lipid Membrane Models

Background/Objectives: BRI is a synthetic arylamide polymer designed to mimic the electrostatic and amphiphilic features of defensin-type antimicrobial peptides (AMPs), although its molecular organization and activity have not been experimentally validated. This study presents the first integrated computational and experimental characterization of BRI to define the physicochemical basis of its AMP-like behavior and membrane interactions. Methods: Molecular modelling was used to evaluate the structural and electrostatic properties of BRI. Model lipid membranes were used to study membrane interactions. Fluorescence spectroscopy, electrochemical measurements, and ζ-potential analyses were performed to characterize membrane insertion, aggregation, ionic conductance, and membrane resistance. Microbiology assays evaluating synergy with azole were also assessed. Results: Molecular modelling showed that BRI is a flexible molecule with cationic and hydrophobic surfaces, a strong amphiphilic dipole, and a dominant +4 charge state, explaining its sensitivity to ionic strength and membrane interactions. BRI displayed two membrane-dependent mechanisms of action. In zwitterionic phospholipid membranes, BRI resembled canonical AMPs, showing membrane insertion, pore formation, and increased ionic conductance. In anionic ergosterol-containing membranes mimicking fungal cells, BRI exhibited sterol-dependent insertion, in-plane aggregation, and modulation of membrane resistance without pore formation. Fluorescence, electrochemical, and ζ-potential measurements supported BRI-BRI interactions at the membrane interface and sensitivity to lipid domain organization. BRI also synergized with azole antifungal drugs, suggesting a mechanistic role for ergosterol in its antifungal activity. Conclusions: These findings reveal a sterol- and domain-mediated mechanism for arylamide polymers and identify lipid organization as a key determinant of antifungal activity. The dependence of BRI activity on ergosterol content provides a mechanistic explanation for its synergy with azole antifungals and supports further investigation of BRI as a membrane-active antifungal agent.