Antifungal and anti-biofilm activity of doxepin against Candida spp. mediated by oxidative stress and modulation of adhesion-related genes

Candida species are associated with invasive infections characterized by causing high rates of mortality and morbidity, particularly when linked to biofilm formation. The present study investigated the in vitro antifungal activity of doxepin (DOX) against fluconazole-resistant strains of Candida spp., including 10 clinical isolates, 2 ATCC strains and 1 CDC strain. Antifungal susceptibility assays were performed on planktonic cells and biofilms, followed by cytometric analyses, including reactive oxygen species (ROS) production, mitochondrial depolarization, phosphatidylserine externalization, and cell viability. Molecular analyses involved docking and gene expression by qRT-PCR. DOX exhibited fungicidal activity against all strains, with MIC50 values ranging from 64-256 μg/mL, as well as significant reduction of biofilm formation. In combination with amphotericin B, a predominantly synergistic effect was observed, with increased efficacy at lower concentrations. Mechanistically, DOX induced oxidative stress, mitochondrial dysfunction, and apoptosis, reducing fungal viability. The in silico ADME profile showed high gastrointestinal absorption, indicating potential for oral bioavailability, compatible with systemic use. Furthermore, it established stable interactions with the Als3 and Sap5 proteins, also promoting changes in the expression of virulence-related genes, such as als3, sap5, ece1, and hwp1. These findings indicate that DOX exhibits promising antifungal activity for the treatment of Candida infections, particularly highlighting its potential against biofilms.