Atividade antifúngica in vitro da doxepina em células planctônicas e biofilme de Candida spp.: determinação do possível mecanismo de ação e expressão gênica

Abstract

Invasive fungal infections represent a significant public health issue due to high mortality rates, particularly those caused by Candida spp., the leading cause of bloodstream infections in healthcare settings. Rising antifungal resistance has spurred the search for novel therapeutic strategies, including drug repurposing and synergistic combinations with existing antifungals. In this context, this study aimed to investigate the in vitro antifungal activity of the tricyclic antidepressant doxepin (DOX) against fluconazole-resistant Candida spp. strains, evaluating both planktonic and biofilm forms. DOX's inhibitory effect was assessed via broth microdilution to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), alongside checkerboard assays to evaluate drug interactions with antifungals and the drug's impact on biofilms. These tests were followed by flow cytometry and in vitro cytotoxicity assessments using human cells. Molecular analyses included docking studies and gene expression analysis via qRT-PCR. DOX demonstrated fungicidal activity against all strains—with MIC50 values ranging from 64 to 256 μg/mL— and significantly reduced biofilm formation. When combined with amphotericin B, a synergistic effect predominated, resulting in increased efficacy at lower concentrations compared to the drugs used alone. Mechanistically, DOX induced oxidative stress, mitochondrial dysfunction, and apoptosis, thereby reducing fungal viability. In silico ADME profiling indicated high lipophilicity, favoring the molecule's interaction with polymeric surfaces such as catheters, while assays using human cells demonstrated low cytotoxicity. Furthermore, interactions with Als3 and Sap5 proteins were observed, alongside alterations in the expression of virulence-related genes, such as ALS3, SAP5, ECE1, and HWP1. These findings indicate that DOX exhibits promising antifungal activity in the treatment of Candida infections, highlighting its potential against biofilms