Antifungal and molecular analysis of gene expression caused by haloperidol in Candida spp

ABSTRACT

Candidiasis, caused by yeasts of the Candida genus, is increasingly characterized by a high prevalence of clinical isolates resistant to conventional antifungals, rendering the development of novel therapeutic strategies paramount. Drug repurposing has emerged as a key strategy, utilizing established pharmaceuticals for indications beyond their original design; notably, haloperidol (HAL) has shown promising antimicrobial potential. In this context, the present study evaluates the activity of haloperidol, both as a monotherapy and in combination with conventional antifungals, against fluconazole-susceptible and fluconazole-resistant Candida spp. clinical strains. Furthermore, we investigate the underlying mechanisms of its antifungal action. Experimental approaches included broth microdilution assays to determine the Minimum Inhibitory Concentration (MIC), checkerboard assays for synergistic analysis, and cellular assessments via flow cytometry and fluorescence microscopy. Haloperidol displayed MIC values between 26.67 and 256 μg/mL. Synergistic interactions were identified between haloperidol and the azoles fluconazole and itraconazole, alongside a 2.5 % synergy rate with amphotericin B. Additionally, mechanistic assays confirmed that haloperidol induces programmed cell death (apoptosis) in C. albicans and C. auris strains. The oxidative stress caused by haloperidol altered Ca2+ homeostasis, followed by mitochondrial membrane depolarization, reduced ATP production, cytochrome c release into the cytosol and metacaspase activation, reduced viability, phosphatidylserine externalization, promoted fragmentation, damage and methylation of DNA. It also induced expression of genes related to oxidative stress. It reduced mitochondrial depolarization and decreased the reduction of glutathione (GSH), causing morphological alterations. The results suggest the apoptotic pathway as the main antifungal mechanism of haloperidol.