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Introduction. Candida spp. are commensal fungal pathogens of humans, but when there is an imbalance in the microbiota, or weak host immunity, these yeasts can become pathogenic, generating high medical costs. Gap Statement. With the increase in resistance to conventional antifungals, the development of new therapeutic strategies is necessary. This study evaluated the in vitro antifungal activity of chlorogenic acid against fluconazole-resistant strains of Candida spp. Mechanism of action through flow cytometry and in silico analyses, as well as molecular docking assays with ALS3 and SAP5, important proteins in the pathogenesis of Candida albicans associated with the adhesion process and biofilm formation. Results. The chlorogenic acid showed in vitro antifungal activity against the strains tested, causing reduced cell viability, increased potential for mitochondrial depolarization and production of reactive oxygen species, DNA fragmentation and phosphatidylserine externalization, indicating an apoptotic process. Concerning the analysis through docking, the complexes formed between chlorogenic acid and the targets Thymidylate Kinase , CYP51, 1 Yeast Cytochrome BC1 Complex e Exo -B-(1,3)- glucanase demonstrated more favourable binding energy. In addition, chlorogenic acid presented significant interactions with the ALS3 active site residues of C. albicans, important in the adhesion process and resistance to fluconazole. Regarding molecular docking with SAP5, no significant interactions were found between chlorogenic acid and the active site of the enzyme. Conclusion. We concluded that chlorogenic acid has potential use as an adjuvant in antifungal therapies, due to its anti- Candida activity and ability to interact with important drug targets.

The genus Candida spp. has been highlighted as one of the main etiological agents causing fungal infections, with Candida albicans being the most prominent, responsible for most cases of candidemia. Due to its capacity for invasion and tissue adhesion, it is associated with the formation of biofilms, mainly in the environment and hospital devices, decreasing the effectiveness of available treatments. The repositioning of drugs, which is characterized by the use of drugs already on the market for other purposes, together with molecular-docking methods can be used aiming at the faster development of new antifungals to combat micro-organisms. This study aimed to evaluate the antifungal effect of diazepam on mature C. albicans biofilms in vitro and its action on biofilm in formation, as well as its mechanism of action and interaction with structures related to the adhesion of C. albicans , ALS3 and SAP5. To determine the MIC, the broth microdilution test was used according to protocol M27-A3 (CLSI, 2008). In vitro biofilm formation tests were performed using 96-well plates, followed by molecular-docking protocols to analyse the binding agent interaction with ALS3 and SAP5 targets. The results indicate that diazepam has antimicrobial activity against planktonic cells of Candida spp. and C. albicans biofilms, interacting with important virulence factors related to biofilm formation (ALS3 and SAP5). In addition, treatment with diazepam triggered a series of events in C. albicans cells, such as loss of membrane integrity, mitochondrial depolarization and increased production of EROs, causing DNA damage and consequent cell apoptosis.