Antifungal activity of 2-aryloxazoline derivatives and miltefosine on Candida spp. and inhibition of Candida albicans virulence factors

New antifungal molecules are being researched, addressing new targets and mechanisms of action. Therefore, the present study aims to evaluate the antifungal activity, the morphophysiological and molecular effects of 2-aryloxazoline derivatives (4i and 9i), and miltefosine (MFS) on Candida spp. The growth of 73 Candida spp. was inhibited by compounds 4i and 9i at minimum inhibitory concentrations (MIC) 2 &#956g/mL accumulating budding yeast. Although these compounds did not inhibit the metabolic activity in Candida spp., they could inhibit up to 50% of the total biomass of Candida albicans biofilms in the first 12h. Furthermore, 4i and 9i inhibited C. albicans proteinase and phospholipase activity. In the in vivo assay on the Galleria mellonella model, 4i (100 mg/kg) did not demonstrate toxicity, however, it did notan show antifungal effect at the doses tested (20-40 mg/kg). Interestingly, compounds 4i and 9i reduced hyphal formation in C. albicans and completely inhibited filamentation in C. tropicalis in RPMI, Lee and Spider medium. In the gene expression assay, some genes were downregulated after treatment of yeast with compound 4i, mainly adhesins (HWP1 and ALS3), candidalysin (ECE1), transcription factors TEC1, EFG1 and CEK1. Due to the downregulation of Efg1 and Tec1 targets, belonging to the cAMP pathway and the downregulation of Cek1, belonging to the MAPK pathway, in treated cells, we suggest that compound 4i is inhibiting morphogenesis by inhibiting these two pathways resulting in reduced expression of genes expressed during filamentation. MFS inhibited the growth of all C. albicans isolates, including strains resistant to fluconazole (MIC=1-2 &#956g/mL), and also showed a fungicidal effect. At MIC and 2xMIC values, MFS significantly inhibits adherence up to 50%. MFS inhibited fungal growth and hyphal formation in all inducing media used (RPMI, Spider (solid and liquid), Lee and SM-GlcNAc). Furthermore, all isolates had their biofilms inhibited in both stages of development, in the formation of biofilms (4-8 &#956g/mL) and in the mature biofilm (16-32 &#956g/mL). In the expression of virulence genes in C. albicans, we observed downregulation of genes in the cAMP pathway (RAS1, CYR1, EFG1 and TEC1) and MAPK (CEK1), in addition to downregulation in genes for adhesins (HWP1 and ALS3). and candidalysin (ECE1). We conclude that the 2-aryloxazoline derivatives (4i and 9i) and MFS have strong potential as antifungal agents against C. albicans and other species. Furthermore, these compounds inhibit the morphogenesis of C. albicans and/or C. tropicalis resulting in the inhibition of other virulence factors associated with the filamentation process, negatively regulating the cAMP and MAPK pathways. (AU)