Chiral Fluorescent Antifungal Azole Probes Detect Resistance, Uptake Dynamics, and Subcellular Distribution in <i>Candida</i> Species

Azoles are essential for fungal infection treatment, yet the increasing resistance highlights the need for innovative diagnostic tools and strategies to revitalize this class of antifungals. We developed two enantiomers of a fluorescent antifungal azole probe (<b>1</b> _<i>S</i> and <b>1</b> _<i>R</i> ), analyzing 60 <i>Candida</i> strains via live-cell microscopy. A database of azole distribution images in strains of <i>Candida albicans</i>, <i>Candida glabrata</i>, and <i>Candida parapsilosis</i>, among the most important pathogenic <i>Candida</i> species, was established and analyzed. This analysis revealed distinct populations of yeast cells based on the correlation between fluorescent probe uptake and cell diameter. Varied uptake levels and subcellular distribution patterns were observed in <i>C. albicans</i>, <i>C. glabrata</i>, and <i>C. parapsilosis</i>, with the latter displaying increased localization to lipid droplets. Comparison of the more potent fluorescent antifungal azole probe enantiomer <b>1</b> _<i>S</i> with the moderately potent enantiomer <b>1</b> _<i>R</i> highlighted time-dependent differences in the uptake profiles. The former displayed a marked elevation in uptake after approximately 150 min, indicating the time required for significant cell permeabilization to occur and its association with the azole's antifungal activity potency. Divergent uptake levels between susceptible and high efflux-based azole-resistant strains were detected, offering a rapid diagnostic approach for identifying azole resistance. This study highlights unique insights achievable through fluorescent antifungal azole probes, unraveling the complexities of azole resistance, subcellular dynamics, and uptake within fungal pathogens.