Real-Time Imaging of the Azole Class of Antifungal Drugs in Live Candida Cells

Abstract

Azoles are the most commonly used class of antifungal drugs, yet where they localize within fungal cells and how they are imported remain poorly understood. Azole antifungals target lanosterol 14α-demethylase, a cytochrome P450, encoded by ERG11 in Candida albicans, the most prevalent fungal pathogen. We report the synthesis of fluorescent probes that permit visualization of antifungal azoles within live cells. Probe 1 is a dansyl dye-conjugated azole, and probe 2 is a Cy5-conjugated azole. Docking computations indicated that each of the probes can occupy the active site of the target cytochrome P450. Like the azole drug fluconazole, probe 1 is not effective against a mutant that lacks the target cytochrome P450. In contrast, the azole drug ketoconazole and probe 2 retained some antifungal activity against mutants lacking the target cytochrome P450, implying that both act against more than one target. Both fluorescent azole probes colocalized with the mitochondria, as determined by fluorescence microscopy with MitoTracker dye. Thus, these fluorescent probes are useful molecular tools that can lead to detailed information about the activity and localization of the important azole class of antifungal drugs.

Localizing Antifungal Drugs to the Correct Organelle Can Markedly Enhance their Efficacy

“…[11,29] Given the key role of azoles in the limited armamentarium of anti-Candida drugs,and that drug tolerance may be associated with increased persistence of infections, [30] the development of azoles that reduce tolerance is ah ighly desirable pharmacological property.T oa sk if the efficacyofazole 3 has any role in preventing the appearance of drug-tolerant subpopulations over time,w ee valuated residual fungal cell growth after 48 and 72 hours by measuring the average cell density (OD 600 )inbroth microdilution assays at drug concentrations above the MIC (supra-MIC). [32] Subsequently,w ec ompared the viability of mammalian cells exposed to azoles 2 and 3 or to FLC using both the human embryonic kidney cell line HEK 293 as well as primary human skin fibroblasts cells,w hich more closely mimic the physiological state of cells in vivo.L ike FLC and azole 2, [25] azole 3 did not significantly affect the viability of the tested mammalian cells (IC 50 @ 25 mgmL À1 ;f or data see the Supporting Information, Section 7C,F igures S1 and S2) even at the maximal concentration tested (25 mgmL À1 ), which was two to three orders of magnitude above the MIC range of azole 3 (Table 1). [31] Thec ells treated with FLC or the dansyl-based azole 2 showed evidence of drug tolerance,a st hey continued to grow,a lbeit slowly,a td rug concentrations beyond the MIC determined at 24 h( arrows, Figure 3A).…”

“…[25] Nonetheless,t he activity of both probes required the presence of ERG11,t he gene encoding the cytochrome P-450 DM ,i ndicating that this enzyme is the target of these fluorescent probes. [25] Nonetheless,t he activity of both probes required the presence of ERG11,t he gene encoding the cytochrome P-450 DM ,i ndicating that this enzyme is the target of these fluorescent probes.…”

Clearing the FoG: Antifungal tolerance is a subpopulation effect that is distinct from resistance and is associated with persistent candidemia

“…A recently developed fluorescent azole probe, FLC-Cy5 81 , provides a powerful tool to monitor intracellular drug levels and uptake rates using flow cytometry (Fig. 3a).…”

Guiding Drugs to Target‐Harboring Organelles: Stretching Drug‐Delivery to a Higher Level of Resolution

“…We therefore developed two inherently fluorescent antifungal azoles 1 and 2 ( Figure 4B)a nd studied their subcellular distribution in live yeast cells of different species of the Candida genus,t he most prevalent cause for fungal infections. [81] Azoles 1 and 2 effectively inhibited growth of awild-type Candida but not the growth of astrain deficient in CYP51, indicating that this enzyme is at arget for the fluorescent azoles.I nterestingly,b oth azoles primarily localized to mitochondria during the first hours after exposure to yeast cells ( Figure 4B)a sc onfirmed through co-localization experiments.T hese data suggested that, depending on the specific structure,s ome antifungal azole drugs may not efficiently reach the ER, where the target enzyme CYP51 resides.…”