Echinocandins Localized to the Target-Harboring Cell Surface Are Not Degraded but Those Entering the Vacuole Are

Abstract

Echinocandin antifungal drugs have a broad spectrum of activities and excellent safety profiles. These agents noncompetitively inhibit the formation of the major polysaccharide component of the fungal cell wall, a reaction catalyzed by the membrane-bound β-glucan synthase (GS) protein complex. We have developed fluorescent probes of three echinocandin drugs: caspofungin (CSF), anidulafungin (ANF), and rezafungin (RZF). Fluorescent echinocandins had the same spectrum of activities as the parent echinocandins, supporting the fact that conjugation of the dye did not alter their mode of action. Of the three echinocandins, ANF has the most potent in vitro activity. Investigation of the subcellular distribution of the fluorescent echinocandins in live Candida yeast cells revealed that despite their high structural similarity, each of the drug probes had a unique subcellular distribution pattern. Fluorescent CSF, which is the least potent of the three echinocandins, accumulated in Candida vacuoles; fluorescent ANF localized in the extracellular environment and on the yeast cell surface where the target GS resides; and fluorescent RZF was partitioned between the surface and the vacuole over time. Recovery of fluorescent CSF from Candida cells revealed substantial degradation over time; functional vacuoles were necessary for this degradation. Under the same conditions, fluorescent ANF was not degraded. This study supports the “target-oriented drug subcellular localization” principle. In the case of echinocandins, localization to the cell surface can contribute to improved potency and accumulation in vacuoles induces degradation leading to drug deactivation.

Deciphering the Biological Activities of Antifungal Agents with Chemical Probes

“…anidulafungin, and rezafungin. [46,47] Imaging of live Candida cells incubated with the fluorescent echinocandins revealed that those derived from caspofungin accumulate in vacuoles of the yeast cells, and those derived from anidulafungin localize to the yeast cell surface; rezafungin-derived probes appeared on the yeast cell surface shortly after their introduction but accumulated in vacuoles after a longer incubation period (Figure 3). Since Fks is a membranebound protein, accumulation of the echinocandin on the cell surface increases its local concentration in proximity to the target.…”

“…To study the subcellular distributions of echinocandins, their mechanisms of entry into fungal cells, and the relationship between their uptake and antifungal activity, Fridman and co‐workers prepared fluorescently labeled caspofungin, anidulafungin, and rezafungin [46, 47] . Imaging of live Candida cells incubated with the fluorescent echinocandins revealed that those derived from caspofungin accumulate in vacuoles of the yeast cells, and those derived from anidulafungin localize to the yeast cell surface; rezafungin‐derived probes appeared on the yeast cell surface shortly after their introduction but accumulated in vacuoles after a longer incubation period (Figure 3).…”

“…This likely results, at least in part, in the superior in vitro activity of anidulafungin compared to those of caspofungin and rezafungin. Interestingly, flow cytometry and fluorescent image analysis revealed that the level of fluorescent caspofungin uptake into yeast cells was significantly higher in a collection of echinocandin‐resistant strains than in parental echinocandin‐susceptible Candida strains [46] . Monitoring the intracellular uptake of fluorescent caspofungin, therefore, provides a rapid and simple method for prediction of echinocandin resistance.…”

Deciphering the Biological Activities of Antifungal Agents with Chemical Probes

“…[45] To study the subcellular distributions of echinocandins, their mechanisms of entry into fungal cells, and the relationship between their uptake and antifungal activity, Fridman and co-workers prepared fluorescently labeled caspofungin, anidulafungin, and rezafungin. [46,47] Imaging of live Candida cells incubated with the fluorescent echinocandins revealed that those derived from caspofungin accumulate in vacuoles of the yeast cells, and those derived from anidulafungin localize to the yeast cell surface; rezafungin-derived probes appeared on the yeast cell surface shortly after their introduction but accumulated in vacuoles after a longer incubation period (Figure 3). Since Fks is a membranebound protein, accumulation of the echinocandin on the cell surface increases its local concentration in proximity to the target.…”

Reshaping Echinocandin Antifungal Drugs To Circumvent Glucan Synthase Point‐Mutation‐Mediated Resistance

“…[6][7][8] The efficacy, tolerability, and favorable pharmacokinetic profile of echinocandins have contributed to their widespread adoption, particularly in managing infections caused by azole-resistant Candida pathogens. [8][9][10] Four echinocandins have been approved for clinical use: caspofungin, micafungin, anidulafungin, and, most recently (March of 2023), rezafungin. (Figure 1A).…”