Zinc and Iron Homeostasis: Target-Based Drug Screening as New Route for Antifungal Drug Development

Abstract

The incidence of fungal diseases is on the rise and the number of fatalities is still unacceptably high. While advances into antifungal drug development have been made there remains an urgent need to develop novel antifungal agents targeting as-yet unexploited pathways, such as metal ion homeostasis. Here we report such an approach by developing a metal sensor screen in the opportunistic human fungal pathogen Candida albicans. Using this reporter strain, we screened a library of 1,200 compounds and discovered several active compounds not previously described as chemical entities with antifungal properties. Two of these, artemisinin and pyrvinium pamoate, have been further characterized and their interference with metal homeostasis and potential as novel antifungal compounds validated. Lastly, we demonstrate that the same strain can be used to report on intracellular conditions within host phagocytes, paving the way toward the development of novel screening platforms that could identify compounds with the potential to perturb ion homeostasis of the pathogen specifically within host cells.

Keywords: Candida; antifungals; artemisinin; high throughput drug screening; iron homeostasis; pyrvinium pamoate; zinc homeostasis.

Figure 1

A culture of Candida albicans containing the Zrt2prom-GFP promoter-reporter construct was adjusted to a cell density OD₆₀₀ = 0.05 and grown in either (A) RPMI, pH 5.6 at 30°C or (B) RPMI, pH 7.2 at 37°C. Cells were grown in absence or presence of indicated metal chelator and cell density and GFP expression was measured every 2 h.

Figure 2

Candida albicans containing the metal promoter-reporter construct or a control plasmid were adjusted to a cell density OD₆₀₀ = 0.05 and grown in RPMI, pH 5.6 at room temperature with 5 μM TPEN for 24 h. Images were taken by JuLI Stage Real-Time Cell History Recorder (NanoEnTek, Korea) every hour. Images shown were taken at 16 h after treatment.

Figure 3

Summary of high throughput metal sensor screen. The total hit distribution and therapeutic classes for the zinc sensor (A,C) and iron sensor (B,D) are shown.

Figure 4

A culture of the promoter-reporter strains of Candida albicans were adjusted to a cell density OD₆₀₀ = 0.05 and grown in RPMI, pH 7.2 at 37°C for 16 h with indicated concentrations of (A) Artemisinin or (B) Pyrvinium pamoate when cell density (OD₆₀₀) and GFP fluorescence was measured. The values are the means ± standard deviation (SD) of at least 3 independent experiments.

Figure 5

Cell cultures of A549 epithelial cells were treated with indicated concentrations of Artemisinin and Pyrvinium pamoate for 48 h. Metabolic activity was determined by CellTiterBlue assay. The values are the means ± SD of at least 3 independent experiments.

Figure 6

Candida albicans cultures were adjusted to a cell density OD₆₀₀ = 0.01 and grown for 16 h in YNB with indicated compound concentrations and total cellular (A) zinc and (B) iron content was measure via ICP-OES. The values are the means ± SD of at least 2 independent experiments with 2 technical replicates. Changes of total metal content was compared to control conditions and p-values were calculate by unpaired, two tailed t-test (ns p > 0.05, ^*p ≤ 0.01, ^**p ≤ 0.001, ^***p < 0.001).

Figure 7

Candida albicans were adjusted to a cell density OD₆₀₀ = 0.05 and grown in RPMI, pH 5.6 at 30°C for 16 h with (A) Artemisinin (10μM—orange, 5μM—blue, 1 μM—green, or DMSO—red) or (B) Pyrvinium pamoate (1μM—orange, 0.5μM—blue, 0.1μM—green, or DMSO—red). Cells were stained with 5 μM FluoZin-3AM and labile zinc concentration was measured via flow cytometry.

Figure 8

The phagocytosis of Candida albicans transformed with (A) the Zrt2prom-GFP, (B) Hap43prom-GFP or (C) 1:1 mixture of Candida albicans transformed with either Hap43prom-dTomato or Zrt2prom-GFP was monitored at 1 h after exposure to J774 cells and images were obtained with a Zeiss Axio microscope using a 40 × objective.