High-Throughput Screen for Identifying Small Molecules That Target Fungal Zinc Homeostasis

Claudia Simm,Eric Weiss,Thomas O'Halloran,Chi-Hao Luan,Ping Wang

doi:10.1371/journal.pone.0025136

Abstract

Resistance to traditional antifungal drugs has increased significantly over the past three decades, making identification of novel antifungal agents and new targets an emerging priority. Based on the extraordinary zinc requirement of several fungal pathogens and their well-established sensitivity to zinc deprivation, we developed an efficient cell-based screen to identify new antifungal drugs that target the zinc homeostasis machinery. The screen is based on the zinc-regulated transcription factor Zap1 of Saccharomyces cerevisiae, which regulates transcription of genes like the high-affinity zinc transporter ZRT1. We generated a genetically modified strain of S. cerevisae that reports intracellular zinc deficiency by placing the coding sequence of green fluorescent protein (GFP) under the control of the Zap1-regulated ZRT1 promoter. After showing that the GFP fluorescence signal correlates with low intracellular zinc concentrations in this strain, a protocol was developed for screening small-molecule libraries for compounds that induce Zap1-dependent GFP expression. Comparison of control compounds and known modulators of metal metabolism from the library reveals a robust screen (Z′ = 0.74) and validates this approach to the discovery of new classes of antifungal compounds that interfere with the intracellular zinc homeostasis. Given that growth of many pathogenic organisms is significantly impaired by zinc limitation; these results identify new types of antifungal drugs that target critical nutrient acquisition pathways.

Highlights

Candida species are pathogenic organisms that account for most fungal infections in humans [1,2]
Yeast transformants report zinc status In order to develop a high-throughput screen for agents that disrupt zinc homeostasis we generated a yeast strain that expresses green fluorescent protein (GFP) under conditions of zinc deprivation. We accomplished this by fusing the sequence of the promoter region of the ZRT1 transporter gene with GFP
Analyzing identified drug leads in Candida albicans We have shown that the zinc responsive elements (ZRE)-GFP promoter-reporter construct was able to identify agents that interfere with zinc homeostasis in the non-pathogenic yeast S. cerevisiae

Summary

Introduction

Candida species are pathogenic organisms that account for most fungal infections in humans [1,2]. The first class targets enzymes of the ergosterol biosynthesis pathway. The second class, Echinocandins, is used in severe cases of Candidiasis. They inhibit bglucan synthase and interfere with cell wall biosynthesis [6]. The last class of antifungal drugs are nucleoside analogues such as 5-Flucytosine. This compound is imported into the cell and modified like cytosine, which results in production of 5-fluoro-UMP, a DNA synthesis inhibitor, and 5fluoro-UTP, which disrupts protein synthesis when incorporated into RNA. Since pathogenic organisms require extraordinary concentrations of zinc and are highly susceptible to zinc deprivation, we have turned our attention to the high affinity pathways involved in zinc acquisition and utilization

Methods

Results

Conclusion