Relationship between Chloroquine Toxicity and Iron Acquisition in Saccharomyces cerevisiae

Abstract

Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drug's mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing.55 FeCl 3 accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive. These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.Chloroquine (CQ) is commonly used for treatment of malaria; however, its mode of action and the mechanism of resistance are still not fully understood. Better knowledge of CQ's mode of action may make it possible to identify new drugs that target similar pathways or to reverse existing resistant phenotypes.CQ has been shown to interact in both mammalian cells and Plasmodium spp. with a number of different pathways, including changes in vacuolar (or lysosomal) pH (9,22,39,56), binding to DNA and RNA (3,11,41,53), binding to heme and ␤-hematin in Plasmodium falciparum (1, 55; reviewed in reference 20). In the case of P. falciparum, CQ was an effective drug whose efficacy has been severely compromised by the emergence of drug-resistant parasites (8,20).To analyze the mechanism of CQ action, we used transcriptional profiling in a model eukaryotic system, Saccharomyces cerevisiae. Differential transcriptional profiling using microarray analysis is based on detecting differences in expression of mRNAs in cells treated under different conditions. No information other than the genomic sequence and the open reading frame (ORF) predictions is necessary to assay mRNA expression. Such whole-genome analysis allows the determination of expression profiles without preselection of genes. For the experiments described here, we used the Affymetrix...

Synergistic Activity of Chloroquine with Fluconazole against Fluconazole-Resistant Isolates of Candida Species

“…highlights the importance of developing new antifungal compounds. Studies have shown that CQ and caspofungin exhibited synergistic activity against C. albicans, C. glabrata, Aspergillus fumigatus, and Saccharomyces cerevisiae in vitro (29), and CQ inhibited the growth of yeast species such as Histoplasma capsulatum and S. cerevisiae because of iron deprivation (17,30,31). The iron deprivation would cause a decrease in the level of membrane sterols and then cause an increase in membrane fluidity, which could enhance the passive diffusion of drugs and result in heightening drug susceptibility (32).…”

Transcriptomic Profiling of theSaccharomyces cerevisiaeResponse to Quinine Reveals a Glucose Limitation Response Attributable to Drug-Induced Inhibition of Glucose Uptake

“…(7,20,50). Previous studies of yeast have identified a number of cellular processes that are affected by quinoline antimalarial drugs (7,53,54). It has been shown by our group that quinidine strongly impairs potassium uptake and accumulation in yeast cells, providing a physiological advantage for cells under quinidine-induced stress (53).…”

“…It was also demonstrated that quinidine affects the homeostasis of intracellular pH, whose main-tenance within a permissive range for the functionality of pHsensitive cellular processes is crucial to cell physiology (53,54). Another interesting study of yeast uncovered a relationship between chloroquine toxicity and iron acquisition and demonstrated that chloroquine is a competitive inhibitor of iron uptake and accumulation (7). Furthermore, genes of the iron uptake pathway are activated at the diauxic shift under the control of the Snf1 kinase (16), one of the main regulators of glucose-repressed genes.…”

“…Genome-wide expression analyses in yeast have been successfully used to assess the cellular and molecular changes imposed by antimalarials, anticancer drugs, fungicides, herbicides, etc. (7,20,50). Previous studies of yeast have identified a number of cellular processes that are affected by quinoline antimalarial drugs (7,53,54).…”

Genomewide Expression Profiling of Cryptolepine-Induced Toxicity in Saccharomyces cerevisiae

“…Millimolar concentrations of chloroquine can prevent iron uptake in yeast (6), resulting in the upregulation of Aft1p-regulated genes and growth defects under iron-limiting conditions. The apparently opposite effect of micromolar concentrations of cryptolepine on the same regulon should have a synergistic effect, because the combination of low expression of iron-assimilating genes and prevention of iron assimilation would produce a very severe effect on the oxidative metabolism of the affected cells.…”