Genetic and transcriptional profile analyses in Saccharomyces cerevisiae reveal role for quinoline-ring antimalarial drugs in iron uptake

Plasmodium falciparum, the major cause of severe human malaria infects over 200 million people per year and resistance is an increasing problem. Resistance to chloroquine, one of the most effective antimalarials is widespread, yet we do not fully understand either its 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 have utilized Saccharomyces cerevisiae as a model eukaryotic system. We took two parallel approaches for this study. First, based on evidence that proteins of the ABC-transporter super-family play a role in chloroquine resistance in malaria, we directly tested this hypothesis using the yeast system. Second, to aid in the discovery of potential drug targets we applied the method of transcriptional profiling to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Using genetic and biochemical approaches, we demonstrated that yeast strains deficient in the ABC-transporter gene PDR5 have increased sensitivity to the quinoline drug chloroquine and exhibit increased accumulation of 14C-chloroquine. The association of chloroquine sensitivity and increased accumulation of drug is consistent with an efflux-mediated mechanism of resistance, as previously demonstrated for PDR5 and cycloheximide in other laboratories. These data clearly indicate a role for the PDR5 ABC-transporter in mediating chloroquine sensitivity in yeast. This is consistent with the hypothesis that a similar transporter, PfMDR1 , plays a role in mediating quinoline drug resistance in P. falciparum. To investigate the possibility that the toxicity of chloroquine is due to effects on multiple targets within the cell, we embarked on an analysis of the genes whose expression level respond to chloroquine treatment using microarrays and transcriptional profiling. Among those genes which were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, 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. These data implicated a change in iron availability as a possible outcome of chloroquine treatment. The transcriptional response of ABC-transporters was surprisingly minimal. We investigated the role of chloroquine in iron trafficking using a variety of approaches including pharmacological, genetic and biochemical techniques. For these experiments, we have utilized yeast lacking the major iron uptake pathways (FET3, FET4) and, in addition, yeast deficient in SIT1, the major up regulated iron siderophore transporter. Our experiments demonstrate that yeast genetically or environmentally limited in iron availability have increased sensitivity to chloroquine in pharmacological assays and the addition of iron rescues these cells from chloroquine killing. 55FeCl₃ 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.