Discovering Thiamine Transporters as Targets of Chloroquine Using a Novel Functional Genomics Strategy

Abstract

Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7T287N) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets.

iSeq: A New Double-Barcode Method for Detecting Dynamic Genetic Interactions in Yeast

“…Repeated bottlenecks would likely select different segregant representatives for each barcode, allowing one to assay the impact of genetic heterogeneity on each GI. Once a pool is constructed, it can be screened repeatedly in multiple environments to quickly and cheaply detect dynamic GIs (Darby et al 2012; Huang et al 2012). However, it is difficult to apply these methods more widely, assaying deletion combinations beyond a handful of query genes against a large array of target genes.…”

Cell Wall Perturbation Sensitizes Fungi to the Antimalarial Drug Chloroquine

“…(10). Recently, CQ was shown to inhibit thiamine transporters in both yeast and human cells (16). Our study indicates that the fungal cell wall acts as a barrier to help preclude the toxic action(s) of CQ and therefore that the potential of CQ as an antifungal agent (12-15) is enhanced considerably by cell wall perturbation.…”

“…The mechanism is thought to involve alkalinization of the host environment of the fungi, with associated iron deprivation in some cases. CQ has also been shown to inhibit thiamine transport in yeast as well as human cells (16).…”

Reply to “Chloroquine, an Antifungal but Also a Fertility Drug”

“…In the case of H. capsulatum, CQ acted by pH-dependent iron deprivation (2), and a similar mechanism was suggested for the model yeast Saccharomyces cerevisiae (5). More recently, however, thiamine transporters were found to be key targets of CQ action in S. cerevisiae as well as in cultured human cell lines (6). The latter mode of action may be particularly relevant to the findings we reported as it relies on direct interaction between CQ and the target organism, which should be exacerbated by increased CQ uptake in fungi with perturbed cell walls (1), rather than indirectly through an effect of CQ on host environment.…”