In this study, we demonstrated that CQ and other quinoline-containing antimalarials inhibit thiamine transporters in yeast. We also showed that such a MOA is conserved between yeast and humans. In particular, the human thiamine transporter SLC19A3 was completely inhibited by CQ when expressed in yeast cells (). This MOA is likely medically relevant. First, much like the thi3Δ
yeast mutant, human cells completely depend on exogenously supplied thiamine for survival, and the thiamine transporters play essential roles in this process. Second, at 20 µM, a concentration achievable in human patients, CQ completely inhibited growth of the thi3Δ thi7Δ
double mutant (). The concentration need to significantly inhibit the thi3Δ
single mutant was about 10 times higher, but yeast cells are generally known to be more resistant to many drugs than mammalian cells due to the presence of cell wall and potent drug pumps. Third, the concentration of thiamine in human serum is in the 10–20 nM range 
, more than two-magnitude lower than those used in this study. The putatively competitive relationship between CQ and thiamine suggests that inhibition of thiamine uptake in human body is achievable using CQ concentrations much lower than those used in this study. Fourth, CQ accumulates in certain tissues (e.g. the retina) at high concentrations, an observation particularly relevant to retinopathy caused by CQ-based medications 
. In this regard, there is already a connection between thiamine deficiency and retinopathy in diabetic patients 
, and diabetic retinopathy can be prevented with thiamine supplementation in a rodent animal model 
. In addition, thiamine deficiency and CQ treatment both lead to neurological and cardiovascular disorders 
. Based on these, it will be interesting to investigate whether thiamine deficiency might underlie some of the CQ-induced adverse effects and whether these can be prevented with concomitant thiamine supplementation.
This study also demonstrated SL/DS as a novel and effective functional genomics strategy for discovering drug targets. This strategy starts with identifying mutants that are hypersensitive specifically to a drug treatment with a genome-wide DGSL screen (). Such a drug-hypersensitive mutant (e.g, thi3Δ
) is then used as a key to directly discover drug target(s) with a genome-wide GGSL or DS screen, or both (). Discovering a drug target with a subsequent GGSL screen is based on the premise that genetic and pharmacological inactivation of a drug target produce similar effects (e.g., fitness defect in the hypersensitive mutant) (). Discovering a drug target with a subsequent DS screen is based on the principle that overexpressing a drug target confers drug resistance 
, in this case, in a hypersensitive mutant (). That both GGSL and DS screens identified Thi7 greatly simplified its selection as a high likelihood candidate CQ target for validation. We note that the particular DS screen reported in this study was performed in the thi3Δ thi7Δ
double mutant, with an intention of using a low dose of CQ to potentially minimize inhibition of additional targets to increase pathway specificity. Such a DS screen would probably have also succeeded if a thi3Δ
single mutant had been used.
A diagram and concept of discovering drug target using the SL/DS strategy.
Most existing in vivo
target identification methods such as haploinsufficiency profiling 
, outright dosage suppression 
, and discovering resistance mutations with genome-sequencing or high throughput complementation 
typically rely on a drug's ability to completely or severely inhibit growth of wild-type cells. In contrast, SL/DS does not have such a requirement and thus can be used to discover non-essential genes as drug targets, as shown with Thi7 in this study. This feature is very significant considering that >80% of all proteins encoded by the yeast genome are non-essential. As a result, this method will offer much broader opportunity than the existing methods for discovering drug targets, especially with drugs that inhibit the growth of certain mutants but not wild-type cells.
SL/DS should also be useful in discovering essential proteins as drug targets. In this regard, it may not be as straightforward as the other methods. However, we have found that the existing methods fail to discover targets of many cytotoxic drugs (unpublished). A possible reason for that is that some drugs simultaneously inhibit multiple targets and that, consequentially, overexpressing or mutating any single target gene does not confer drug resistance in an otherwise wild-type strain background. In such a case, SL/DS could be effective because it is always possible to first discover drug-hypersensitive deletion mutants using a DGSL screen and subsequently identify the drug targets using GGSL and DS screens in these mutant backgrounds. The DS screen in a hypersensitive mutant could work because a lower drug dose can be used to minimize inhibition of other target pathways.
The SL/DS methodology seems to be similar to but is distinctly different from a previously described compendium approach, where targets of novel drugs are inferred from comparing a large compendium of genome-wide DGSL profiles of old drug treatments and GGSL profiles of genetic perturbation for similarities 
. Like SL/DS, this compendium approach could identify both essential and non-essential proteins as drug targets using the DGSL and GGSL profiles 
. However, it does not directly identify drug targets but instead infers candidate targets from profile similarity. A potential limitation is that perturbations in potentially many components of a given drug target pathway typically produce similar profiles, making it difficult to determine the actual drug target. Its discovery scope is also limited to the available DGSL or GGSL reference profiles, which are very difficult to generate at a large scale in higher eukaryotes. In contrast, SL/DS directly identifies a drug's target with only three genome-wide screens: DGSL followed with GGSL and DS. It does not rely on DGSL profiles of other drugs or GGSL profiles of other genetic perturbation as references. A similar SL/DS strategy will likely also be useful for drug target identification in human cells, where genome-wide DGSL, GGSL, and DS screens are now possible