Malaria is a serious public health burden that causes an estimated 1–2 millions deaths and 300–500 million infections each year1. There is no effective vaccine available, and parasites resistant to almost all antimalarial drugs currently in use have been reported, including reduced sensitivity to derivatives of the traditional Chinese medicine qinghaosu (artemisinin, 2)2. Development of new drugs and a better understanding of the targets of antimalarial drugs and drug resistance are urgently needed.
Phenotypic characterization of human malaria parasites is limited because pathogenic stages live within red blood cells (RBCs) and laboratory models for in vivo investigations are often unsatisfactory. Changes in response to antimalarial drugs, differences in growth rate, or variations in virulence are among the few phenotypes typically accessible3. When phenotypes are available, genetic mapping is a powerful tool to assign them to particular determinants, and various high-throughput genotyping methods, including microarrays for detecting single nucleotide polymorphism (SNPs) and microsatellites (MS), have been developed for studies of Plasmodium falciparum traits4. However, there are ~5,400 predicted genes in the parasite genome, and the function of the majority of these genes remains unknown5. Characterizing phenotypic differences in malaria parasites and identifying the genes affecting the differences may provide important information for investigating gene function.
A challenge in understanding drug action and mechanisms of drug resistance is to identify the relevant molecular target. One useful strategy is to synthesize an active compound and use it to affinity purify the protein target(s) to which the compound binds6. This approach, however, generally requires compounds that have high affinity for their targets. Another strategy employs genetic mapping to link chromosomal loci that affect parasite responses to compounds, allowing molecular targets to be identified after fine mapping and functional characterizations of candidate genes. In addition to discovering potential new antimalarial compounds, these strategies can detect and define differential chemical phenotypes (DCPs) that show distinct signature responses to compounds among a variety of parasite isolates.
Here we demonstrate a strategy for identifying targets of chemical compounds in malaria parasites by integrating quantitative high-throughput screening (qHTS) with genetic mapping (Fig. 1). We tested seven P. falciparum lines, including parents of three genetic crosses7–9, for their responses to 1,279 bioactive compounds and identified candidate genes for three DCPs using progeny from a genetic cross and genetic transfection methods of allelic replacement. These results show that differential responses of small molecules between parasite lines can be reliable phenotypes for exploring molecular mechanisms of pharmacologic interest. This study also provides an effective approach for investigating drug action and resistance mechanisms in diseases other than malaria.