Modeling mutant PfCRT-mediated drug transport to predict the emergence of piperaquine-resistant Plasmodium falciparum malaria

PROJECT SUMMARY The protozoan parasite Plasmodium falciparum causes over 400,000 malaria deaths each year, mostly in young African children. With no effective vaccine, chemotherapy remains the cornerstone of malaria treatment and control. Malaria eradication efforts have been hindered by the rise of resistance to first-line antimalarials in

Southeast Asia, including piperaquine (PPQ, used in combination with dihydroartemisinin). Piperaquine resistance (PPQ-R) and chloroquine resistance are primarily mediated by mutations in the P. falciparum Chloroquine Resistance Transporter (PfCRT). Mutant PfCRT transports drug away from its site of action in the

asexual blood stage parasite’s digestive vacuole. This acidic organelle degrades endocytosed host hemoglobin and extrudes globin-derived peptides for parasite protein synthesis. Less understood, however, is how certain amino acid substitutions confer this efflux mechanism and how they impact native transporter function. To

investigate the effect of these mutations, I propose in Aim 1 to purify contemporary PPQ-R PfCRT isoforms and perform binding and transport assays with PPQ, other clinically used antimalarials, and positively-charged peptides as proposed natural substrates. These studies will provide a comprehensive functional characterization

of PPQ-R PfCRT that is currently lacking. It is also important to predict how PPQ-R could spread to or emerge in other malaria endemic regions. The PfCRT mutations found in SE Asia have yet to be seen on African and South American backgrounds; however, PPQ is being used in these areas including as first-line treatment or for

uncomplicated malaria. To predict whether the contemporary amino acid substitutions seen in SE Asia could emerge in other regions to achieve PPQ-R, I propose in Aim 2 to engineer these mutations onto African and South American PfCRT isoforms in P. falciparum parasites from these regions. Assays will quantify the

susceptibility of these lines to PPQ and other antimalarials, and determine the relative fitness of each line. The degree of resistance conferred and the fitness cost imposed, along with the regional drug regimen, will be important in determining which pfcrt alleles predominate and which can emerge and spread in areas of PPQ

use. These aims are predicated on the hypotheses that (1) PPQ-R mutations in PfCRT alter transport of drugs and natural substrates and that (2) PPQ-R can arise in Africa and South America via the emergence of single amino acid substitutions observed in SE Asia in mutant PfCRT. These data will also identify whether a gain of

PPQ-R restores parasite susceptibility to chloroquine, as has been seen with most mutations in SE Asia, thereby creating therapeutic opportunities for new combination therapies. These studies are expected to yield important new insights into the molecular basis for antimalarial drug resistance, and to leverage that knowledge to predict

the emergence of novel PPQ-R PfCRT isoforms in distinct geographic regions. This will guide the development of treatment strategies to reduce the global impact and spread of drug-resistant malaria.