Artemisinin-based combination therapies have been pivotal in achieving major reductions in the global burden of Plasmodium falciparum (Pf) malaria. Pf parasite resistance to artemisinin, which first emerged in Cambodia through mutations in K13, has placed increased selection pressure on its partner drugs. Recent reports document clinical treatment failures with the first-line therapy dihydroartemisinin+piperaquine (PPQ) in Cambodia. PPQ resistance is also emerging in French Guiana. The absence of fully effective alternatives underscores the need to define the molecular basis of PPQ resistance as a means to curtail its spread. Whole-genome sequence (WGS) analysis and phenotypic studies of PPQ-resistant Cambodian isolates have identified candidate genes whose putative functions relate to hemoglobin (Hb) metabolism and solute transport in the parasite's digestive vacuole (DV). Our central hypothesis is that PPQ resistance is mediated by altered Hb metabolism in the DV and accompanying changes in transporters that regulate DV physiology.
In Aim 1 we address the hypothesis that Cambodian isolates achieve resistance primarily via amplification of plasmepsins 2 and 3, which encode Hb-digesting proteases. We will also genetically assess the evidence implicating mutations in the Hb peptidase DPAP1 and the DV solute and multidrug transporters PfCRT and PfMDR1. To test this hypothesis, parasites will be engineered through transgene expression and Cas9 or zinc-finger nuclease-mediated gene editing. Changes in resistance will be examined using PPQ survival and dose-response assays.
In Aim 2 we tackle the emerging problem of PPQ resistance in French Guiana by implementing genetic crosses with humanized FRG-NOD mice that are receptive to Pf liver and blood stages. WGS and phenotypic analysis of the progeny will identify loci linked to resistance. Parallel WGS studies on PPQ-resistant isolates will help converge on the selection of resistance candidates, whose role will be assessed using transfection.
In Aim 3 we define the functional basis of PPQ resistance and its impact on other antimalarials. Using clinical isolates and recombinant lines, we will assess whether reduced PPQ accumulation and heme binding is a hallmark of resistance. We will also evaluate whether resistance results in increased levels of toxic free heme resulting from Hb metabolism. Metabolomic studies will delineate the relationship between PPQ resistance and Hb digestion. Finally, we will assess the impact of resistance on other heme-binding antimalarials, with an emphasis on identifying drugs that retain or even increase their activity against PPQ-resistant parasites. We believe that this project provides powerful and scientifically innovative approaches to elucidate the molecular basis of PPQ resistance, yield genetic markers to monitor its emergence and spread, and identify optimal means of treatment. This project will also provide important new insights into Hb metabolism, which continues to provide a rich source of antimalarials for future clinical use.
Piperaquine is a vital drug in the fight against malaria, yet resistance is emerging independently in Asia and South America. We propose to identify the molecular basis of Plasmodium falciparum parasite resistance to this heme-binding drug using field isolates and genetic strategies to validate candidate resistance mediators. This project is expected to yield molecular markers to detect the emergence and spread of piperaquine resistance, define the role of hemoglobin metabolism, and identify the impact of resistance on the efficacy of other clinical antimalarials.
|Ross, Leila S; Dhingra, Satish K; Mok, Sachel et al. (2018) Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine. Nat Commun 9:3314|
|Schuh, Anna Katharina; Rahbari, Mahsa; Heimsch, Kim C et al. (2018) Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum. ACS Infect Dis 4:1601-1612|
|Lee, Andrew H; Dhingra, Satish K; Lewis, Ian A et al. (2018) Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter. Sci Rep 8:13578|
|Witkowski, Benoit; Duru, Valentine; Khim, Nimol et al. (2017) A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype-genotype association study. Lancet Infect Dis 17:174-183|
|Agrawal, Sonia; Moser, Kara A; Morton, Lindsay et al. (2017) Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity. J Infect Dis 216:468-476|
|Blasco, Benjamin; Leroy, Didier; Fidock, David A (2017) Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic. Nat Med 23:917-928|
|Dhingra, Satish K; Redhi, Devasha; Combrinck, Jill M et al. (2017) A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine. MBio 8:|
|Tilley, Leann; Straimer, Judith; Gnädig, Nina F et al. (2016) Artemisinin Action and Resistance in Plasmodium falciparum. Trends Parasitol 32:682-696|
|Veiga, M Isabel; Dhingra, Satish K; Henrich, Philipp P et al. (2016) Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun 7:11553|