TARGETING MITOCHONDRIAL METABOLISM AS A KEY VULNERABILITY IN ARTEMISININ-RESISTANT PLASMODIUM FALCIPARUM MALARIA

Our Discovery Award application addresses the Fiscal Year 2018 Peer Reviewed Medical Research Program topic of Malaria, focusing on the investigation of mechanisms of artemisinin (ART) resistance in the human malaria parasite Plasmodium falciparum (Pf), and leveraging our findings to discover new therapeutic approaches to treating drug-resistant malaria in U.S. military personnel.Rationale: ART)-resistant strains of Pf have rapidly disseminated throughout Southeast Asia, compromising the efficacy of ART-based combination therapies (ACTs) that are used to treat local cases of malaria including in U.S. military personnel deployed to this region. If ART resistance were to spread to South America and Africa, it would foreshadow the global loss of first-line antimalarial therapies, with devastating consequences. Mutations in K13 constitute the primary genetic determinant of ART resistance in clinical isolates. Our multipronged systems biology-based studies have converged on mitochondrial metabolism and purine salvage as key mediators of how parasites arrest their development and thus survive ART treatment.Hypothesis: We posit that mitochondria dysregulation underpins ART resistance by priming the cells to enter quiescence upon ART exposure, allowing them to survive and subsequently exit quiescence and resume development after the drug is cleared. We hypothesize that ART resistance in K13 mutant Pf parasites can be overcome by inhibiting parasite mitochondrial functions or purine metabolism.Innovative Aspects: Our study proposes innovative hypotheses regarding the role of the mitochondria in mediating ART resistance and pioneers the exploitation of recently observed alterations in mitochondrial function seen in K13 mutant parasites as a novel approach to overcome ART resistance. We will use a variety of biochemical and gene-editing approaches to carefully characterize the basal mitochondrial metabolic state and activity in resistant and sensitive parasites. We will also identify new therapeutic strategies to overcome resistance by using inhibitors to target mitochondrial metabolism and DNA precursor synthesis and salvage pathways.Specific Aims and Objectives: In Aim 1, we will test the hypothesis that mutant K13 achieves ART resistance by altering its mitochondrial functionalities. In Aim 1.1, we will test the hypothesis that cellular respiration and energy metabolism, through the mitochondrial tricarboxylic acid (TCA) cycle or the electron transport chain (ETC), are involved in K13-mediated ART resistance and post-treatment cell survival. This will be extended in Aim 1.2 to test whether defects in the mitochondrial heme synthesis pathway can phenocopy ART resistance. Our Aim 2, of practical application, is to examine whether K13 mutant lines are hypersensitized to inhibitors that target mitochondrial functions or purine salvage and identify compounds that prevent the reactivation of quiescent parasites as a means to overcome ART resistance.Study Design: In Aim 1, we will characterize changes in the metabolic, redox, and cellular respiratory profiles of the mitochondrial TCA cycle and the ETC in a panel of K13 edited mutant and isogenic wild-type (WT) lines. We will measure the metabolic profile by quantifying isotope-labeled TCA cycle intermediates through tandem mass spectrometry. Our hypothesis that K13 mutations minimize ART-induced mitochondrial redox perturbations will be tested using genetically encoded mitochondria-targeting fluorescent redox biosensor probes. A possible contribution of altered respiratory rates in ART-resistant parasites will be examined by monitoring oxygen consumption in cultured parasites tested ± the active ART metabolite dihydroartemisinin (DHA). We will also biochemically assess whether K13 mutant ring-stage parasites alter their synthesis of labile heme in the mitochondria as a mechanism to prevent ART activation, using 13C-aminolevulinic acid isotope-labeling and measuring the levels of intracellular porphyrins, and employing knockout lines with specific defects in heme biosynthesis. In Aim 2, we will test the sensitivity of K13 mutant and WT parasites against a panel of known inhibitors that target the TCA cycle, the ETC, pyrimidine synthesis, and purine salvage pathways. Data will be collected using 72-hour dose-response assays as well as 4-hour DHA-pulsed ring stage survival assays. Isobologram analyses will also be used to identify compounds that synergize with DHA against K13 mutant parasites.Expected Results and Impact: This study is expected to transform our understanding of how K13 mutant parasites exploit changes in mitochondrial function and DNA precursor availability to survive ART exposure. We expect that this innovative line of research will illustrate how to exploit vulnerabilities in the ART resistance mechanism and identify novel means of protecting the U.S. military against drug-resistant malaria.