PROJECT SUMMARY Plasmodium falciparum (Pf) malaria cases have recently surged, with an estimated 627,000 deaths in 2020, mostly in young African children. Efforts to reduce the burden of disease in Africa include both chemoprevention strategies to protect vulnerable populations and treatment of symptomatic infections with effective artemisinin- based combination therapies. Clinical studies have identified piperaquine (PPQ) as an ideal chemoprevention and treatment partner drug due to its potency against asexual blood stage parasites, long plasma half-life, and good safety profile in children and pregnant women. However, both drugs in the first-line combination of dihydroartemisinin-PPQ have encountered widespread resistance in Southeast Asia. Given the recent emergence in Rwanda and Uganda of artemisinin-resistant Pf parasites, there is a pressing need to investigate whether PPQ resistance can emerge and spread in Africa, identify its genetic mediators, and develop new ways to neutralize resistance. In Aim 1, our studies will focus on novel mutations that emerged on the background of chloroquine-resistant isoforms of the Pf chloroquine resistance transporter PfCRT, as well as the amplification of plasmepsins 2 and 3 (pm2/3), both of which contribute to PPQ resistance and treatment failure in Southeast Asia. Using gene editing and overexpression approaches, we will determine whether PPQ resistance can be generated in African lines by introducing individual mutations into African-specific PfCRT isoforms and assessing whether increased pm2/3 copy numbers augment resistance. We will also assess whether pm2/3 amplifications afford a fitness benefit to PfCRT mutant parasites, which could help establish and maintain PPQ resistance in high-transmission African settings. In Aim 2, we will examine the role of inhibition of heme detoxification and concentration-dependent drug efflux in PPQ action and resistance, respectively. Our studies will also test the hypothesis that PPQ-resistant PfCRT isoforms perturb intracellular hemoglobin-derived peptide levels, which may be corrected by pm2/3 amplification. In Aim 3, we propose that directly targeting PfCRT-mediated resistance represents a high-value approach to retaining PPQ efficacy. Leveraging insights regarding opposing selective pressures, we will test whether combining PPQ with other quinoline-based drugs (chloroquine, amodiaquine or mefloquine) can eliminate PPQ-resistant parasites and prevent the recrudescence of resistant variants. We will also screen for inhibitors that reverse resistance by blocking PPQ efflux via mutant PfCRT. Assays will include ZY19489, an antimalarial in human clinical trials that inhibits PfCRT-mediated drug efflux and hypersensitizes Pf parasites to PPQ. Concurrently, we will conduct a high-throughput screen designed to identify novel PPQ resistance reversal agents. This proposal, which aligns with NIAID’s priority of supporting research on antimicrobial drug resistance, is desi...