Mutant PfCRT Can Mediate Piperaquine Resistance in African Plasmodium falciparum With Reduced Fitness and Increased Susceptibility to Other Antimalarials.

BACKGROUND: Additional therapeutic strategies could benefit efforts to reverse the recent increase in malaria cases in sub-Saharan Africa, which mostly affects young children. A primary candidate is dihydroartemisinin + piperaquine (DHA + PPQ), which is effective for uncomplicated malaria treatment, seasonal malaria chemoprevention, and intermittent preventive treatment. In Southeast Asia, Plasmodium falciparum parasites acquired PPQ resistance, mediated primarily by mutations in the P falciparum chloroquine resistance transporter PfCRT. The recent emergence in Africa of DHA-resistant parasites creates an imperative to assess whether PPQ resistance could emerge in African parasites with distinct PfCRT isoforms. METHODS: We edited 2 PfCRT mutations known to mediate high-grade PPQ resistance in Southeast Asia into GB4 parasites from Gabon. Gene-edited clones were profiled in antimalarial concentration-response and fitness assays. RESULTS: The PfCRT F145I mutation mediated moderate PPQ resistance in GB4 parasites but with a substantial fitness cost. No resistance was observed with the PfCRT G353V mutant. Both edited clones became significantly more susceptible to amodiaquine, chloroquine, and quinine. CONCLUSIONS: A single PfCRT mutation can mediate PPQ resistance in GB4 parasites, but with a growth defect that may preclude its spread without further genetic adaptations. Our findings support regional use of drug combinations that exert opposing selective pressures on PfCRT.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance.

Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion.