Detailing the mode-of-action of novel antimalarial drugs is important to predict and optimize their treatment efficacy. To complement standardized in vivo studies, an in vitro pharmacodynamic characterization of the Plasmodium elongation factor 2 inhibitor cabamiquine has been undertaken on Plasmodium falciparum-infected red blood cells. Parasite growth arrest and killing kinetics were assessed using a MitoTracker assay, two genetically engineered luminescent parasite lines expressing firefly luciferase or NanoLuc (BRRoK assays), standard growth inhibition assays, and long-term live cell imaging. The results confirm that cabamiquine is a potent and fast-acting inhibitor of parasite protein translation, inducing complete arrest of the translational machinery within 6 h of drug exposure. Rate-of-kill assays showed a >100-fold difference in potency between the block on translation (luciferase-based BRRoK IC50 = 0.9 nM) and overall parasite killing (6 h-treatment, ring-stage standard growth inhibition assay IC50 > 100 nM), highlighting that translational arrest occurs well before parasite death. Prolonged inhibition of protein synthesis drives parasite exhaustion, followed by irreversible cell death within 48 h, preceding the parasite clearance observed in patients. These in vitro findings help contextualize why the clinically observed lag in parasite clearance may be temporally distinct from symptomatic improvement, including fever resolution. Here, cabamiquine presents itself as a fast-acting, slow clearing antimalarial drug.

Abstract New antimalarial drugs are needed to combat the current emergence and spread of Plasmodium falciparum parasite resistance to artemisinin-based combination therapies. Here, we characterize ZY19489, a triaminopyrimidine presently in a Phase Ib clinical trial. Asexual blood-stage parasites pressured with ZY19489 acquire low-grade resistance, mediated by a novel mutation in the P. falciparum chloroquine resistance transporter (PfCRT) that causes slow growth rates and a substantial fitness cost. ZY19489-resistant parasites lose their chloroquine resistance status and become hypersusceptible to piperaquine (PPQ), an artemisinin-based combination partner drug. Uptake studies in proteoliposomes loaded with drug-resistant PfCRT isoforms demonstrate that ZY19489 can block mutant PfCRT-mediated PPQ and chloroquine transport. In parasites, PfCRT mutant variants can mediate PPQ and chloroquine resistance via their efflux out of the digestive vacuole. Our findings evoke a scenario of an evolutionary trap whereby resistance to ZY19489 can block PPQ and chloroquine efflux and thereby restore their activity. Metabolomic studies show that ZY19489 leads to significantly reduced intracellular levels of short hemoglobin-derived peptides (a natural substrate of PfCRT) and accumulation of pyrimidine deoxynucleotides. Our results present a possible marker for tracking the evolution of clinical resistance to ZY19489 and a rationale for pairing this molecule with PPQ to generate a resistance-refractory combination.

New antimalarial drugs are essential to combat the current emergence and spread of Plasmodium falciparum parasite resistance to first-line artemisinin-based combination therapies. Here, we identify a mechanism of parasite resistance to ZY19489, a triaminopyrimidine currently in a Phase IIb clinical trial. Low-grade resistance was mediated by a novel mutation in the P. falciparum chloroquine resistance transporter PfCRT, which caused a major reduction in asexual blood stage parasite growth rates and a substantial fitness cost. Parasites resistant to ZY19489 lost their chloroquine resistance status and became hypersusceptible to the artemisinin-based combination partner drug piperaquine. All three agents were shown to interfere with parasite-mediated catabolism of host hemoglobin. Uptake studies in PfCRT-containing proteoliposomes provide evidence that ZY19489 can block mutant PfCRT-mediated efflux of piperaquine and chloroquine, creating a scenario of an evolutionary trap whereby resistance to ZY19489 blocks PfCRT efflux-mediated resistance and restores susceptibility to piperaquine and chloroquine. Metabolomic studies revealed that ZY19489 significantly reduces intracellular levels of short hemoglobin-derived peptides (a natural substrate of PfCRT) and leads to higher accumulation of pyrimidine deoxynucleotides. Our data present a marker for tracking the evolution of clinical resistance to ZY19489 and a rationale for pairing this with piperaquine to generate a novel resistance-refractory combination.

Surveillance of drug resistance and the discovery of novel targets—key objectives in the fight against malaria—rely on identifying resistance-conferring mutations in Plasmodium parasites. Current approaches, while successful, require laborious experimentation or large sample sizes. To elucidate shared determinants of antimalarial resistance that can empower in silico inference, we examined the genomes of 724 Plasmodium falciparum clones, each selected in vitro for resistance to one of 118 compounds. We identified 1448 variants in 128 recurrently mutated genes, including drivers of antimalarial multidrug resistance. In contrast to naturally occurring variants, those selected in vitro are more likely to be missense or frameshift, involve bulky substitutions, and occur in conserved, ordered protein domains. Collectively, our dataset reveals mutation features that predict drug resistance in eukaryotic pathogens.

ABSTRACTMalaria elimination efforts in Southeast Asia have been hindered by multidrug-resistant Plasmodium falciparum. High-grade resistance to piperaquine (PPQ, used in combination with dihydroartemisinin) is associated with PfCRT mutations that arose in strains expressing the PfCRT Dd2 isoform, which mediates resistance to the related 4-aminoquinoline chloroquine (CQ). The PPQ-resistant PfCRT haplotype Dd2 + F145I mediates the highest level resistance but causes a significant growth defect in intra-erythrocytic parasites. Recently, three separate mutations (F131C, I347T and C258W) have been observed on Dd2 + F145I PfCRT either during extended parasite culture or in Southeast Asian isolates no longer subject to PPQ pressure. Competitive growth assays with pfcrt-edited parasites reveal that these compensatory mutations reduce the fitness defect caused by F145I. PPQ survival assays on edited lines show a loss of PPQ resistance in two of the three variants, including the field mutant (C258W). The latter restores CQ resistance. None of these variants alter parasite susceptibility to the first-line partner drug, mefloquine. Utilizing drug transport assays with purified PfCRT isoforms reconstituted into proteoliposomes, we identify differences in mutant PfCRT-mediated transport of PPQ and CQ. Molecular dynamics energy minimization calculations predict that these same mutations cause small but significant conformational changes in PfCRT regions implicated in drug interactions. Metabolomic analyses of isogenic parasite lines reveal differences in hemoglobin-derived peptide accumulation as a hallmark of PfCRT variation. These studies highlight the transient nature of PPQ resistance upon removal of drug pressure and suggest a strategy for employing this drug as part of multiple first-line therapies.IMPORTANCEOur study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance in vitro and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping using 34 recombinant haplotypes, and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

Background Cabamiquine is a novel antimalarial that inhibits Plasmodium falciparum translation elongation factor 2. We investigated the causal chemoprophylactic activity and dose-exposure-response relationship of single oral doses of cabamiquine following the direct venous inoculation (DVI) of P falciparum sporozoites in malaria-naive, healthy volunteers.Methods This was a phase 1b, randomised, double-blind, placebo-controlled, adaptive, dose-finding, single-centre study performed in Leiden, Netherlands. Malaria-naive, healthy adults aged 18-45 years were divided into five cohorts and randomly assigned (3:1) to receive cabamiquine or placebo. Randomisation was done by an independent statistician using codes in a permuted block schedule with a block size of four. Participants, investigators, and study personnel were masked to treatment allocation. A single, oral dose regimen of cabamiquine (200, 100, 80, 60, or 30 mg) or matching placebo was administered either at 2 h (early liver-stage) or 96 h (late liver-stage) after DVI. The primary endpoints based on a per-protocol analysis set were the number of participants who developed parasitaemia within 28 days of DVI, time to parasitaemia, number of participants with documented parasite blood-stage growth, clinical symptoms of malaria, and exposure-efficacy modelling. The impact of cabamiquine on liver stages was evaluated indirectly by the appearance of parasitaemia in the blood. The Clopper-Pearson CI (nominal 95%) was used to express the protection rate. The secondary outcomes were safety and tolerability, assessed in those who had received DVI and were administered one dose of the study intervention. The trial was prospectively registered on ClinicalTrials.gov (NCT04250363).Findings Between Feb 17, 2020 and April 29, 2021, 39 healthy participants were enrolled (early liver-stage: 30 mg [n=3], 60 mg [n=6], 80 mg [n=6], 100 mg [n=3], 200 mg [n=3], pooled placebo [n=6]; late liver-stage: 60 mg [n=3], 100 mg [n=3], 200 mg [n=3], pooled placebo [n=3]). A dose-dependent causal chemoprophylactic effect was observed, with four (67%) of six participants in the 60 mg, five (83%) of six participants in the 80 mg, and all three participants in the 100 and 200 mg cabamiquine dose groups protected from parasitaemia up to study day 28, whereas all participants in the pooled placebo and 30 mg cabamiquine dose group developed parasitaemia. A single, oral dose of 100 mg cabamiquine or higher provided 100% protection against parasitaemia when administered during early or late liver-stage malaria. The median time to parasitaemia in those with early liver-stage malaria was prolonged to 15, 22, and 24 days for the 30, 60, and 80 mg dose of cabamiquine, respectively, compared with 10 days for the pooled placebo. All participants with positive parasitaemia showed documented blood-stage parasite growth, apart from one participant in the pooled placebo group and one participant in the 30 mg cabamiquine group. Most participants did not exhibit any malaria symptoms in both the early and late liver-stage groups, and those reported were mild in severity. A positive dose-exposure-efficacy relationship was established across exposure metrics. The median maximum concentration time was 1-6 h, with a secondary peak observed between 6 h and 12 h in all cabamiquine dose groups (early liver-stage). All cabamiquine doses were safe and well tolerated. Overall, 26 (96%) of 27 participants in the early liver-stage group and ten (833%) of 12 participants in the late liver-stage group reported at least one treatment-emergent adverse event (TEAE) with cabamiquine or placebo. Most TEAEs were of mild severity, transient, and resolved without sequelae. The most frequently reported cabamiquine-related TEAE was headache. No dose-related trends were observed in the incidence, severity, or causality of TEAEs.Interpretation The results from this study show that cabamiquine has a dose-dependent causal chemoprophylactic activity. Together with previously demonstrated activity against the blood stages combined with a half-life of more than 150 h, these results indicate that cabamiquine could be developed as a single-dose monthly regimen for malaria prevention.

Background: M5717 is a novel antimalarial that inhibits Plasmodium falciparum translation elongation factor 2. We investigated the chemoprophylactic activity and dose–exposure–response relationship of single oral doses of M5717 following the direct venous inoculation (DVI) of Plasmodium falciparum sporozoites in healthy volunteers.Methods: This was a phase 1b, randomised, double-blind, placebo-controlled, adaptive, dose-finding, single-centre study (NCT04250363). Eligible, malaria-naïve healthy adults aged 18–45 years were divided into five cohorts and randomised in a 3:1 ratio to receive M5717 or a placebo. A single oral dose of M5717 (200, 100, 80, 60, or 30 mg) or matching placebo was administered either at 2 hours (early liver-stage) or 96 hours (late liver-stage) after DVI. The primary outcomes were the assessments of chemoprophylactic activity and dose–exposure–response relationship. The secondary outcomes were safety and tolerability.Findings: Thirty-nine healthy participants were enrolled from 17 February 2020 to 7 June 2021. A single oral dose of ≥ 100 mg M5717 provided 100% protection against early and late liver-stage malaria. The median time to parasitaemia in early liver-stage malaria was prolonged to 15, 22, and 24 days for the 30, 60, and 80 mg dose of M5717, respectively, compared with 10 days for the pooled placebo. A positive dose–exposure–efficacy relationship was established across exposure metrics. All M5717 doses were safe and well tolerated.Interpretation: The potent, dose-dependent chemoprophylactic activity of M5717 indicates its potential as a single-dose antimalarial agent.Funding Information: This study was sponsored by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).Declaration of Interests: MB, ÖY, AT, AS, and AK are employed by the healthcare business of Merck KGaA, Darmstadt, Germany, the study sponsor. DB is employed by Merck Pty Ltd (an affiliate of Merck KGaA, Darmstadt, Germany), Modderfontein, South Africa. CO and TS are employed by the Global Health Institute of Merck, Ares Trading S.A. (a subsidiary of Merck KGaA, Darmstadt, Germany), Eysins, Switzerland. WB is a former (retired) employee of the Merck Institute for Pharmacometrics, Merck Serono S.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Lausanne, Switzerland. JW received funding for consulting with the healthcare business of Merck KGaA, Darmstadt, Germany, during the course of this study. All other authors declare no competing interests.Ethics Approval Statement: The independent medical ethics committee Stichting Beoordeling Ethiek Biomedisch Onderzoek (Assen, The Netherlands) approved the study prior to the start of any study procedure. The study was conducted in accordance with the Declaration of Helsinki, the International Council on Harmonisation guidelines for Good Clinical Practice, and applicable laws and regulations in The Netherlands.

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter’s central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13- propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2 , earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.

BackgroundM5717 is the first plasmodium translation elongation factor 2 inhibitor to reach clinical development as an antimalarial. We aimed to characterise the safety, pharmacokinetics, and antimalarial activity of M5717 in healthy volunteers.MethodsThis first-in-human study was a two-part, single-centre clinical trial done in Brisbane, QLD, Australia. Part one was a double-blind, randomised, placebo-controlled, single ascending dose study in which participants were enrolled into one of nine dose cohorts (50, 100, 200, 400, 600, 1000, 1250, 1800, or 2100 mg) and randomly assigned (3:1) to M5717 or placebo. A sentinel dosing strategy was used for each dose cohort whereby two participants (one assigned to M5717 and one assigned to placebo) were initially randomised and dosed. Randomisation schedules were generated electronically by independent, unblinded statisticians. Part two was an open-label, non-randomised volunteer infection study using the Plasmodium falciparum induced blood-stage malaria model in which participants were enrolled into three dose cohorts. Healthy men and women of non-childbearing potential aged 18–55 years were eligible for inclusion; individuals in the volunteer infection study were required to be malaria naive. Safety and tolerability (primary outcome of the single ascending dose study and secondary outcome of the volunteer infection study) were assessed by frequency and severity of adverse events. The pharmacokinetic profile of M5717 was also characterised (primary outcome of the volunteer infection study and secondary outcome of the single ascending dose study). Parasite clearance kinetics (primary outcome of the volunteer infection study) were assessed by the parasite reduction ratio and the corresponding parasite clearance half-life; the incidence of recrudescence up to day 28 was determined (secondary outcome of the volunteer infection study). Recrudescent parasites were tested for genetic mutations (exploratory outcome). The trial is registered with ClinicalTrials.gov (NCT03261401).FindingsBetween Aug 28, 2017, and June 14, 2019, 221 individuals were assessed for eligibility, of whom 66 men were enrolled in the single ascending dose study (eight per cohort for 50–1800 mg cohorts, randomised three M5717 to one placebo, and two in the 2100 mg cohort, randomised one M5717 to one placebo) and 22 men were enrolled in the volunteer infection study (six in the 150 mg cohort and eight each in the 400 mg and 800 mg cohorts). No adverse event was serious; all M5717-related adverse events were mild or moderate in severity and transient, with increased frequency observed at doses above 1250 mg. In the single ascending dose study, treatment-related adverse events occurred in three of 17 individuals in the placebo group; no individual in the 50 mg, 100 mg, or 200 mg groups; one of six individuals in each of the 400 mg, 1000 mg, and 1250 mg groups; two of six individuals in the 600 mg group; and in all individuals in the 1800 mg and 2100 mg groups. In the volunteer infection study, M5717-related adverse events occurred in no participants in the 150 mg or 800 mg groups and in one of eight participants in the 400 mg group. Transient oral hypoesthesia (in three participants) and blurred vision (in four participants) were observed in the 1800 mg or 2100 mg groups and constituted an unknown risk; thus, further dosing was suspended after dosing of the two sentinel individuals in the 2100 mg cohort. Maximum blood concentrations occurred 1–7 h after dosing, and a long half-life was observed (146–193 h at doses ≥200 mg). Parasite clearance occurred in all participants and was biphasic, characterised by initial slow clearance lasting 35–55 h (half-life 231·1 h [95% CI 40·9 to not reached] for 150 mg, 60·4 h [38·6 to 138·6] for 400 mg, and 24·7 h [20·4 to 31·3] for 800 mg), followed by rapid clearance (half-life 3·5 h [3·1 to 4·0] for 150 mg, 3·9 h [3·3 to 4·8] for 400 mg, and 5·5 h [4·8 to 6·4] for 800 mg). Recrudescence occurred in three (50%) of six individuals dosed with 150 mg and two (25%) of eight individuals dosed with 400 mg. Genetic mutations associated with resistance were detected in four cases of parasite recrudescence (two individuals dosed with 150 mg and two dosed with 400 mg).InterpretationThe safety, pharmacokinetics, and antimalarial activity of M5717 support its development as a component of a single-dose antimalarial combination therapy or for malaria prophylaxis.FundingWellcome Trust and the healthcare business of Merck KGaA, Darmstadt, Germany.

The emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites, driven by K13 mutations, has led to widespread antimalarial treatment failure in Southeast Asia. In Africa, our genotyping of 3,299 isolates confirms the emergence of the K13 R561H variant in Rwanda and reveals the continuing dominance of wild-type K13 across 11 countries. We show that this mutation, along with M579I and C580Y, confers varying degrees of in vitro ART resistance in African parasites. C580Y and M579I cause substantial fitness costs, which may counter-select against their dissemination in high-transmission settings. We also define the impact of multiple K13 mutations on ART resistance and fitness in multiple Southeast Asian strains. ART susceptibility is unaltered upon editing point mutations in ferrodoxin or mdr2, earlier resistance markers. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

Antimalarial drug resistance in the Plasmodium falciparum parasite poses a constant challenge for drug development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early phases of development is essential in developing the targeted parasitological and clinical profile of the final drug product. Here, we have studied the combination of M5717, a P. falciparum translation elongation factor 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of in vitro isobolograms as well as in vivo studies in the P. falciparum severe combined immunodeficient (SCID) mouse model. We also analyzed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was able to suppress the selection of M5717-resistant mutants, as well as significantly delay the recrudescence of parasites both with suboptimal and optimal dosing regimens.

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide 1 . Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy 2 . Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite 3 – 9 . Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance 5 , but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively 6 , 9 , reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.

23 Antimalarial drug resistance in the Plasmodium falciparum parasite poses a constant challenge for drug 24 development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. 25 Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early 26 phases of development is essential in developing the targeted parasitological and clinical profile of the final drug 27 product. Here, we have studied the combination of M5717, a Plasmodium falciparum translation elongation factor 28 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of in vitro 29 isobolograms as well as in vivo studies in the P. falciparum severe combined immunodeficient (SCID) mouse 30 model. We also analysed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of 31 recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and 32 pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was 33 able to suppress the selection of M5717-resistant mutants as well as significantly delay the recrudescence of 34 parasites both with suboptimal and optimal dosing regimens. Here we describe the pharmacokinetic and pharmacodynamic properties of the combination of M5717 and pyronaridine, both in vitro through asexual blood-stage P. falciparum parasites as well as in vivo by means of the 73 well-established P. falciparum SCID mouse model. 20 The objective of the studies was to evaluate and describe the parasitological behaviour and pharmacokinetic parameters of the two compounds when used as a combination.

Our study defines the allelic distribution of pfcrt , an important mediator of multidrug resistance in Plasmodium falciparum , in Africa and Asia. We leveraged whole-genome sequence analysis and gene editing to demonstrate how current drug combinations can select different allelic variants of this gene and shape region-specific parasite population structures. We document the ability of PfCRT mutations to modulate parasite susceptibility to current antimalarials in dissimilar, pfcrt allele-specific ways. This study underscores the importance of actively monitoring pfcrt genotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.

The Articles by William Hamilton and colleagues1Hamilton WL Amato R van der Pluijm RW et al.Evolution and expansion of multidrug-resistant malaria in southeast Asia: a genomic epidemiology study.Lancet Infect Dis. 2019; 19: 943-951Summary Full Text Full Text PDF PubMed Scopus (102) Google Scholar and Rob van der Pluijm and colleagues2van der Pluijm RW Imwong M Chau NH et al.Determinants of dihydroartemisinin–piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological and genetic study.Lancet Infect Dis. 2019; 19: 952-961Summary Full Text Full Text PDF PubMed Scopus (122) Google Scholar illustrate the plummeting clinical efficacy of dihydroartemisinin–piperaquine as a first-line treatment for Plasmodium falciparum malaria in southeast Asia. These authors also report a rapid regional spread of clonal parasite lineages harbouring novel variants of the P falciparum chloroquine resistance transporter PfCRT (emerging on the chloroquine-resistant Dd2 isoform). These lineages exclusively harboured the Cys580Tyr mutation in the K13 gene that is associated with decreased artemisinin efficacy. These studies raise important questions about whether these novel PfCRT variants cause piperaquine resistance, how they effect other antimalarials, and whether changes in prevalence over time reflect differences in parasite fitness. Here, we show that the now predominant PfCRT Thr93Ser and Ile218Phe mutations, which of all mutations have expanded the most rapidly in the past 5 years,1Hamilton WL Amato R van der Pluijm RW et al.Evolution and expansion of multidrug-resistant malaria in southeast Asia: a genomic epidemiology study.Lancet Infect Dis. 2019; 19: 943-951Summary Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 2van der Pluijm RW Imwong M Chau NH et al.Determinants of dihydroartemisinin–piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological and genetic study.Lancet Infect Dis. 2019; 19: 952-961Summary Full Text Full Text PDF PubMed Scopus (122) Google Scholar confer piperaquine resistance when individually edited into the pfcrt locus of Dd2 parasites. Assays with cultured intra-erythrocytic parasites show 8–13% survival at piperaquine concentrations ranging from 200 nM to 1600 nM (figure; appendix p 5). The Phe145Ile mutation was highly piperaquine-resistant, with 57–69% survival at these elevated concentrations, consistent with previous findings.3Witkowski B Duru V Khim N et al.A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype-genotype association study.Lancet Infect Dis. 2017; 17: 174-183Summary Full Text Full Text PDF PubMed Scopus (180) Google Scholar Parental or control Dd2 parasites with an edited pfcrt showed less than 1% survival, reflecting background rates. Quantitative PCR assays showed a single copy of plasmepsin II in all edited parasites (data not shown), indicating that amplification of this initial resistance marker4Amato R Lim P Miotto O et al.Genetic markers associated with dihydroartemisinin-piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype-phenotype association study.Lancet Infect Dis. 2017; 17: 164-173Summary Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 5Ross LS Dhingra SK Mok S et al.Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.Nat Commun. 2018; 93314Crossref PubMed Scopus (93) Google Scholar was not required for piperaquine resistance. Whole-genome sequence data of 84 Cambodian isolates support the expansion of Thr93Ser and Ile218Phe between 2013 and 2016, overtaking the prevalence of Phe145Ile (appendix p 4). Recent years showed a reduced percentage of parasites harbouring four or more copies of plasmepsins II and III, although parasites with two to three copies remained the majority (appendix p 4). pfmdr1 amplification, a marker of reduced susceptibility to lumefantrine and mefloquine, became less common over time (appendix p 4). Survival rates of piperaquine-treated cultured parasites increased over the years (appendix p 4), mirroring increasing dihydroartemisinin–piperaquine clinical failure rates. Emerging PfCRT mutations also increased P falciparum susceptibility to chloroquine, amo-​diaquine, quinine, pyronaridine, and ferroquine, with the Phe145Ile mutation causing the greatest sensitisation (appendix pp 6, 7). Dihydroartemisinin, lumefantrine, and mefloquine were unchanged. These data highlight the broad effect of PfCRT mutations on multiple antimalarials. To test for differences in parasite fitness between mutants, we used a competitive growth rate assay in which each parasite line was individually cocultured with an isogenic green fluorescent protein (GFP)-positive Dd2 line. Parasites expressing the Thr93Ser allele showed a negligible fitness cost compared with control Dd2 pfcrt-edited parasites, with both lines out-proliferating GFP-positive Dd2 parasites (figure). The Ile218Phe mutation showed a mild growth attenuation. Phe145Ile parasites showed a substantial fitness cost, potentially explaining why this allele is ceding ground to the less resistant but fitter Thr93Ser and Ile218Phe mutations. The data support a key role for PfCRT mutations in driving the recent expansion of highly piperaquine-resistant parasites in southeast Asia and highlight the need for vigilance in screening for novel PfCRT mutations in other malaria-endemic regions, notably in Africa or South America where piperaquine use has been increasing. We declare no competing interests. Download .pdf (1.49 MB) Help with pdf files Evolution and expansion of multidrug-resistant malaria in southeast Asia: a genomic epidemiology studyAfter emerging and circulating for several years within Cambodia, the P falciparum KEL1/PLA1 co-lineage diversified into multiple subgroups and acquired new genetic features, including novel crt mutations. These subgroups have rapidly spread into neighbouring countries, suggesting enhanced fitness. These findings highlight the urgent need for elimination of this increasingly drug-resistant parasite co-lineage, and the importance of genetic surveillance in accelerating malaria elimination efforts. Full-Text PDF Open Access