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Malaria is a critical public health issue in the tropical world, causing extensive morbidity and mortality. Infection by unicellular, obligate intracellular Plasmodiumparasites causes malaria. The emergence of resistance to current antimalarial drugs necessitates the development of novel therapeutics. A potential novel drug target is the purine import transporter. Because Plasmodiumparasites are purine auxotrophic, they must import purines from their host to fulfill metabolic requirements. They import purines via equilibrative nucleoside transporter 1 (ENT1) homologs. Recently, we used a yeast-based high-throughput screen to identify inhibitors of the P. falciparumENT1 (PfENT1) that kill P. falciparumparasites in culture. P. bergheiinfection of mice is an animal model for human malaria. Because P. bergheiENT1 (PbENT1) shares only 60% amino acid sequence identity with PfENT1, we sought to characterize PbENT1 and its sensitivity to our PfENT1 inhibitors. We expressed PbENT1 in purine auxotrophic yeast and used radiolabeled substrate uptake to characterize its function. We showed that PbENT1 transports both purines and pyrimidines. It preferred nucleosides compared with nucleobases. Inosine (IC50= 3.7 µM) and guanosine (IC50= 21.3 µM) had the highest affinities. Our recently discovered PfENT1 inhibitors were equally effective against both PbENT1- and PfENT1-mediated purine uptake. The PfENT1 inhibitors are at least 10-fold more potent against PfENT1 than human hENT1. They kill P. bergheiparasites in 24-hour ex vivo culture. Thus, the P. bergheimurine malaria model may be useful to evaluate the efficacy of PfENT1 inhibitors in vivo and their therapeutic potential for treatment of malaria.

Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparumis primarily conferred by mutations in the “chloroquine resistance transporter” (PfCRT). The resistance-conferring form of PfCRT (PfCRTCQR) mediates CQ resistance by effluxing the drug from the parasite’s digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRTCQRcan also decrease the parasite’s susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRTCQRand a series of dimeric quinine molecules using a Xenopus laevisoocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H+ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1and 6were also examined in vitro(against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo(against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRTCQRreported to date. Furthermore, the lead compounds (1and 6) were not effluxed by PfCRTCQRfrom the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1and 6exhibited in vitroantiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1and 6in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRTCQR. This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.

ABSTRACTThe increasing prevalence in Southeast Asia of Plasmodium falciparuminfections with delayed parasite clearance rates, following treatment of malaria patients with the artemisinin derivative artesunate, highlights an urgent need to identify which of the currently available artemisinin-based combination therapies (ACTs) are most suitable to treat populations with emerging artemisinin resistance. Here, we demonstrate that the rodent Plasmodium bergheiSANA strain has acquired artemisinin resistance following drug pressure, as defined by reduced parasite clearance and early recrudescence following daily exposure to high doses of artesunate or the active metabolite dihydroartemisinin. Using the SANA strain and the parental drug-sensitive N strain, we have interrogated the antimalarial activity of five ACTs, namely, artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, dihydroartemisinin-piperaquine, and the newest combination artesunate-pyronaridine. By monitoring parasitemia and outcome for 30 days following initiation of treatment, we found that infections with artemisinin-resistant P. bergheiSANA parasites can be successfully treated with artesunate-pyronaridine used at doses that are curative for the parental drug-sensitive N strain. No other partner drug combination was as effective in resolving SANA infections. Of the five partner drugs tested, pyronaridine was also the most effective at suppressing the recrudescence of SANA parasites. These data support the potential benefit of implementing ACTs with pyronaridine in regions affected by artemisinin-resistant malaria.

ABSTRACTEvidence of emerging Plasmodium falciparumresistance to artemisinin-based combination therapies, documented in western Cambodia, underscores the continuing need to identify new antimalarial combinations. Given recent reports of the resurgence of chloroquine-sensitive P. falciparumparasites in Malawi, after the enforced and prolonged withdrawal of this drug, and indications of a possible synergistic interaction with the macrolide azithromycin, we sought to further characterize chloroquine-azithromycin combinations for their in vitroand in vivoantimalarial properties. In vitro96-h susceptibility testing of chloroquine-azithromycin combinations showed mostly additive interactions against freshly cultured P. falciparumfield isolates obtained from Mali. Some evidence of synergy, however, was apparent at the fractional 90% inhibitory concentration level. Additional in vitrotesting highlighted the resistance reversal properties of amlodipine for both chloroquine and quinine. In vivoexperiments, using the Peters 4-day suppressive test in a P. yoeliimouse model, revealed up to 99.9% suppression of parasitemia following treatment with chloroquine-azithromycin plus the Renantiomer of amlodipine. This enantiomer was chosen because it does not manifest the cardiac toxicities observed with the racemic mixture. Pharmacokinetic/pharmacodynamic analyses in this rodent model and subsequent extrapolation to a 65-kg adult led to the estimation that 1.8 g daily of R-amlodipine would be required to achieve similar efficacy in humans, for whom this is likely an unsafe dose. While these data discount amlodipine as an additional partner for chloroquine-based combination therapy, our studies continue to support azithromycin as a safe and effective addition to antimalarial combination therapies.

The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

ABSTRACTThe microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculideveloping spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

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, pfcrtallele-specific ways. This study underscores the importance of actively monitoring pfcrtgenotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.

ABSTRACTCurrent efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparumresistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates.IMPORTANCEThe global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium. Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparumresistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparumchloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance.

MMV390048, a member of a new class of inhibitors of the Plasmodiumphosphatidylinositol 4-kinase, shows potential for both treatment and prophylaxis.