Detailing organelle division and segregation in Plasmodium falciparum.

The malaria causing parasite, Plasmodium falciparum , replicates through a tightly orchestrated process termed schizogony, where approximately 32 daughter parasites are formed in a single infected red blood cell and thousands of daughter cells in mosquito or liver stages. One-per-cell organelles, such as the mitochondrion and apicoplast, need to be properly divided and segregated to ensure a complete set of organelles per daughter parasites. Although this is highly essential, details about the processes and mechanisms involved remain unknown. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D-imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during the last stage of schizogony. Analysis of focused ion beam scanning electron microscopy (FIB-SEM) data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques (CPs) in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation.

Unique Properties of Apicomplexan Mitochondria.

Apicomplexan parasites constitute more than 6,000 species infecting a wide range of hosts. These include important pathogens such as those causing malaria and toxoplasmosis. Their evolutionary emergence coincided with the dawn of animals. Mitochondrial genomes of apicomplexan parasites have undergone dramatic reduction in their coding capacity, with genes for only three proteins and ribosomal RNA genes present in scrambled fragments originating from both strands. Different branches of the apicomplexans have undergone rearrangements of these genes, with Toxoplasma having massive variations in gene arrangements spread over multiple copies. The vast evolutionary distance between the parasite and the host mitochondria has been exploited for the development of antiparasitic drugs, especially those used to treat malaria, wherein inhibition of the parasite mitochondrial respiratory chain is selectively targeted with little toxicity to the host mitochondria. We describe additional unique characteristics of the parasite mitochondria that are being investigated and provide greater insights into these deep-branching eukaryotic pathogens.

Metabolic responses in blood-stage malaria parasites associated with increased and decreased sensitivity to PfATP4 inhibitors.

BACKGROUND: Spiroindolone and pyrazoleamide antimalarial compounds target Plasmodium falciparum P-type ATPase (PfATP4) and induce disruption of intracellular Na+ homeostasis. Recently, a PfATP4 mutation was discovered that confers resistance to a pyrazoleamide while increasing sensitivity to a spiroindolone. Transcriptomic and metabolic adaptations that underlie this seemingly contradictory response of P. falciparum to sublethal concentrations of each compound were examined to understand the different cellular accommodation to PfATP4 disruptions. METHODS: A genetically engineered P. falciparum Dd2 strain (Dd2A211V) carrying an Ala211Val (A211V) mutation in PfATP4 was used to identify metabolic adaptations associated with the mutation that results in decreased sensitivity to PA21A092 (a pyrazoleamide) and increased sensitivity to KAE609 (a spiroindolone). First, sublethal doses of PA21A092 and KAE609 causing substantial reduction (30-70%) in Dd2A211V parasite replication were identified. Then, at this sublethal dose of PA21A092 (or KAE609), metabolomic and transcriptomic data were collected during the first intraerythrocytic developmental cycle. Finally, the time-resolved data were integrated with a whole-genome metabolic network model of P. falciparum to characterize antimalarial-induced physiological adaptations. RESULTS: Sublethal treatment with PA21A092 caused significant (p < 0.001) alterations in the abundances of 91 Plasmodium gene transcripts, whereas only 21 transcripts were significantly altered due to sublethal treatment with KAE609. In the metabolomic data, a substantial alteration (≥ fourfold) in the abundances of carbohydrate metabolites in the presence of either compound was found. The estimated rates of macromolecule syntheses between the two antimalarial-treated conditions were also comparable, except for the rate of lipid synthesis. A closer examination of parasite metabolism in the presence of either compound indicated statistically significant differences in enzymatic activities associated with synthesis of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. CONCLUSION: The results of this study suggest that malaria parasites activate protein kinases via phospholipid-dependent signalling in response to the ionic perturbation induced by the Na+ homeostasis disruptor PA21A092. Therefore, targeted disruption of phospholipid signalling in PA21A092-resistant parasites could be a means to block the emergence of resistance to PA21A092.

Transcriptional changes in Plasmodium falciparum upon conditional knock down of mitochondrial ribosomal proteins RSM22 and L23.

The mitochondrion of malaria parasites is an attractive antimalarial drug target, which require mitoribosomes to translate genes encoded in the mitochondrial (mt) DNA. Plasmodium mitoribosomes are composed of highly fragmented ribosomal RNA (rRNA) encoded in the mtDNA. All mitoribosomal proteins (MRPs) and other assembly factors are encoded in the nuclear genome. Here, we have studied one putative assembly factor, RSM22 (Pf3D7_1027200) and one large subunit (LSU) MRP, L23 (Pf3D7_1239100) in Plasmodium falciparum. We show that both proteins localize to the mitochondrion. Conditional knock down (KD) of PfRSM22 or PfMRPL23 leads to reduced cytochrome bc1 complex activity and increased sensitivity to bc1 inhibitors such as atovaquone and ELQ-300. Using RNA sequencing as a tool, we reveal the transcriptomic changes of nuclear and mitochondrial genomes upon KD of these two proteins. In the early phase of KD, while most mt rRNAs and transcripts of putative MRPs were downregulated in the absence of PfRSM22, many mt rRNAs and several MRPs were upregulated after KD of PfMRPL23. The contrast effects in the early phase of KD likely suggests non-redundant roles of PfRSM22 and PfMRPL23 in the assembly of P. falciparum mitoribosomes. At the late time points of KD, loss of PfRSM22 and PfMRPL23 caused defects in many essential metabolic pathways and transcripts related to essential mitochondrial functions, leading to parasite death. In addition, we enlist mitochondrial proteins of unknown function that are likely novel Plasmodium MRPs based on their structural similarity to known MRPs as well as their expression profiles in KD parasites.

Mitochondrially targeted proximity biotinylation and proteomic analysis in Plasmodium falciparum.

Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.

Dramatic Consequences of Reducing Erythrocyte Membrane Cholesterol on Plasmodium falciparum.

Cholesterol is the most abundant lipid in the erythrocyte. During its blood-stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in the intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-ß-cyclodextrin (MßCD) was shown to inhibit parasite invasion and growth. In addition, MßCD treatment of trophozoite-stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that MßCD treatment for just 30 min causes dramatic expulsion of the trophozoite-stage parasites. This forceful expulsion occurs within 10 s. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented MßCD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of the infected erythrocyte plasma membrane. IMPORTANCE Malaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBCs), leading to their destruction. The parasite depends upon its host for much of its nutritional needs. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malaria parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here, we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in the physiology of malaria parasites.

Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure.

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol-a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

Atypical Molecular Basis for Drug Resistance to Mitochondrial Function Inhibitors in Plasmodium falciparum.

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc1 complex, which is an essential component of the mitochondrial electron transport chain (mtETC) required for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc1 was readily attained, resulting in a parasite strain (SB1-A6) that was panresistant to both mtETC and DHODH inhibitors. Here, we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line, which lacks the common cyt bc1 point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation (∼2×) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified single nucleotide polymorphism (SNP) using CRISPR-Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 and atovaquone resistance, SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the panresistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc1 complex or de novo pyrimidine synthesis that could help guide future antimalarial combination therapies and reduce the rapid development of drug resistance in the field.

Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages.

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite's susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.

Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis.

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Diverse Chemical Compounds Target Plasmodium falciparum Plasma Membrane Lipid Homeostasis.

Lipid homeostasis is essential to the maintenance of life. We previously reported that disruptions of the parasite Na+ homeostasis via inhibition of PfATP4 resulted in elevated cholesterol within the parasite plasma membrane as assessed by saponin sensitivity. A large number of compounds have been shown to target the parasite Na+ homeostasis. We screened 800 compounds from the Malaria and Pathogen Boxes to identify chemotypes that disrupted the parasite plasma membrane lipid homeostasis. Here, we show that the compounds disrupting parasite Na+ homeostasis also induced saponin sensitivity, an indication of parasite lipid homeostasis disruption. Remarkably, 13 compounds were identified that altered the plasma membrane lipid composition independently of the Na+ homeostasis disruption. Further studies suggest that these compounds target the Plasmodium falciparum Niemann-Pick type C1-related (PfNCR1) protein, which is hypothesized to be involved in maintaining plasma membrane lipid composition. PfNCR1, like PfATP4, appears to be targeted by multiple chemotypes with the potential for drug discovery.

The mitochondrial ribosomal protein L13 is critical for the structural and functional integrity of the mitochondrion in Plasmodium falciparum.

The phylum Apicomplexa contains a group of protozoa causing diseases in humans and livestock. Plasmodium spp., the causative agent of malaria, contains a mitochondrion that is very divergent from that of their hosts. The malarial mitochondrion is a clinically validated target for the antimalarial drug atovaquone, which specifically blocks the electron transfer activity of the bc1 complex of the mitochondrial electron transport chain (mtETC). Most mtETC proteins are nuclear-encoded and imported from the cytosol, but three key protein subunits are encoded in the Plasmodium mitochondrial genome: cyt b, COXI, and COXIII. They are translated inside the mitochondrion by mitochondrial ribosomes (mitoribosomes). Here, we characterize the function of one large mitoribosomal protein in Plasmodium falciparum, PfmRPL13. We found that PfmRPL13 localizes to the parasite mitochondrion and is refractory to genetic knockout. Ablation of PfmRPL13 using a conditional knockdown system (TetR-DOZI-aptamer) caused a series of adverse events in the parasite, including mtETC deficiency, loss of mitochondrial membrane potential (Δψm), and death. The PfmRPL13 knockdown parasite also became hypersensitive to proguanil, a drug proposed to target an alternative process for maintaining Δψm Surprisingly, transmission EM revealed that PfmRPL13 disruption also resulted in an unusually elongated and branched mitochondrion. The growth arrest of the knockdown parasite could be rescued with a second copy of PfmRPL13, but not by supplementation with decylubiquinone or addition of a yeast dihydroorotate dehydrogenase gene. In summary, we provide first and direct evidence that mitoribosomes are essential for malaria parasites to maintain the structural and functional integrity of the mitochondrion.

Alkoxycarbonate Ester Prodrugs of Preclinical Drug Candidate ELQ-300 for Prophylaxis and Treatment of Malaria.

ELQ-300 is a preclinical antimalarial drug candidate that is active against liver, blood, and transmission stages of Plasmodium falciparum. While ELQ-300 is highly effective when administered in a low multidose regimen, poor aqueous solubility and high crystallinity have hindered its clinical development. To overcome its challenging physiochemical properties, a number of bioreversible alkoxycarbonate ester prodrugs of ELQ-300 were synthesized. These bioreversible prodrugs are converted to ELQ-300 by host and parasite esterase action in the liver and bloodstream of the host. One such alkoxycarbonate prodrug, ELQ-331, is curative against Plasmodium yoelii with a single low dose of 3 mg/kg in a murine model of patent malaria infection. ELQ-331 is at least as fully protective as ELQ-300 in a murine malaria prophylaxis model when delivered 24 h before sporozoite inoculation at an oral dose of 1 mg/kg. Here, we show that ELQ-331 is a promising prodrug of ELQ-300 with improved physiochemical and metabolic properties and excellent potential for clinical formulation.

Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes.

The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.

Caged Garcinia Xanthones, a Novel Chemical Scaffold with Potent Antimalarial Activity.

Caged Garcinia xanthones (CGXs) constitute a family of natural products that are produced by tropical/subtropical trees of the genus Garcinia CGXs have a unique chemical architecture, defined by the presence of a caged scaffold at the C ring of a xanthone moiety, and exhibit a broad range of biological activities. Here we show that synthetic CGXs exhibit antimalarial activity against Plasmodium falciparum, the causative parasite of human malaria, at the intraerythrocytic stages. Their activity can be substantially improved by attaching a triphenylphosphonium group at the A ring of the caged xanthone. Specifically, CR135 and CR142 were found to be highly effective antimalarial inhibitors, with 50% effective concentrations as low as ∼10 nM. CGXs affect malaria parasites at multiple intraerythrocytic stages, with mature stages (trophozoites and schizonts) being more vulnerable than immature rings. Within hours of CGX treatment, malaria parasites display distinct morphological changes, significant reduction of parasitemia (the percentage of infected red blood cells), and aberrant mitochondrial fragmentation. CGXs do not, however, target the mitochondrial electron transport chain, the target of the drug atovaquone and several preclinical candidates. CGXs are cytotoxic to human HEK293 cells at the low micromolar level, which results in a therapeutic window of around 150-fold for the lead compounds. In summary, we show that CGXs are potent antimalarial compounds with structures distinct from those of previously reported antimalarial inhibitors. Our results highlight the potential to further develop Garcinia natural product derivatives as novel antimalarial agents.

Antiparasitic and disease-modifying activity of Nyctanthes arbor-tristis Linn. in malaria: An exploratory clinical study.

BACKGROUND: An unceasing threat of drug resistance continuously poses demand for new antimalarial drugs. A scientific assessment of traditionally used antimalarial plants through reverse pharmacology is crucial for a fast track drug discovery. An Ayurvedic plant Nyctanthes arbor-tristis Linn. - (Parijat) is being used in clinical practice and had shown antimalarial activity, with a parasite clearance in 76.6% of 120 patients, in an earlier clinical study. OBJECTIVE: To further explore antimalarial potential of the plant through additional objective markers. MATERIALS AND METHODS: An open-labelled observational study was conducted at M.A. Podar Hospital - Ayurveda (MAPH-A) after ethics committee approval. Administration of a paste of 5 fresh leaves, thrice a day for a week was a standard practice for management of malaria at MAPH-A. Clinical activity of N. arbor-tristis was evaluated by monitoring pyrexia, parasitemia and morbidity score (MS) in twenty patients. In addition, immune and biochemical markers and organ functions were monitored for objective markers of response. Student's paired-'t' test was applied to assess statistical significance. RESULTS: Ten out of 20 patients showed both fever and parasite clearance, which was confirmed by polymerase chain reaction. Remaining ten patients had persistent but decreasing parasitemia. Four of them needed chloroquine as a fail-safe procedure. Irrespective of the degree of parasitemia all the patients showed decrease in MS. There was also an increase in platelet count and normalization of plasma lactic acid. There was a good clinical tolerability and an improvement in organ function. The inflammatory cytokines showed a reduction; particularly in TNF-α within a day. CONCLUSIONS: At the given dosage, N. arbor-tristis showed disease-modifying activity; early clinical recovery with a decline of TNF-α and a gradual parasite clearance. Further studies with a standardised formulation for dose-searching and optimizing the treatment schedule are needed in a larger sample size. CLINICAL TRIAL REGISTRATION NO: The process of trial registration had not begun when the study was conducted in 2000.

Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria.

Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV.