The role of Plasmodium V-ATPase in vacuolar physiology and antimalarial drug uptake.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs.

The structure of Plasmodium falciparum multidrug resistance protein 1 reveals an N-terminal regulatory domain.

Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), an adenosine triphosphate (ATP)-binding cassette (ABC) transporter on the digestive vacuole (DV) membrane of the parasite, is associated with the resistance to antimalarial drugs. To understand the mechanisms of PfMDR1, we determined the cryo-electron microscopy structures of this transporter in different states. The transporter in the apo state shows an inward-facing conformation with a large cavity opening to the cytoplasm. Upon ATP binding and dimerization of the nucleotide-binding domains (NBDs), PfMDR1 displays an outward-facing conformation with a cavity toward the DV lumen. Drug resistance-associated mutations were investigated in both structures for their effects, and Y184F was identified as an allosteric activity-enhancing mutation. The amphiphilic substrate-binding site of PfMDR1 was revealed by the complex structure with the antimalarial drug mefloquine and confirmed by mutagenesis studies. Remarkably, a helical structure was found to hinder NBD dimerization and inhibit PfMDR1 activity. The location of this regulatory domain in the N terminus is different from the well-studied R domain in the internal linker region of other ABC transporter family members. The lack of the phosphorylation site of this domain also suggests a different regulation mechanism.

Clearing of hemozoin crystals in malaria parasites enables whole-cell STED microscopy.

Malaria is a devastating mosquito-borne parasitic disease that manifests when Plasmodium parasites replicate within red blood cells. During the development within the red blood cell, the parasite digests hemoglobin and crystalizes the otherwise toxic heme. The resulting hemozoin crystals limit imaging by STED nanoscopy owing to their high light-absorbing capacity, which leads to immediate cell destruction upon contact with the laser. Here, we establish CUBIC-P-based clearing of hemozoin crystals, enabling whole-cell STED nanoscopy of parasites within red blood cells. Hemozoin-cleared infected red blood cells could reliably be stained with antibodies, and hence proteins in the hemozoin-containing digestive vacuole membrane, as well as in secretory vesicles of gametocytes, could be imaged at high resolution. Thus, this process is a valuable tool to study and understand parasite biology and the potential molecular mechanisms mediating drug resistance. This article has an associated First Person interview with the first author of the paper.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites.

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs-modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

Amodiaquine Nonspecifically Binds Double Stranded and Three-Way Junction DNA Structures.

The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of µM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.

Inhibitors of ApiAP2 protein DNA binding exhibit multistage activity against Plasmodium parasites.

Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified putative AP2-EXP interacting compounds. Four compounds were found to block DNA binding by AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that interact with AP2 DNA binding domains. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.

GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.

Piperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages.

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.

Cladosporin, A Highly Potent Antimalaria Drug?

Cladosporin, a unique natural product from the fungus Cladosporium cladosporioides, exhibits nanomolar inhibitory activity against Plasmodium falciparum by targeting its cytosolic lysyl-tRNA synthetase (PfKRS) to inhibit protein biosynthesis. Due to its exquisite selectivity towards pathogenic parasites, cladosporin has become a very promising lead compound for developing antiparasitic drugs to treat drug-resistant malaria and cryptosporidiosis infections. Here we review the recent research progress of cladosporin covering aspects of the chemical synthesis, biosynthesis, bioactivity, cellular target and structure-activity relationship.

Key insights into secondary metabolites from various Chaetomium species.

Endophytic fungi have proved to be a major source of secondary metabolites, wherein the genus Chaetomium has emerged as a source of multifarious bioactive natural compounds belonging to diverse classes such as chaetoglobosins, epipolythiodioxopiperazines, azaphilones, xanthones, anthraquinone, chromones, depsidones, terpenoids, and steroids. The objective of this review is to encapsulate recent findings on various Chaetomium strains, such as C. globosum, C. cupreum, C. elatum, C. subspirale, C. olivaceum, C. indicum, and C. nigricolor known for production of beneficial secondary metabolites, with an insight into their origin and function. A thorough literature survey was conducted for obtaining Chaetomium-derived secondary metabolites, with a scope of future application into drug development efforts. More than 100 secondary metabolites, with various beneficial properties such as antitumor, cytotoxic, antimalarial, and enzyme inhibitory activities, were enlisted. We believe this review will enhance the understanding of beneficial effects conferred by various Chaetomium-derived secondary metabolites and emphasize their potential in serving novel drug development efforts. KEY POINTS: • Identified Chaetomium-derived metabolites with potential for drug development. • More than 100 beneficial metabolites are enlisted. • Benefits include anti-cancerous, antimalarial, and anti-enzymatic properties.

Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage.

Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B "pauses" the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.

Biotechnological Approaches for Production of Artemisinin, an Anti-Malarial Drug from Artemisia annua L.

Artemisinin is an anti-malarial sesquiterpene lactone derived from Artemisia annua L. (Asteraceae family). One of the most widely used modes of treatment for malaria is an artemisinin-based combination therapy. Artemisinin and its associated compounds have a variety of pharmacological qualities that have helped achieve economic prominence in recent years. So far, research on the biosynthesis of this bioactive metabolite has revealed that it is produced in glandular trichomes and that the genes responsible for its production must be overexpressed in order to meet demand. Using biotechnological applications such as tissue culture, genetic engineering, and bioreactor-based approaches would aid in the upregulation of artemisinin yield, which is needed for the future. The current review focuses on the tissue culture aspects of propagation of A. annua and production of artemisinin from A. annua L. cell and organ cultures. The review also focuses on elicitation strategies in cell and organ cultures, as well as artemisinin biosynthesis and metabolic engineering of biosynthetic genes in Artemisia and plant model systems.

The Antagonizing Role of Heme in the Antimalarial Function of Artemisinin: Elevating Intracellular Free Heme Negatively Impacts Artemisinin Activity in Plasmodium falciparum.

The rich source of heme within malarial parasites has been considered to underly the action specificity of artemisinin. We reasoned that increasing intraparasitic free heme levels might further sensitize the parasites to artemisinin. Various means, such as modulating heme synthesis, degradation, polymerization, or hemoglobin digestion, were tried to boost intracellular heme levels, and under several scenarios, free heme levels were significantly augmented. Interestingly, all results arrived at the same conclusion, i.e., elevating heme acted in a strongly negative way, impacting the antimalarial action of artemisinin, but exerted no effect on several other antimalarial drugs. Suppression of the elevated free heme level by introducing heme oxygenase expression effectively restored artemisinin potency. Consistently, zinc protoporphyrin IX/zinc mesoporphyrin, as analogues of heme, drastically increased free heme levels and, concomitantly, the EC50 values of artemisinin. We were unable to effectively mitigate free heme levels, possibly due to an unknown compensating heme uptake pathway, as evidenced by our observation of efficient uptake of a fluorescent heme homologue by the parasite. Our results thus indicate the existence of an effective and mutually compensating heme homeostasis network in the parasites, including an uncharacterized heme uptake pathway, to maintain a certain level of free heme and that augmentation of the free heme level negatively impacts the antimalarial action of artemisinin. Importance: It is commonly believed that heme is critical in activating the antimalarial action of artemisinins. In this work, we show that elevating free heme levels in the malarial parasites surprisingly negatively impacts the action of artemisinin. We tried to boost free heme levels with various means, such as by modulating heme synthesis, heme polymerization, hemoglobin degradation and using heme analogues. Whenever we saw elevation of free heme levels, reduction in artemisinin potency was also observed. The homeostasis of heme appears to be complex, as there exists an unidentified heme uptake pathway in the parasites, nullifying our attempts to effectively reduce intraparasitic free heme levels. Our results thus indicate that too much heme is not good for the antimalarial action of artemisinins. This research can help us better understand the biological properties of this mysterious drug.

Malaria parasite plasmepsins: More than just plain old degradative pepsins.

Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion, and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some (e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsin V and a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism.

Metabolic Retroversion of Piperaquine (PQ) via Hepatic Cytochrome P450-Mediated N-Oxidation and Reduction: Not an Important Contributor to the Prolonged Elimination of PQ.

As a partner antimalarial with an extremely long elimination half-life (∼30 days), piperaquine (PQ) is mainly metabolized into a pharmacologically active N-oxide metabolite [piperaquine N-oxide (PN1)] in humans. In the present work, the metabolic retroversion of PQ and PN1, potentially associated with decreased clearance of PQ, was studied. The results showed that interconversion existed for PQ and its metabolite PN1. The N-oxidation of PQ to PN1 was mainly mediated by CYP3A4, and PN1 can rapidly reduce back to PQ via cytochrome P450 (P450)/flavin-containing monooxygenase enzymes. In accordance with these findings, the P450 nonselective inhibitor (1-ABT) or CYP3A4 inhibitor (ketoconazole) inhibited the N-oxidation pathway in liver microsomes (>90%), and the reduction metabolism was inhibited by 1-ABT (>90%) or methimazole (∼50%). Based on in vitro physiologic and enzyme kinetic studies, quantitative prediction of hepatic clearance (CLH) of PQ was performed, which indicated its negligible decreased elimination in humans in the presence of futile cycling, with the unbound CLH decreasing by 2.5% (0.069 l/h per kilogram); however, a minor decrease in unbound CLH (by 12.8%) was found in mice (0.024 l/h per kilogram). After an oral dose of PQ (or PN1) to mice, the parent form predominated in the blood circulation, and PN1 (or PQ) was detected as a major metabolite. Other factors probably associated with delayed elimination of PQ (intestinal metabolism and enterohepatic circulation) did not play a key role in PQ elimination. These data suggested that the metabolic interconversion of PQ and its N-oxide metabolite contributes to but may not significantly prolong its duration in humans. SIGNIFICANCE STATEMENT: This paper investigated the interconversion metabolism of piperaquine (PQ) and its N-oxide metabolite in vitro as well as in mice. The metabolic profiles of PQ were reestablished by this futile cycling, which contributes to but may not significantly prolong its elimination in humans. Enzyme phenotyping indicated a low possibility of interaction of PQ during artemisinin drug-based combination therapy treatment.

Balancing glucose and oxygen uptake rates to enable high amorpha-4,11-diene production in Escherichia coli via the methylerythritol phosphate pathway.

Amorpha-4,11-diene (AMD4,11) is a precursor to artemisinin, a potent antimalarial drug that is traditionally extracted from the shrubs of Artemisia annua. Despite significant prior efforts to produce artemisinin and its precursors through biotechnology, there remains a dire need for more efficient biosynthetic routes for its production. Here, we describe the optimization of key process conditions for an Escherichia coli strain producing AMD4,11 via the native methylerythritol phosphate (MEP) pathway. By studying the interplay between glucose uptake rates and oxygen demand, we were able to identify optimal conditions for increasing carbon flux through the MEP pathway by manipulating the availability of NADPH required for terpenoid production. Installation of an optimal qO2 /qglucose led to a 6.7-fold increase in product titers and a 6.5-fold increase in carbon yield.

Structure guided development of potent piperazine-derived hydroxamic acid inhibitors targeting falcilysin.

The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria and is estimated to kill 400,000 people a year. The parasite infects and replicates in host red blood cells (RBCs), where it expresses an array of proteases to carry out multiple essential processes. We are investigating the function of falcilysin (FLN), a protease known to be required for parasite development in the RBC. We previously developed a piperazine-based hydroxamic acid scaffold to generate the first inhibitors of FLN, and the current study reports the optimization of the lead compound from that series. A range of substituents were tested at the N1 and N4 positions of the piperazine core, and inhibitors with significantly improved potency against purified FLN and cultured P. falciparum were identified. Computational studies were also performed to understand the mode of binding for these compounds, and predicted a binding model consistent with the biochemical data and the distinctive SAR observed at both the N1 and N4 positions.

Multiple mutations in RNA polymerase ß-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) ß-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.