Artemisinin resistance in the malaria parasite, Plasmodium falciparum, originates from its initial transcriptional response.

The emergence and spread of artemisinin-resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria elimination ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016-2018. A specific artemisinin resistance-associated transcriptional profile was identified that involves a broad but discrete set of biological functions related to proteotoxic stress, host cytoplasm remodelling, and REDOX metabolism. The artemisinin resistance-associated transcriptional profile evolved from initial transcriptional responses of susceptible parasites to artemisinin. The genetic basis for this adapted response is likely to be complex.

Malaria Parasite Metabolic Pathways (MPMP) Upgraded with Targeted Chemical Compounds.

Malaria Parasite Metabolic Pathways (MPMP) is the website for the functional genomics of intraerythrocytic Plasmodium falciparum. All the published information about targeted chemical compounds has now been added. Users can find the drug target and publication details linked to a drug database for further information about the medicinal properties of each compound.

Antiplasmodial properties of acyl-lysyl oligomers in culture and animal models of malaria.

Our previous analysis of antiplasmodial properties exhibited by dodecanoyl-based oligo-acyl-lysyls (OAKs) has outlined basic attributes implicated in potent inhibition of parasite growth and underlined the critical role of excess hydrophobicity in hemotoxicity. To dissociate hemolysis from antiplasmodial effect, we screened >50 OAKs for in vitro growth inhibition of Plasmodium falciparum strains, thus revealing the minimal requirements for antiplasmodial potency in terms of sequence and composition, as confirmed by efficacy studies in vivo. The most active sequence, dodecanoyllysyl-bis(aminooctanoyllysyl)-amide (C(12)K-2α(8)), inhibited parasite growth at submicromolar concentrations (50% inhibitory concentration [IC(50)], 0.3 ± 0.1 µM) and was devoid of hemolytic activity (<0.4% hemolysis at 150 µM). Unlike the case of dodecanoyl-based analogs, which equally affect ring and trophozoite stages of the parasite developmental cycle, the ability of various octanoyl-based OAKs to distinctively affect these stages (rings were 4- to 5-fold more sensitive) suggests a distinct antiplasmodial mechanism, nonmembranolytic to host red blood cells (RBCs). Upon intraperitoneal administration to mice, C(12)K-2α(8) demonstrated sustainable high concentrations in blood (e.g., 0.1 mM at 25 mg/kg of body weight). In Plasmodium vinckei-infected mice, C(12)K-2α(8) significantly affected parasite growth (50% effective dose [ED(50)], 22 mg/kg) but also caused mortality in 2/3 mice at high doses (50 mg/kg/day × 4).

A call for using natural compounds in the development of new antimalarial treatments - an introduction.

Natural compounds, mostly from plants, have been the mainstay of traditional medicine for thousands of years. They have also been the source of lead compounds for modern medicine, but the extent of mining of natural compounds for such leads decreased during the second half of the 20th century. The advantage of natural compounds for the development of drugs derives from their innate affinity for biological receptors. Natural compounds have provided the best anti-malarials known to date. Recent surveys have identified many extracts of various organisms (mostly plants) as having antiplasmodial activity. Huge libraries of fractionated natural compounds have been screened with impressive hit rates. Importantly, many cases are known where the crude biological extract is more efficient pharmacologically than the most active purified compound from this extract. This could be due to synergism with other compounds present in the extract, that as such have no pharmacological activity. Indeed, such compounds are best screened by cell-based assay where all potential targets in the cell are probed and possible synergies identified. Traditional medicine uses crude extracts. These have often been shown to provide many concoctions that deal better with the overall disease condition than with the causative agent itself. Traditional medicines are used by ~80 % of Africans as a first response to ailment. Many of the traditional medicines have demonstrable anti-plasmodial activities. It is suggested that rigorous evaluation of traditional medicines involving controlled clinical trials in parallel with agronomical development for more reproducible levels of active compounds could improve the availability of drugs at an acceptable cost and a source of income in malaria endemic countries.

Analysis of additivity and synergism in the anti-plasmodial effect of purified compounds from plant extracts.

In the search for antimalarials from ethnobotanical origin, plant extracts are chemically fractionated and biological tests guide the isolation of pure active compounds. To establish the responsibility of isolated active compound(s) to the whole antiplasmodial activity of a crude extract, the literature in this field was scanned and results were analysed quantitatively to find the contribution of the pure compound to the activity of the whole extract. It was found that, generally, the activity of isolated molecules could not account on their own for the activity of the crude extract. It is suggested that future research should take into account the "drugs beside the drug", looking for those products (otherwise discarded along the fractionation process) able to boost the activity of isolated active compounds.

Modulation of cerebral malaria by fasudil and other immune-modifying compounds.

Malaria continues to cause millions of deaths annually. No specific effective treatment has yet been found for cerebral malaria, one of the most severe complications of the disease. The pathology of cerebral malaria is considered to be primarily immunological. We examined a number of compounds with known effects on the immune system, in a murine model of cerebral malaria. Of the compounds tested, only fasudil and curcumin had significant effects on the progression of the disease. Although neither drug caused a reduction in parasitemia, survival of the treated mice was significantly increased, and the development of cerebral malaria was either delayed or prevented. Our results support the hypothesis that an immunomodulator efficient in preventing CM should be administered together with anti-plasmodial drugs to prevent severe malaria disease; curcumin and fasudil should be further investigated to determine efficiency and feasibility of treatment.

Membrane transport proteins of the malaria parasite.

The malaria parasite-infected erythrocyte is a multi-compartment structure, incorporating numerous different membrane systems. The movement of nutrients, metabolites and inorganic ions into and out of the intraerythrocytic parasite, as well as between subcellular compartments within the parasite, is mediated by transporters and channels - integral membrane proteins that facilitate the movement of solutes across the membrane bilayer. Proteins of this type also play a key role in antimalarial drug resistance. Genes encoding transporters and channels account for at least 2.5% of the parasite genome. However, ascribing functions and physiological roles to these proteins, and defining their roles in drug resistance, is not straightforward. For any given membrane transport protein, a full understanding of its role(s) in the parasitized erythrocyte requires a knowledge of its subcellular localization and substrate specificity, as well as some knowledge of the effects on the parasite of modifying the sequence and/or level of expression of the gene involved. Here we consider recent work in this area, describe a number of newly identified transport proteins, and summarize the likely subcellular localization and putative substrate specificity of all of the candidate membrane transport proteins identified to date.

The homeostasis of Plasmodium falciparum-infected red blood cells.

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

Caveat emptor: limitations of the automated reconstruction of metabolic pathways in Plasmodium.

The functional reconstruction of metabolic pathways from an annotated genome is a tedious and demanding enterprise. Automation of this endeavor using bioinformatics algorithms could cope with the ever-increasing number of sequenced genomes and accelerate the process. Here, the manual reconstruction of metabolic pathways in the functional genomic database of Plasmodium falciparum--Malaria Parasite Metabolic Pathways--is described and compared with pathways generated automatically as they appear in PlasmoCyc, metaSHARK and the Kyoto Encyclopedia for Genes and Genomes. A critical evaluation of this comparison discloses that the automatic reconstruction of pathways generates manifold paths that need an expert manual verification to accept some and reject most others based on manually curated gene annotation.

The transcriptome of Plasmodium vivax reveals divergence and diversity of transcriptional regulation in malaria parasites.

Plasmodium vivax causes over 100 million clinical infections each year. Primarily because of the lack of a suitable culture system, our understanding of the biology of this parasite lags significantly behind that of the more deadly species P. falciparum. Here, we present the complete transcriptional profile throughout the 48-h intraerythrocytic cycle of three distinct P. vivax isolates. This approach identifies strain specific patterns of expression for subsets of genes predicted to encode proteins associated with virulence and host pathogen interactions. Comparison to P. falciparum revealed significant differences in the expression of genes involved in crucial cellular functions that underpin the biological differences between the two parasite species. These data provide insights into the biology of P. vivax and constitute an important resource for the development of therapeutic approaches.

Pharmacogenomic analyses of targeting the AT-rich malaria parasite genome with AT-specific alkylating drugs.

UNLABELLED: Human malaria parasites, including the most lethal Plasmodium falciparum, are increasingly resistant to existing antimalarial drugs. One remarkable opportunity to selectively target P. falciparum stems from the unique AT-richness of its genome (80% A/T, relative to 60% in human DNA). To rationally explore this opportunity, we used drugs (adozelesin and bizelesin) which distinctly target AT-rich minisatellites and an in silico approach for genome-wide analysis previously experimentally validated in human cells [Woynarowski JM, Trevino AV, Rodriguez KA, Hardies SC, Benham CJ. AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs. J Biol Chem 2001;276:40555-66]. Both drugs demonstrate a potent, rapid and irreversible inhibition of the cultured P. falciparum (50% inhibition at 110 and 10+/-2.3 pM, respectively). This antiparasital activity reflects most likely drug binding to specific super-AT-rich regions. Relative to the human genome, the P. falciparum genome shows 3.9- and 7-fold higher frequency of binding sites for adozelesin and bizelesin, respectively. The distribution of these sites is non-random with the most prominent clusters found in large unique minisatellites [median size 3.5 kbp of nearly pure A/T, with multiple converging repeats but no shared consensus other than (A/T)(n)]. Each of the fourteen P. falciparum chromosomes contains only one such "super-AT island" located within approximately 3-7.5 kbp of gene-free and nucleosome-free loci. Important functions of super-AT islands are suggested by their exceptional predicted potential to serve as matrix attachment regions (MARs) and a precise co-localization with the putative centromeres. CONCLUSION: Super-AT islands, identified as unique domains in the P. falciparum genome with presumably crucial functions, offer therapeutically exploitable opportunity for new antimalarial strategies.

Electrophysiological studies of malaria parasite-infected erythrocytes: current status.

The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.

The evolution of the new permeability pathways in Plasmodium falciparum--infected erythrocytes--a kinetic analysis.

Malaria parasites demonstrably increase the permeability of the membrane of the erythrocyte in which they develop and propagate. New permeability pathways (NPPs) generated by parasite activity and identified in the erythrocyte membrane are held responsible for these changes. Here, we present a novel analysis of hemolysis curves of infected cells in iso-osmotic solutions of solutes that penetrate selectively into infected cells, as a function of parasite development. The analysis yields three parameters: the t(1/2) of lysis (reciprocally related to permeability), the maximal lysis, and a parameter that expresses the variation of the cell population. Different developmental stages of the parasite were obtained either by sampling synchronized cultures with time or by the fractionation of asynchronous cultures on a Percoll-sorbitol density gradient. While the results confirm previous reports on the stage-dependent evolution of NPPs, they also reveal that the evolution of NPPs is not synchronous: NPPs evolve differentially throughout the ring stage and only at the mid-trophozoite stage they are fully deployed in the majority of the infected cells, but not in all. This leads to desynchronization in the culture and to less than the maximal possible rate of multiplication.

Progress in in silico functional genomics: the malaria Metabolic Pathways database.

Genomic, transcriptomic and proteomic data can be turned into biologically meaningful information if they are synthesized into processes. Such amalgamation has been done for the most virulent malaria parasite Plasmodium falciparum in the Metabolic Pathways database. The dialectics of construction of metabolic pathways and other biological processes using this database is presented here. Additional features, such as links to other databases and the incorporation of transcriptomic clocks, are elucidated. Comparison of Metabolic Pathways to other similar databases is analyzed.

Arylpiperazines displaying preferential potency against chloroquine-resistant strains of the malaria parasite Plasmodium falciparum.

Arylpiperazines in which the terminal secondary amino group is unsubstituted were found to display a mefloquine-type antimalarial behavior in being significantly more potent against the chloroquine-resistant (W2 and FCR3) strains of Plasmodium falciparum than against the chloroquine-sensitive (D10 and NF54) strains. Substitution of the aforementioned amino group led to a dramatic drop in activity across all strains as well as abolition of the preferential potency against resistant strains that was observed for the unsubstituted counterparts. The data suggest that unsubstituted arylpiperazines are not well-recognized by the chloroquine resistance mechanism and may imply that they act mechanistically differently from chloroquine. On the other hand, 4-aminoquinoline-based heteroarylpiperazines in which the terminal secondary amino group is also unsubstituted, were found to be equally active against the chloroquine-resistant and chloroquine-sensitive strains, suggesting that chloroquine cross-resistance is not observed with these two 4-aminoquinolines. In contrast, two 4-aminoquinoline-based heteroarylpiperazines are positively recognized by the chloroquine resistance mechanism. These studies provide structural features that determine the antimalarial activity of arylpiperazines for further development, particularly against chloroquine-resistant strains.

Should chloroquine be laid to rest?

Chloroquine (CQ) has been the front line antimalarial drug due to its efficacy, low cost and scanty side effects, until resistance has evolved. Although its use has been officially discontinued in most malaria-affected countries, it is still widely used. Practical and pharmacological considerations indicate that it could be still used in semi-immune adults and that more efficient treatment protocols could be devised to treat even patients infected with CQ-resistant parasite strains. Since its antimalarial activity is pleiotropic, drug resistance may be due to different mechanisms, each amenable to reversal by drug combination.

How many functional transport pathways does Plasmodium falciparum induce in the membrane of its host erythrocyte?

Intraerythrocytic malaria parasites induce considerable change in the permeability of the membrane of their host cell. Using classical techniques of radiolabel uptake and iso-osmotic lysis, the permeability characteristics of the host-cell membrane have been determined. In a recent analysis of these results, we concluded that there are at least two types of channel that conform to the data: a low copy number (four channels per cell) type that mediates the transport of cations, anions and most other osmolytes that were tested, and a high copy number (300-400 channels per cell) type that is an anion channel that could also mediate the translocation of purine nucleosides. Patch-clamping experiments using cells infected with Plasmodium falciparum reveal 200-1000 anion channels of more than one type that are of host-cell endogenous provenance. Recent reports show that parasites can grow normally in erythrocytes that lack these endogenous agencies and in which the anion channels are not expressed, although their osmolyte permeability is present. We suggest that only the latter type of channel is important for normal development of the parasite.

Data mining of the transcriptome of Plasmodium falciparum: the pentose phosphate pathway and ancillary processes.

The general paradigm that emerges from the analysis of the transcriptome of the malaria parasite Plasmodium falciparum is that the expression clusters of genes that code for enzymes engaged in the same cellular function is coordinated. Here the consistency of this perception is examined by analysing specific pathways that metabolically-linked. The pentose phosphate pathway (PPP) is a fundamental element of cell biochemistry since it is the major pathway for the recycling of NADP+ to NADPH and for the production of ribose-5-phosphate that is needed for the synthesis of nucleotides. The function of PPP depends on the synthesis of NADP+ and thiamine pyrophosphate, a co-enzyme of the PPP enzyme transketolase. In this essay, the transcription of gene coding for enzymes involved in the PPP, thiamine and NAD(P)+ syntheses are analysed. The genes coding for two essential enzymes in these pathways, transaldolase and NAD+ kinase could not be found in the genome of P. falciparum. It is found that the transcription of the genes of each pathway is not always coordinated and there is usually a gene whose transcription sets the latest time for the full deployment of the pathway's activity. The activity of PPP seems to involve only the oxidative arm of PPP that is geared for maximal NADP+ reduction and ribose-5-phosphate production during the early stages of parasite development. The synthesis of thiamine diphosphate is predicted to occur much later than the expression of transketolase. Later in the parasite cycle, the non-oxidative arm of PPP that can use fructose-6-phosphate and glyceraldehyde-3-phosphate supplied by glycolysis, becomes fully deployed allowing to maximize the production of ribose-5-phosphate. These discrepancies require direct biochemical investigations to test the activities of the various enzymes in the developing parasite. Notably, several transcripts of PPP enzyme-coding genes display biphasic pattern of transcription unlike most transcripts that peak only once during the parasite cycle. The physiological meaning of this pattern requires further investigation.