Antimalarial drugs are key tools for the control and elimination of malaria. Recent decreases in the global malaria burden are likely due, in part, to the deployment of artemisinin-based combination ...therapies. Therefore, the emergence and potential spread of artemisinin-resistant parasites in southeast Asia and changes in sensitivities to artemisinin partner drugs have raised concerns. In recognition of this urgent threat, the International Centers of Excellence for Malaria Research (ICEMRs) are closely monitoring antimalarial drug efficacy and studying the mechanisms underlying drug resistance. At multiple sentinel sites of the global ICEMR network, research activities include clinical studies to track the efficacies of antimalarial drugs, ex vivo/in vitro assays to measure drug susceptibilities of parasite isolates, and characterization of resistance-mediating parasite polymorphisms. Taken together, these efforts offer an increasingly comprehensive assessment of the efficacies of antimalarial therapies, and enable us to predict the emergence of drug resistance and to guide local antimalarial drug policies. Here we briefly review worldwide antimalarial drug resistance concerns, summarize research activities of the ICEMRs related to drug resistance, and assess the global impacts of the ICEMR programs.
Malaria remains a globally prevalent infectious disease that leads to significant morbidity and mortality. While there are a number of drugs approved for its treatment, drug resistance has ...compromised most of them, making the development of new drugs for the treatment and prevention of malaria essential. The completion of the Plasmodium falciparum genome and a growing understanding of parasite biology are fueling the search for novel drug targets. Despite this, few targets have been chemically validated in vivo. The pyrimidine biosynthetic pathway illustrates one of the best examples of successful identification of anti-malarial drug targets. This review focuses on recent studies to exploit the fourth enzyme in the de novo pyrimidine biosynthetic pathway of P. falciparum, dihydroorotate dehydrogenase (PfDHODH), as a new target for drug discovery. Several chemical scaffolds have been identified by high throughput screening as potent inhibitors of PfDHODH and these show strong selectivity for the malarial enzyme over that from the human host. Potent activity against parasites in whole cell models with good correlation between activity on the enzyme and the parasite have also been observed for a number of the identified series. Lead optimization of a triazolopyrimidine-based series has identified an analog with prolonged plasma exposure, that is orally bioavailable, and which shows good efficacy against the in vivo mouse model of the disease. These data provide strong evidence that PfDHODH is a validated target for the identification of new antimalarial chemotherapy. The challenge remains to identify compounds with the necessary combination of potency and metabolic stability to allow identification of a clinical candidate.
More than a century after the discovery of Plasmodium spp. parasites, the pathogenesis of severe malaria is still not well understood. The majority of malaria cases are caused by Plasmodium ...falciparum and Plasmodium vivax, which differ in virulence, red blood cell tropism, cytoadhesion of infected erythrocytes, and dormant liver hypnozoite stages. Cerebral malaria coma is one of the most severe manifestations of P. falciparum infection. Insights into its complex pathophysiology are emerging through a combination of autopsy, neuroimaging, parasite binding, and endothelial characterizations. Nevertheless, important questions remain regarding why some patients develop life-threatening conditions while the majority of P. falciparum-infected individuals do not, and why clinical presentations differ between children and adults. For P. vivax, there is renewed recognition of severe malaria, but an understanding of the factors influencing disease severity is limited and remains an important research topic. Shedding light on the underlying disease mechanisms will be necessary to implement effective diagnostic tools for identifying and classifying severe malaria syndromes and developing new therapeutic approaches for severe disease. This review highlights progress and outstanding questions in severe malaria pathophysiology and summarizes key areas of pathogenesis research within the International Centers of Excellence for Malaria Research program.
Malaria is one of the most serious global infectious diseases. The pyrimidine biosynthetic enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is an important target for antimalarial ...chemotherapy. We describe a detailed analysis of protein–ligand interactions between DHODH and a triazolopyrimidine-based inhibitor series to explore the effects of fluorine on affinity and species selectivity. We show that increasing fluorination dramatically increases binding to mammalian DHODHs, leading to a loss of species selectivity. Triazolopyrimidines bind Plasmodium and mammalian DHODHs in overlapping but distinct binding sites. Key hydrogen-bond and stacking interactions underlying strong binding to PfDHODH are absent in the mammalian enzymes. Increasing fluorine substitution leads to an increase in the entropic contribution to binding, suggesting that strong binding to mammalian DHODH is a consequence of an enhanced hydrophobic effect upon binding to an apolar pocket. We conclude that hydrophobic interactions between fluorine and hydrocarbons provide significant binding energy to protein–ligand interactions. Our studies define the requirements for species-selective binding to PfDHODH and show that the triazolopyrimidine scaffold can alternatively be tuned to inhibit human DHODH, an important target for autoimmune diseases.
Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote ...the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A critical assessment of new opportunities for drug discovery to treat malaria
Malaria parasites, carried by mosquitoes and transmitted to humans, infect ∼200 million individuals and cause ∼500,000 ...deaths each year (
1
). Fifteen years after identifying the genome sequence of a malaria-causing parasite,
Plasmodium falciparum
, malaria treatments still rely heavily on chemicals derived from natural products that were used centuries ago (
2
). With cell-based functional assays, the gap between genome sequences of
Plasmodium
spp. and the identification of valuable new therapeutics may be reduced by determining which genes are essential for parasite propagation in the disease-causing blood stage of the parasite life-cycle. The most potent and clinically useful antimalarial drugs rapidly eliminate parasites growing in red blood cells (RBCs). On page 506 of this issue, Zhang
et al.
(
3
) report a mutagenesis screen on
P. falciparum
cultured in human RBCs, identifying 2680 indispensable parasite genes, which may contain antimalarial drug targets. However, it is important to critically assess what fraction of these essential genes will be good drug targets and how one should prioritize such targets for drug discovery.
Due to the spread of resistance to front-line artemisinin derivatives worldwide, there is a need for new antimalarials. Tartrolon E (TrtE), a secondary metabolite of a symbiotic bacterium of marine ...bivalve mollusks, is a promising antimalarial because it inhibits the growth of sexual and asexual blood stages of
at sub-nanomolar levels. The potency of TrtE warrants further investigation into its mechanism of action, cytotoxicity, and ease with which parasites may evolve resistance to it.
Malaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in ...the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase (
Gyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear-encoded
GyrA was disrupted using CRISPR/Cas9 gene editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of
ΔGyrA was possible in the presence of IPP.
GyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis, but there is a need for a more detailed description of the mode of action of ciprofloxacin in malaria parasites. As predicted,
ΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin but not to the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued
ΔGyrA, possibly suggesting that ciprofloxacin may have an additional nonapicoplast target in P. falciparum. Overall, we confirm that
GyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.
Summary
Splenic filtration of infected red blood cells (RBCs) may contribute to innate immunity and variable outcomes of malaria infections. We show that filterability of individual RBCs is well ...predicted by the minimum cylindrical diameter (MCD) which is calculated from a RBC's surface area and volume. The MCD describes the smallest diameter tube or smallest pore that a cell may fit through without increasing its surface area. A microfluidic device was developed to measure the MCD from thousands of individual infected RBCs (IRBCs) and uninfected RBCs (URBCs). Average MCD changes during the blood‐stage cycle of Plasmodium falciparum were tracked for the cytoadherent strain ITG and the knobless strain Dd2. The MCD values for IRBCs and URBCs raise several new intriguing insights into how the spleen may remove IRBCs: some early‐stage ring‐IRBCs, and not just late‐stage schizont‐IRBCs, may be highly susceptible to filtration. In addition, knobby parasites may limit surface area expansions and thus confer high MCDs on IRBCs. Finally, URBCs, in culture with IRBCs, show higher surface area loss which makes them more susceptible to filtration than naive URBCs. These findings raise important basic questions about the variable pathology of malaria infections and metabolic process that affect volume and surface area of IRBCs.
Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of ...infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-µm widths were readily traversed by the 8 µm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-µm channels; some that passed through the 4-µm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-µm channels, trophozoites mimicked "pitting," a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-µm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-µm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.
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