Azithromycin disrupts apicoplast biogenesis in replicating and dormant liver stages of the relapsing malaria parasites Plasmodium vivax and Plasmodium cynomolgi.

The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapsed infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, although their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need.

Transfection Models to Investigate -Type Dormant Liver Stage Parasites.

causes the second highest number of malaria morbidity and mortality cases in humans. Several biological traits of this parasite species, including the formation of dormant stages (hypnozoites) that persist inside the liver for prolonged periods of time, present an obstacle for intervention measures and create a barrier for the elimination of malaria. Research into the biology of hypnozoites requires efficient systems for parasite transmission, liver stage cultivation and genetic modification.

Antimalarial Agents Targeting Plasmodium falciparum Carbonic Anhydrase: Towards Artesunate Hybrid Compounds with Dual Mechanism of Action.

Malaria continues to be a major public health challenge worldwide and, as part of the global effort toward malaria eradication, plasmodium carbonic anhydrases (CAs) have recently been proposed as potential targets for malaria treatment. In this study, a series of eight hybrid compounds combining the Artesunate core with a sulfonamide moiety were synthesized and evaluated for their inhibition potency against the widely expressed human (h) CAs I, II and the isoform from P. falciparum (PfCA).

Epigenetically regulated RNA-binding proteins signify malaria hypnozoite dormancy.

Dormancy enables relapsing malaria parasites, such as Plasmodium vivax and cynomolgi, to survive unfavorable conditions. It is enabled by hypnozoites, parasites remaining quiescent inside hepatocytes before reactivating and establishing blood-stage infection. We integrate omics approaches to explore gene-regulatory mechanisms underlying hypnozoite dormancy.

Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi.

Background: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection.

Sterile protection against relapsing malaria with a single-shot vaccine.

Vaccine development for Plasmodium vivax, an important human relapsing malaria, is lagging behind. In the case of the most deadly human malaria P. falciparum, unprecedented high levels of protection have been obtained by immunization with live sporozoites under accompanying chemoprophylaxis, which prevents the onset of blood-stage malaria.

Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Anti-relapse Compounds.

Malaria hypnozoites are dormant parasite stages that reside inside hepatocytes. Upon activation, these stages can resume growth, causing new episodes of blood stage malaria infection. This chapter describes a fast and sensitive protocol for the detection of bioluminescent (BL) hypnozoites in vitro.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) () is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard -rhesus monkey () model, but in a small clinical trial, it had no efficacy against hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and liver-stage activity of and its metabolites. Compound had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the -desmethyl, combined -desmethyl/-desethyl, and -didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of in rhesus monkeys vs that seen in humans.

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite.

Novel bis-1,2,4-triazine compounds with potent activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites and .

Modeling Relapsing Malaria: Emerging Technologies to Study Parasite-Host Interactions in the Liver.

Recent studies of liver stage malaria parasite-host interactions have provided exciting new insights on the cross-talk between parasite and its mammalian (predominantly rodent) host. We review the latest state of the art and and zoom in on new technologies that will provide the tools necessary to investigate host-parasite interactions of relapsing parasites. Interactions between hypnozoites and hepatocytes are particularly interesting because the parasite can remain in a quiescent state for prolonged periods of time and triggers for reactivation have not been irrefutably identified.

Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Antirelapse Compounds.

Efforts to eradicate malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic parasite line that contains Nanoluc driven by the constitutive promoter, as well as firefly luciferase driven by the schizont-specific promoter.

A dual fluorescent reporter line reveals in vitro malaria hypnozoite reactivation.

malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P.

Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite during progression into dormancy.

Relapses of dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al.

A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite .

liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite .

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P.

Antimalarial efficacy of MMV390048, an inhibitor of phosphatidylinositol 4-kinase.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all life cycle stages, apart from late hypnozoites in the liver.

Plasmodium knowlesi: a relevant, versatile experimental malaria model.

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase.

A Triazolopyrimidine-Based Dihydroorotate Dehydrogenase Inhibitor with Improved Drug-like Properties for Treatment and Prevention of Malaria.

The emergence of drug-resistant malaria parasites continues to hamper efforts to control this lethal disease. Dihydroorotate dehydrogenase has recently been validated as a new target for the treatment of malaria, and a selective inhibitor (DSM265) of the Plasmodium enzyme is currently in clinical development. With the goal of identifying a backup compound to DSM265, we explored replacement of the SF-aniline moiety of DSM265 with a series of CF-pyridinyls while maintaining the core triazolopyrimidine scaffold.

High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria.

In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format.

PI4 Kinase Is a Prophylactic but Not Radical Curative Target in Plasmodium vivax-Type Malaria Parasites.

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally.

Lead optimization of imidazopyrazines: a new class of antimalarial with activity on Plasmodium liver stages.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P.

Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures.

Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi.

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax).