An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase.

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

Author Correction: Arylmethylamino steroids as antiparasitic agents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies.

BACKGROUND: Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. METHODS: Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. RESULTS: Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. CONCLUSION: The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum.

Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction-oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54- attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- and long-term effects of selected antimalarial agents. Of note, the single components of the redox probes themselves did not influence the redox balance of the parasites. Our analyses revealed comparable results for both the hGrx1-roGFP2 and sfroGFP2 probes, with sfroGFP2 exhibiting a more pronounced fluorescence intensity in cellulo. Accordingly, the sfroGFP2 probe was employed to monitor the fluorescence signals throughout the parasites' asexual life cycle. Through the use of stable genomic integration, we demonstrate a means of overcoming the limitations of transient transfection, allowing more detailed in-cell studies as well as high-throughput analyses using plate reader-based approaches.

Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes.

Redox balance is essential for the survival, growth and multiplication of malaria parasites and oxidative stress is involved in the mechanism of action of many antimalarial drugs. Hydrogen peroxide (H2O2) plays an important role in redox signalling and pathogen-host cell interactions. For monitoring intra- and subcellular redox events, highly sensitive and specific probes are required. Here, we stably expressed the ratiometric H2O2 redox sensor roGFP2-Orp1 in the cytosol and the mitochondria of Plasmodium falciparum (P. falciparum) NF54-attB blood-stage parasites and evaluated its sensitivity towards oxidative stress, selected antimalarial drugs, and novel lead compounds. In both compartments, the sensor showed reproducible sensitivity towards H2O2 in the low micromolar range and towards antimalarial compounds at pharmacologically relevant concentrations. Upon short-term exposure (4 h), artemisinin derivatives, quinine and mefloquine impacted H2O2 levels in mitochondria, whereas chloroquine and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor affected the cytosol; 24 h exposure to arylmethylamino steroids and G6PD inhibitors revealed oxidation of mitochondria and cytosol, respectively. Genomic integration of an H2O2 sensor expressed in subcellular compartments of P. falciparum provides the basis for studying complex parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity, and sets the stage for high-throughput approaches to identify antimalarial agents perturbing redox equilibrium.

H2O2 dynamics in the malaria parasite Plasmodium falciparum.

Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity.

Arylmethylamino steroids as antiparasitic agents.

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC50 1-5 nM) as well as against gametocytes. In P. berghei-infected mice, oral administration of 1o drastically reduces parasitaemia and cures the animals. Furthermore, 1o efficiently blocks parasite transmission from mice to mosquitoes. The steroid compounds show low cytotoxicity in mammalian cells and do not induce acute toxicity symptoms in mice. Moreover, 1o has a remarkable activity against the blood-feeding trematode parasite Schistosoma mansoni. The steroid and the hydroxyarylmethylamino moieties are essential for antimalarial activity supporting a chelate-based quinone methide mechanism involving metal or haem bioactivation. This study identifies chemical scaffolds that are rapidly internalized into blood-feeding parasites.

Determination of glutathione redox potential and pH value in subcellular compartments of malaria parasites.

The malaria parasite Plasmodium falciparum is exposed to multiple sources of oxidative challenge during its complex life cycle in the Anopheles vector and its human host. In order to further elucidate redox-based parasite host cell interactions and mechanisms of drug action, we targeted the genetically encoded glutathione redox sensor roGFP2 coupled to human glutaredoxin 1 (roGFP2-hGrx1) as well as the ratiometric pH sensor pHluorin to the apicoplast and the mitochondrion of P. falciparum. Using live cell imaging, this allowed for the first time the determination of the pH values of the apicoplast (7.12±0.40) and mitochondrion (7.37±0.09) in the intraerythrocytic asexual stages of the parasite. Based on the roGFP2-hGrx1 signals, glutathione-dependent redox potentials of -267mV and -328mV, respectively, were obtained. Employing these novel tools, initial studies on the effects of redox-active agents and clinically employed antimalarial drugs were carried out on both organelles.

Detection of thiol-based redox switch processes in parasites - facts and future.

Malaria and African trypanosomiasis are tropical diseases caused by the protozoa Plasmodium and Trypanosoma, respectively. The parasites undergo complex life cycles in the mammalian host and insect vector, during which they are exposed to oxidative and nitrosative challenges induced by the host immune system and endogenous processes. Attacking the parasite's redox metabolism is a target mechanism of several known antiparasitic drugs and a promising approach to novel drug development. Apart from this aspect, oxidation of cysteine residues plays a key role in protein-protein interaction, metabolic responses to redox events, and signaling. Understanding the role and dynamics of reactive oxygen species and thiol switches in regulating cellular redox homeostasis is crucial for both basic and applied biomedical approaches. Numerous techniques have therefore been established to detect redox changes in parasites including biochemical methods, fluorescent dyes, and genetically encoded probes. In this review, we aim to give an insight into the characteristics of redox networks in the pathogens Plasmodium and Trypanosoma, including a comprehensive overview of the consequences of specific deletions of redox-associated genes. Furthermore, we summarize mechanisms and detection methods of thiol switches in both parasites and discuss their specificity and sensitivity.