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.

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.

Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase is a potential drug target.

The malarial parasite Plasmodium falciparum is exposed to substantial redox challenges during its complex life cycle. In intraerythrocytic parasites, haemoglobin breakdown is a major source of reactive oxygen species. Deficiencies in human glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate pathway (PPP), lead to a disturbed redox equilibrium in infected erythrocytes and partial protection against severe malaria. In P. falciparum, the first two reactions of the PPP are catalysed by the bifunctional enzyme glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase (PfGluPho). This enzyme differs structurally from its human counterparts and represents a potential target for drugs. In the present study we used epitope tagging of endogenous PfGluPho to verify that the enzyme localises to the parasite cytosol. Furthermore, attempted double crossover disruption of the PfGluPho gene indicates that the enzyme is essential for the growth of blood stage parasites. As a further step towards targeting PfGluPho pharmacologically, ellagic acid was characterised as a potent PfGluPho inhibitor with an IC50 of 76 nM. Interestingly, pro-oxidative drugs or treatment of the parasites with H2O2 only slightly altered PfGluPho expression or activity under the conditions tested. Furthermore, metabolic profiling suggested that pro-oxidative drugs do not significantly perturb the abundance of PPP intermediates. These data indicate that PfGluPho is essential in asexual parasites, but that the oxidative arm of the PPP is not strongly regulated in response to oxidative challenge.

Characterization of tryptophan aminotransferase 1 of Malassezia furfur, the key enzyme in the production of indolic compounds by M. furfur.

Malassezia yeasts are responsible for the widely distributed skin disease Pityriasis versicolor (PV), which is characterized by a hyper- or hypopigmentation of affected skin areas. For Malassezia furfur, it has been shown that pigment production relies on tryptophan metabolism. A tryptophan aminotransferase was found to catalyse the initial catalytic step in pigment formation in the model organism Ustilago maydis. Here, we describe the sequence determination, recombinant production and biochemical characterization of tryptophan aminotransferase MfTam1 from M. furfur. The enzyme catalyses the transamination from l-tryptophan to keto acids such as α-ketoglutarate with Km values for both substrates in the low millimolar range. Furthermore, MfTam1 presents a temperature optimum at 40°C and a pH optimum at 8.0. MfTam1 activity is highly dependent on pyridoxal phosphate (PLP), whereas compounds interfering with PLP, such as cycloserine (CS) and aminooxyacetate, inhibit the MfTam1 reaction. CS is known to reverse hyperpigmentation in PV. Thus, the results of the present study give a deeper insight into the role of MfTam1 in PV pathogenesis and as potential target for the development of novel PV therapeutics.

Crystal structure of the Plasmodium falciparum thioredoxin reductase-thioredoxin complex.

Over the last decades, malaria parasites have been rapidly developing resistance against antimalarial drugs, which underlines the need for novel drug targets. Thioredoxin reductase (TrxR) is crucially involved in redox homeostasis and essential for Plasmodium falciparum. Here, we report the first crystal structure of P. falciparum TrxR bound to its substrate thioredoxin 1. Upon complex formation, the flexible C-terminal arm and an insertion loop of PfTrxR are rearranged, suggesting that the C-terminal arm changes its conformation during catalysis similar to human TrxR. Striking differences between P. falciparum and human TrxR are a Plasmodium-specific insertion and the conformation of the C-terminal arm, which lead to considerable differences in thioredoxin binding and disulfide reduction. Moreover, we functionally analyzed amino acid residues involved in substrate binding and in the architecture of the intersubunit cavity, which is a known binding site for disulfide reductase inhibitors. Cell biological experiments indicate that P. falciparum TrxR is indeed targeted in the parasite by specific inhibitors with antimalarial activity. Differences between P. falciparum and human TrxR and details on substrate reduction and inhibitor binding provide the first solid basis for structure-based drug development and lead optimization.

Identification and characterization of novel human glucose-6-phosphate dehydrogenase inhibitors.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP(+). Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC(50) values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC(50) values. The most potent hG6PD inhibitors presented IC(50) values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC(50) ~25 µM) more strongly than that of normal MCF10-A cells (IC(50) >50 µM).

Discovery of a Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase inhibitor (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro.

A high-throughput screen of the NIH's MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum , the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC(50) = 889 nM). It is completely selective for the enzyme's human isoform, displays micromolar potency (IC(50) = 2.6 µM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.

Glucose-6-phosphate metabolism in Plasmodium falciparum.

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.

High-throughput screening for small-molecule inhibitors of plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase.

Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites' pentose phosphate pathway. In this study, we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries-namely, LOPAC (Sigma-Aldrich, 1280 compounds), Spectrum (MicroSource Discovery Systems, 1969 compounds), and DIVERSet (ChemBridge, 49 971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC(50) values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.