Enolase Inhibitors as Early Lead Therapeutics against Trypanosoma brucei.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against T. brucei ENO (TbENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC50 value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC50 value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of -6.8 and -7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of -4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against T. brucei, with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC50 values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against T. brucei and that further development of prodrug HEX analogs is warranted.

Hypothetical proteins play a role in stage conversion, virulence, and the stress response in the Entamoeba species.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and amoebic liver abscess in humans, affecting millions of people worldwide. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. As cysts can be ingested from contaminated food and water, this parasite is prevalent in underdeveloped countries and poses a significant health burden. Until recently there was no reliable method for inducing stage conversion in E. histolytica in vitro. As such, the reptilian pathogen, Entamoeba invadens, has long-served as a surrogate. Much remains unclear about stage conversion in these parasites and current treatments for amoebiasis are lacking, as they cause severe side effects. Therefore, new therapeutic strategies are needed. The genomes of these parasites remain enigmatic as approximately 54% of E. histolytica genes and 66% of E. invadens genes are annotated as hypothetical proteins. In this study, we characterized two hypothetical proteins in the Entamoeba species, EIN_059080, in E. invadens, and its homolog, EHI_056700, in the human pathogen, E. histolytica. EHI_056700 has no homolog in the human host. We used an RNAi-based silencing system to reduce expression of these genes in E. invadens and E. histolytica trophozoites. Loss of EIN_059080 resulted in a decreased rate of encystation and an increased rate of erythrophagocytosis, an important virulence function. Additionally, mutant parasites were more susceptible to oxidative stress. Similarly, loss of EHI_056700 in E. histolytica trophozoites resulted in increased susceptibility to oxidative stress and glucose deprivation, but not to nitrosative stress. Unlike the E. invadens mutants, E. histolytica parasites with decreased reduced expression of EHI_056700 exhibited a decreased rate of erythrophagocytosis of and adhesion to host cells. Taken together, these data suggest that these hypothetical proteins play a role in stage conversion, virulence, and the response to stress in the Entamoebae. Since parasites with reduced expression of EHI_056700 show decreased virulence functions and increased susceptibility to physiologically relevant stressors, EHI_056700 may represent a possible therapeutic target for the treatment of amoebiasis.

pH regulation in glycosomes of procyclic form Trypanosoma brucei.

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay.

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2.

The majority of the glycolytic enzymes in the African trypanosome are compartmentalised within peroxisome-like organelles, the glycosomes. Polypeptides harbouring peroxisomal targeting sequences (PTS type 1 or 2) are targeted to these organelles. This targeting is essential to parasite viability, as compartmentalisation of glycolytic enzymes prevents unregulated ATP-dependent phosphorylation of intermediate metabolites. Here, we report the surprising extra-glycosomal localisation of a PTS-2 bearing trypanosomal hexokinase, TbHK2. In bloodstream form parasites, the protein localises to both glycosomes and to the flagellum. Evidence for this includes fractionation and immunofluorescence studies using antisera generated against the authentic protein as well as detection of epitope-tagged recombinant versions of the protein. In the insect stage parasite, distribution is different, with the polypeptide localised to glycosomes and proximal to the basal bodies. The function of the extra-glycosomal protein remains unclear. While its association with the basal body suggests that it may have a role in locomotion in the insect stage parasite, no detectable defect in directional motility or velocity of cell movement were observed for TbHK2-deficient cells, suggesting that the protein may have a different function in the cell.

Glycerol 3-phosphate alters Trypanosoma brucei hexokinase activity in response to environmental change.

The African trypanosome, Trypanosoma brucei, compartmentalizes some metabolic enzymes within peroxisome-like organelles called glycosomes. The amounts, activities, and types of glycosomal enzymes are modulated coincident with developmental and environmental changes. Pexophagy (fusion of glycosomes with acidic lysosomes) has been proposed to facilitate this glycosome remodeling. Here, we report that, although glycosome-resident enzyme T. brucei hexokinase 1 (TbHK1) protein levels are maintained during pexophagy, acidification inactivates the activity. Glycerol 3-phosphate, which is produced in vivo by a glycosome-resident glycerol kinase, mitigated acid inactivation of lysate-derived TbHK activity. Using recombinant TbHK1, we found that glycerol 3-P influenced enzyme activity at pH 6.5 by preventing substrate and product inhibition by ATP and ADP, respectively. Additionally, TbHK1 inhibition by the flavonol quercetin (QCN) was partially reversed by glycerol 3-P at pH 7.4, whereas at pH 6.5, enzyme activity in the presence of QCN was completely maintained by glycerol 3-P. However, glycerol 3-P did not alter the interaction of QCN with TbHK1, as the lone Trp residue (Trp-177) was quenched under all conditions tested. These findings suggest potential novel mechanisms for the regulation of TbHK1, particularly given the acidification of glycosomes that can be induced under a variety of parasite growth conditions.

The anti-trypanosomal agent lonidamine inhibits Trypanosoma brucei hexokinase 1.

Glycolysis is essential to the parasitic protozoan Trypanosoma brucei. The first step in this metabolic pathway is mediated by hexokinase, an enzyme that transfers the gamma-phosphate of ATP to a hexose. The T. brucei genome (TREU927/4 GUTat10.1) encodes two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. Our previous efforts have revealed that TbHK2 is an important regulator of TbHK1 in procyclic form parasites. Here, we have found through RNAi that TbHK1 is essential to the bloodstream form parasite. Silencing the gene for 4 days reduces cellular hexokinase approximately 60% and leads to parasite death. Additionally, we have found that the recombinant enzyme is inhibited by lonidamine (IC(50)=850 microM), an anti-cancer drug that targets tumor hexokinases. This agent also inhibits HK activity from whole parasite lysate (IC(50)=965 microM). Last, lonidamine is toxic to cultured bloodstream form parasites (LD(50)=50 microM) and procyclic form parasites (LD(50)=180 microM). Interestingly, overexpression of TbHK1 protects PF parasites from lonidamine. These studies provide genetic evidence that TbHK1 is a valid therapeutic target while identifying a potential molecular target of the anti-trypanosomal agent lonidamine.

Residues in an ATP binding domain influence sugar binding in a trypanosome hexokinase.

Trypanosoma brucei harbors two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 has not, a finding attributed to differences in the C-termini of the proteins. Sequence analysis suggests that the C-termini of TbHKs are part of a newly identified conserved motif found in other eukaryotic hexokinases. Here, we have explored the role of tail residues in the differences in catalytic activity between TbHK1 and TbHK2. Our studies reveal that tail residues D454, F462, M466, and N469 are essential for HK activity while both I458 and V468 are required for catalysis and substrate specificity. To activate rTbHK2, all of the residues important for activity in rTbHK1 (D454, V458, F462, M466, V468, and N469) were required. These results indicate that the overall structure of the C-terminal tail influences the HK activity of rTbHK1.

Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-amino-acid C-terminal tail.

Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2-/- procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 +/- 0.02 mM and 0.28 +/- 0.1 mM, respectively. The k(cat) value for TbHK1 is 2.9 x 10(4) min(-1). TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 microM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.